研究目的
To develop a target-selective aptamer-based platform for the reliable detection of Escherichia coli (E. coli) bacteria in Phosphate-buffered saline (PBS) using fluorescent microscopy, and to propose a cell-counting application based on the Neubauer Lam method.
研究成果
The aptamer-based platform on Au-micro-squares is highly selective for E. coli detection, as demonstrated by fluorescence microscopy. The method was successfully applied to a Neubauer Lam for accurate cell counting, showing potential for multiplexed detection of different cell types with small sample volumes. This approach offers a reliable and selective biosensing tool for bacterial detection.
研究不足
The study is limited to detection in PBS solution and may not directly apply to complex real-world samples like food or water without further optimization. The use of fluorescence microscopy might have sensitivity constraints, and the platform's scalability and cost for large-scale applications are not addressed.
1：Experimental Design and Method Selection:
The study employs a solid-state platform with an array of Au-micro-squares for immobilizing aptamers and detecting bacteria cells using fluorescence microscopy. The design includes aptamer immobilization, binding to target cells, and validation of selectivity and cell counting.
2：Sample Selection and Data Sources:
E. coli cells (ATCC generic strain 25922) and Pseudomonas bacteria cells were cultured and used. Aptamers were purchased from Bioneer Corporation.
3：List of Experimental Equipment and Materials:
Includes a fluorescence microscope (OLYMPUS BX43), spectrophotometer (Spectrophotometer 259, Sherwood), centrifuge, DC sputtering equipment for gold layer deposition, and various chemicals like dithiothreitol (DTT), sodium acetate, ethyl acetate, and Phosphate-buffered saline (PBS).
4：Experimental Procedures and Operational Workflow:
Aptamers were prepared by dissolving in water, adding DTT, incubating, and extracting with ethyl acetate. Bacteria were cultured, centrifuged, and suspended in PBS. The Au-micro-array platform was fabricated using chemical vapor deposition and etching. Aptamers were immobilized on the array, incubated, washed, and then exposed to bacteria solutions. Fluorescence images were taken before and after binding to assess selectivity and intensity changes. The Neubauer Lam was coated with gold and used for cell counting validation.
5：Data Analysis Methods:
Fluorescence intensity changes were compared visually from microscopic images to confirm binding and selectivity. Cell concentrations were determined using spectrophotometry (OD600 measurements).
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