Preparation of Optical Biosensor Using Immobilized Microalgae 1819

3.2.1. Preparation of Microalgal Membrane

1. Cultivate the microalgae (for example Chlorella vulgaris strain CCAP 211/12) in sterile culture medium.

2. To induce high AP activity, centrifuge the algal cultures and resuspend the pellets in phosphate-free medium for 25 d, which corresponds to maximal enzyme activity. But for the biosensors based on chlorophyll fluorescence measurement cultivate the algae under continuous mode (see Note 4).

3. Filter 2 mL of the resulting aliquot on a glass microfiber filter (GF/C, Whatman).

5. Keep the glass microfiber filter in a desiccator for 30 min before fitting to the optical transducer.

3.2.2. Construction of Algal Optical Biosensor

1. Place the algal membrane in a 1-mL flow cell (see Fig. 3).

2. Place a bifurcated bundle of randomized optical fibers opposite to the membrane. The orientation should be in such a way that the incident light after hitting the upper part of the membrane should be transmitted to the Fluorometer.

3.2.3. Operation of Algal Optical Biosensor

1. Test the biosensor by adding the MUP solution into the microplate wells containing Chlorella vulgaris (see Notes 5 and 6). Testing can also be done by measuring chlorophyll fluorescence directly.

2. Feed the sensor with toxic-free solution, called reference, at first.

3. Then introduce herbicide/heavy metal solution and measure the biosensor response.

4. Analyze the response of the sensor to pollutants by comparing the peak heights.

5. After running the toxic compounds, reversibility of the biosensor should be tested each time (see Note 7).

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