The Polonator Flow Cell
The Polonator flow cell has been carefully designed to permit sequential biochemistry cycles to be performed upon the beads bound to the underside of the cover slip during its arraying protocol (above). It has also been designed to maximize the imaging area (number of beads), minimize reagent consumption, and provide good thermal conductivity to minimize the time required for thermal cycling. A detailed drawing of the Polonator flow cell is shown below:
Each flow cell is individually loaded into the flow cell carrier. Depending on their throughput goals, users can elect to perform either a single or a dual flow cell sequencing run. When a single flow cell sequencing run is planned, that flow cell, designated as flow cell 0, is loaded into the left-hand side of the carrier. If a dual flow cell run is planned, a second flow cell, designated as flow cell 1, is added; it is loaded into the right-hand side of the carrier. To load a flow cell, loosen the locking knob at the front of the flow cell carrier (Fig 2), lift the front preload bar up and out of the way, and slide the flow cell into the carrier. The glass side faces up, and the entry and exit ports are to the rear. Friction will increase as the docking ports slide into the flow cell body; press firmly until the flow cell bottoms out on the docking ports. The front preload bar is then lowered into place, and the locking knob is tightened until the preload bar is in contact with the front of the carrier.
Reagents enter the flow cell one at a time, via the entry port at the center rear of the flow cell. The reagents are routed upwards to the entry bifurcations, which divide the reagents equally among eight parallel lanes. Lane 0 is the leftmost lane of each flow cell, and lane 7 is the rightmost. Biochemistry takes place between the reagents and DNA on the surface of beads, which are bound to the glass above each lane. After being drawn into the flow cell, reagents are heated or cooled to their optimal reaction temperature by Peltier heating/cooling elements beneath each flow cell, and then reagents are then left to react for a programmed period of time. The reagent is then drawn out through the exit bifurcations and return line, routed down into the flow cell body, and out the exit port on the left rear of the flow cell.
The Polonator acquires 2,180 images per lane in each of four colors for every base that is read. The lane numbering, as well as the layout of images within the lanes is shown in the diagram below:
At the conclusion of a sequencing run, the X-Y stage moves the flow cell carrier to the front and center of travel. The user loosens the captive locking knob, lifts the front preload bar up and out of the way, and removes each flow cell by pulling it out from the rear. The flow cell is a one-use consumable item, and should be discarded in a manner consistent with local regulations.