细胞代谢过程溶液pH值和溶解氧单光纤双参数测量方法及机理研究

Energy metabolism is the basic vital movement for living cells. If the change of the chemical parameter concentration is monitored dynamically,the mechanism of metabolism can be analyzed, the source of diseases can thus be revealed. However, there is still no efficient method for measurement of multi-parameter with high sensitivity for the detection of the parameters in this process nowadays. The main reason is the big size of detectors will induce interference when several detectors work in parallel. A new method for the simultaneous measurement of pH and dissolved oxygen with one single optical fiber is proposed. In this method, particles of Quantum Dot indicator are embedded in a photonic crystal gel to form the cladding of the optical fiber. The technique for modifying different type of indicator onto the surface of the quantum dot and the embedding of quantum dot indicator into the photonic crystal gel are discussed. The mechanism of dissolved oxygen to quantum dot indicator and H+ to photonic crystal gel are also analyzed. The relationship between the quenching time of the indicator and the concentration of dissolved oxygen is established. The principle of the output spectrum and the pH value of the solution is revealed. Size of the holes in the gel is changed by the H+ in the solution and the spectrum of the output light is thus changed. The fluorescence signal of the particle is quenched by the oxygen dissolved in the resolution. The life-time of fluorescence signals from the indicator and the quantum dot are referenced in frequency domain by the proposed method with two modulating frequency.The interference is thus resisted. The method for producing the dual-parameter probe is proposed and the experiment system is established. The measurement accuracy can be better than 0.01 for pH value and 0.01mg per Litre for dissolved oxygen. The proposed method will provide a practical technique for the realization of cellular energy metabolism apparatus.

细胞能量代谢过程中培养液内各种成分的变化是分析代谢机理、揭示疾病发生根源的重要指标，但现存的多个探头并列进行参数同时测量的方法体积大、探头间相互干扰严重。本项目提出一种在单根光纤表面制备包埋量子点指示剂颗粒的光子晶体凝胶包层，实现溶液pH值和溶解氧同时测量的新方法，保证参数间互不干扰。项目研究不同种类指示剂修饰到量子点表面和量子点指示剂颗粒在光子晶体凝胶包层内的固定方法；分析量子点指示剂颗粒对溶解氧和光子晶体凝胶对pH值的各自敏感机理，建立指示剂荧光淬灭时间和溶解氧浓度以及光子晶体输出光谱变化与溶液pH值之间的关系；研究频率域的信号参比处理方法，增强溶解氧测量的抗干扰能力；研究单光纤溶解氧和pH值双参数敏感探头制备方法并进行溶液浓度测量试验验证，保证达到pH0.01和溶解氧0.01mg/L的测量灵敏度。本研究解决单光纤双参数测量的机理和方法问题，为细胞能量代谢实时测定仪的实现奠定基础。

本课题主要完成了以下五方面的研究内容：.（1）完成了稳定的高灵敏度pH、氧双敏感量子点指示剂的生成。.完成稳定的核壳型量子点的制备方法研究，完成在核壳型量子点CdSe/ZnS表面修饰氧敏感指示剂的方法研究。.（2）完成指示剂中量子点荧光强度与溶液pH值的数学模型建立及稳定性分析。.使用标准仪器荧光分光光度计进行量子点荧光强度测量，使用标准pH计对溶液pH进行测量，建立量子点荧光强度与溶液pH值的关系的数学模型，完成量子点稳定性分析，考虑温度等因素的影响，确定所建立数学模型的正确性。.（3）完成指示剂中香豆素荧光强度与溶液DO值的数学模型建立及稳定性分析。.使用标准仪器荧光分光光度计进行香豆素荧光强度测量，使用标准溶氧仪对溶液DO值进行测量。建立香豆素的荧光强度与溶液DO值关系的数学模型，并验证数学模型的准确性。.（4）完成利用指示剂实现溶液pH值、DO值的双参数测量方法。.将以上两个模型进行整合优化，建立所用指示剂的两个不同波长荧光强度与溶液pH值、DO值关系的数学模型，实现通过一种指示剂完成溶液pH值、DO值的双参数测量方法。使用标准仪器荧光分光光度计测量荧光强度，使用标准pH计测量溶液pH值，使用标准溶氧仪测量溶液DO值，验证所提出的双参数测量方法的正确性。.（5）完成细胞代谢双参数检测系统的设计与搭建及其稳定性分析。.为了实现微量溶液的双参数测量，在原方案基础上做出适当调整，将原定的制作光纤探头的思路转为制作台式仪器的思路，以实现极微量溶液的测量。根据所研究的溶液双参数测量方法，设计并搭建细胞代谢双参数检测系统，完成双参数检测系统算法设计，应用此系统进行微量溶液pH值、DO值的测量，完成系统验证及系统算法验证，为形成实用化的细胞代谢参数检测仪奠定基础。经过理论分析和实验验证，该双参数检测系统可以进行微量溶液pH值、DO值的同步在线检测，测量pH值的灵敏度为0.009个pH，测量DO值的灵敏度为0.008mg/L，完成所需目标。