基于可逆加成-断裂链转移聚合的孕酮分子印迹膜制备与检测

Preparation and detection of progesterone by molecularly imprinted film based on reversible addition fragmentation chain transfer polymerization

基于可逆加成-断裂链转移(reversible addition fragmentation chain transfer,RAFT)聚合原理,通过紫外光引发方式,以2-(十二烷基硫代碳酸酯基)-2-甲基丙烯酸(DDMAT)作为唯一的控制试剂,在DDMAT修饰的金膜表面合成了针对孕酮分子的分子印迹物聚合物(molecularly imprinted polymers,MIPs)薄膜,并作为表面等离子体共振(SPR)传感器的识别单元.通过现场原位监控MIPs薄膜生长动力学,使其厚度得到有效控制.聚合动力学研究表明,DDMAT在紫外光引发聚合过程中起到引发剂和链转移试剂双重作用.对MIPs薄膜表面进行接触角、红外光谱、扫面电子显微镜表征,结果表明通过紫外光引发的方式能够有效地将MIPs薄膜接枝到DDMAT修饰的金膜表面,且MIPs薄膜厚度均一,其表面布满的纳米尺寸孔径的孔穴,为识别孕酮分子提供了通道.在pH 7.4的PBS缓冲液中对浓度范围10^(-12)~10^(-7) mol/L的孕酮样品进行检测,结果表明该MIPs薄膜修饰的传感器对孕酮分子具有较高的灵敏度,检测限为3.24×10^(-13) mol/L(信噪比,S/N=3),且具有良好的选择识别性能和重复使用性能;稳定性实验结果显示,MIPs薄膜修饰的SPR传感器芯片在N_2气保护下,室温存储30 d对低浓度的孕酮分子样品仍具有良好的信号响应.实际水体及人工尿液中测得孕酮回收率为92.9%~96.5%,说明此传感器可用于实际样品测定.

Molecularly imprinted technique is a versatile and straightforward way to produce artificial receptors for a given target molecule. The molecularly imprinted polymers （MIPs） have received widespread attention and application, owing to their high physical stability, remarkable robustness and low cost. Despite the tremendous progress made in this field, many changes still remain to be solved. In general, MIPs are prepared by traditional free radical polymerization. However, this approach causes problems such as low-affinity binding, high diffusion barrier, low-rate mass transfer. In particular, it has been demonstrated that most of the MIPs fail to exhibit specific recognition towards the target molecules in aqueous environments although they can be successfully applied to recognize the target molecules in organic solvent media, which severely limits their potential application in some fields. In this work, a new method to prepare MIPs film for the detection of progesterone based on reversible addition-fragmentation chain transfer （RAPT） polymerization is presented. 2-Methyl-2-[（dodecylsulfanylthiocarbonyl）sulfanyl]propanoic acid （DDMAT） was employed as the sole control agent in the UV polymerization. The SPR sensor chip with 50 nm Au was firstly immersed in an ethanol solution of cysteamine （5 mmol/L） for 24 h. The cysteamine modificated chip was washed by pure ethanol solvent repeatedly and dried under a stream of nitrogen. Then the chip was placed in 40 mL acetonitrile solution with 14.6 mg DDMAT and 16.5 mg N,N＂ -dicyclohexylcarbodiimide （DCC） for 24 h. After that, the chip was washed with acetonitrile several times and dried under a stream of nitrogen. The MIPs film was grafted by UV irradiated without adding additional photo initiator or catalyst in the presence of progesterone （31.4 rag）, methacrylic acid （33.4 gL）, ethylene glycol dimethacrylate （338.5pL） and DDMAT （15 mg） in 4 mL acetonitrile. The growth process of the MIPs film was monitored by surface plasmon resonance （SPR） in situ. A mixture of PBS （pH 7.4） buffer and acetic acid （v：v=9： 1） was used as a washing solution to remove the template molecules from the MIPs film. Results indicated that DDMAT acted as both photo initiator and chain transfer agent during the polymerization. The MIPs film was characterized by contact angle measurements, infrared spectroscopy and scanning electron microscope （SEM）. Analysis of SPR spectroscopy showed that the MIPs film coated sensor displayed good sensitivity and selectivity to progesterone compared to other analytes with a similar structure. The response of the progesterone ranged from 10-12 to 10-7 mol]L with a limit of detection （LOD） of 3.24x10-13 mol/L （S/N=3） in PBS buffer （pH 7.4）. The MIPs film coated sensor also showed good reusability and stability, and kept about 81% of its original response after 30 d of storage under N2 protection at room temperature. In addition, the SPR sensor was successfully used to determine progesterone in tap water, underground water and artificial urine samples with recoveries from 92.9% to 96.5%, which indicated the SPR sensor can be applied for the real sample detection. This method will expand the surface modification of the MIPs film on the surface of different sensors and is a great candidate for rapid, simple and label-free detection of molecules in aqueous solution. Finally, this study opens a door to re-know the roles of chain transfer agents （DDMAT, in this study） in synthesizing MIPs and we believe that the properties of various chain transfer agents will be excavated and utilized in future study.