Roshid Md Harun Or, Moraskie Michael, O'Connor Gregory, Dikici Emre, Zingg Jean-Marc, Deo Sapna, Bachas Leonidas G, Daunert Sylvia
Department of Chemistry, University of Miami, Miami, FL 33146.
Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136.
Microchem J. 2023 Oct;193. doi: 10.1016/j.microc.2023.109088. Epub 2023 Jul 17.
A portable, field deployable whole-cell biosensor was developed that can withstand the complex matrices of soil and requires minimal to no sample preparation to monitor bioavailable concentrations of the essential micronutrient copper (II). Conventional measurement of micronutrients is often complex, laboratory-based, and not suitable for monitoring their bioavailable concentration. To address this need, we developed a fluorescence based microbial whole-cell biosensing (MWCB) system encoding for a Cu-responsive protein capable of generating a signal upon binding to Cu. The sensing-reporting protein was designed by performing circular permutation on the green fluorescent protein (GFP) followed by insertion of a Cu binding motif into the structure of GFP. The design included insertion of several binding motifs and creating plasmids that encoded the corresponding sensing proteins. The signal generated by the sensing-reporting protein is directly proportional to the concentration of Cu in the sample. Evaluation of the resulting biosensing systems carrying these plasmids was performed prior to selection of the optimal fluorescence emitting Cu-binding protein. The resulting optimized biosensing system was encapsulated in polyacrylate-alginate beads and embedded in soil for detection of the analyte. Once exposed to the soil, the beads were interrogated to measure the fluorescence signal emitted by the sensing-reporting protein using a portable imaging device. The biosensor was optimized for detection of Cu in terms of selectivity, sensitivity, matrix effects, detection limits, and reproducibility in both liquid and soil matrices. The limit of detection (LoD) of the optimized encapsulated biosensor was calculated as 0.27 mg/L and 1.26 mg/kg of Cu for Cu in solution and soil, respectively. Validation of the portable imaging tools as a potential biosensing device in the field was performed.
开发了一种便携式、可现场部署的全细胞生物传感器，它能够耐受土壤的复杂基质，并且监测必需微量营养元素铜（II）的生物可利用浓度时所需的样品制备极少甚至无需样品制备。微量营养元素的传统测量方法通常很复杂，需要在实验室进行，且不适用于监测其生物可利用浓度。为满足这一需求，我们开发了一种基于荧光的微生物全细胞生物传感（MWCB）系统，该系统编码一种铜响应蛋白，该蛋白在与铜结合时能够产生信号。通过对绿色荧光蛋白（GFP）进行环状排列，然后将铜结合基序插入GFP结构中，设计了传感报告蛋白。该设计包括插入几个结合基序并创建编码相应传感蛋白的质粒。传感报告蛋白产生的信号与样品中铜的浓度成正比。在选择最佳荧光发射铜结合蛋白之前，对携带这些质粒的生物传感系统进行了评估。将得到的优化生物传感系统封装在聚丙烯酸酯 - 藻酸盐珠中，并嵌入土壤中以检测分析物。一旦暴露于土壤中，就使用便携式成像设备对珠子进行检测，以测量传感报告蛋白发出的荧光信号。该生物传感器在选择性、灵敏度、基质效应、检测限以及在液体和土壤基质中的重现性方面针对铜的检测进行了优化。优化后的封装生物传感器对溶液和土壤中铜的检测限（LoD）分别计算为0.27 mg/L和1.26 mg/kg。对便携式成像工具作为现场潜在生物传感设备进行了验证。
