Biosensor Detection of Cytoplasmic Analytes

3.3.1. Preparation of Detector Cell

Cells to be used as IP3 detectors are prepared as follows (5-8,17):

1. Plate BHK21 cells 24 to 48 h prior to experiments in an open cell chamber made by attaching a silicon "O" ring to a no. 1 glass cover slip.

2. Coat the cover slip with collagen to improve cell adherence to the glass by pipeting 200 ||L collagen solution onto the cover slip followed by air drying. If desired, the coated cover slip can be sterilized by a 70% ethanol wash.

3. Maintain the cells in a humidified 37°C, 5% CO2 incubator in minimum essential medium (MEM) until use.

4. Prior to the experiments, the cells are incubated for 1 h in Mag-fura-2 (25 ||M) and washed.

5. Incubate the cells for 5 min with digitonin (20 |lg/mL) in buffer A with 100 ||M Ca2+ (see Note 6).

6. Wash the cells five times in buffer A with 900 nM Ca2+.

7. Maintain the detector cells in buffer A with 900 nM free Ca2+ during electrophoresis.

3.3.2. Positioning of Capillary Outlet With Respect to the Cell Place the capillary the specified distance above the cell as follows:

1. Bring the upper portion of the cell into focus by visualizing the cell through the microscope eyepiece.

2. Use the micrometer markers on the focusing knob of the microscope to move the focal plane of the microscope lens (100x, 1.3 numerical aperture) above the cell by the required distance.

3. Lower the capillary outlet mounted to a three-axis micromanipulator until the bottom (or outlet) end of the lumen is in focus.

4. Return the micrometer to its previous setting to ensure that the cell remains focused in the original focal plane of the capillary once the capillary outlet is lowered to the specified height above the cell.

3.3.3. Imaging of Detector Cell

1. Mount the dish containing the cells on the stage of an inverted fluorescence microscope.

2. Center a single detector cell under the capillary and illuminate at 380 nm (D380x filter, Chroma Technologies, Brattleboro, VT). The intensity of the excitation light is adjusted so that negligible photobleaching occurs during the time of experiments. Emission light from the cell is spectrally filtered (D510/40 filter, Chroma Technologies) and collected with a photomultiplier tube (PMT) or intensified charged coupled device (CCD) camera. Data points are acquired by integrating the fluorescence signal from the cell over 5 s.

3.3.4. Construction of a Calibration Curve for IP3

The detector cell for IP3 functions as a detector of the mass of eluted IP3 (6). To quantify the amount of IP3 in a sample, a calibration curve must be constructed that correlates the detector cell fluorescence change with the amount of IP3 in the sample.

1. Electrophorese a known quantity of IP3 onto a detector cell and measure the fluorescence increase of the cell (5).

2. After the fluorescence has returned to baseline, add a maximally stimulating concentration of IP3 (5 |M) to the chamber containing the cell. The calibrated response is defined as the ratio of the peak area of the electrophoresed IP3 to the peak height after the 5-|M calibration exposure.

3. Repeat this process using varying quantities of IP3 to establish the calibration curve. The response of an unknown sample is compared to the curve to determine the number of moles of IP3 in the sample. This value is divided by the sample volume (see Subheading 3.1.2., step 4) to calculate the concentration of IP3 in the sample.

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