Modelos Pedagogicos

The BioSpot device was a nano pipetting system for non-contact liquid handling and was fabricated and provided with appropriate software by the company BioFluidix GmbH (Freiburg, Germany). It comprised several parts featuring a) a PipeJet dispenser moving in Z-directions for liquid uptake and delivery, b) a sample slay fixed on a slide rail moving in X- directions and c) a power control box also housing a syringe pump supporting aspiration and dispension of liquid. Onto the sample slay the chip-holder device of the CytoCycler device was installed, carrying a flask-filled reservoir device (figure 10 A). The slide rail was about 70 cm in length and guided the chip-holder with an accuracy of ±50 µm, thus enabling a seamless motion of LOC chips to a sample take-up position close to the microscope, to the dispensing PipeJet module and to the Fluorescence Reader (figure 8).

Figure 8. Overview of the particular units of the whole LOC system. The five modules of the lab-on-a-chip

are shown namely the microscope combining laser microdissection and SPATS transfer, the chip-holder of the CytoCycler fixed on a moving slay, the BioSpot dispensing device and components of the Fluorescence Reader. The slide rail enabled X-directed motion of the chip-holder, thus providing an elegant connection between individual LOC modules. The housing for all the optical components of the Fluorescence Reader as well as this graphical overview picture was designed in a computer aided design (CAD) program (SolidWorks 2006, Solid Works Corp.) and was kindly provided by G. Lieckfeld.

The BioSpot operating unit consisted of three PipeJets named PipeJet1 (PJ1), PipeJet2 (PJ) and PipeJet3 (PJ3) executing all aspiration, dispension and shooting operations. The PipeJets were equipped with a tube reservoir, which was connected to a syringe pump for handling the aspiration and dispension of liquids. PJ1 and PJ2 could handle aqueous liquids up to 50 µl, while PJ3 could handle up to 1 ml of mineral oil for droplet coverage when performing virtual reaction chamber PCR. Each PipeJet housed an elastic polymer tube,

which was actuated by a piezostack driven piston supporting shooting operations. Polymer tubes were of low cost and could be used as disposables. “Shooting” meant a sequential dispension of liquids at tiniest amounts of a few nl. Squeezing the tube via the piston resulted in a fast displacement of the filled liquid to both, the open end of the tube and the end connected to the reservoir (figure 9). Thereby a small droplet of about 22.5 nl of liquid was dispensed to the designated surface or reservoir, forming droplets of a few µl when repeated several times. Dosage volumes could be controlled by the amplitude of the piezo actuator, while other parameters involved in the dispensing process could be defined via the freely programmable software “BioSpot”.

Figure 9. Dosage principle of the BioSpot’s PipeJet modules. The figure shows the piston driven actuation

of a polymer tube as housed in PipeJets PJ1, PJ2 and PJ3, resulting in the bidirectional dispension of nanoliter droplets. Via a principle of fast displacement and slow release, smallest droplet sizes of 22.5 nl could be loaded precisely on the chip surface (BioFluidix GmbH, Freiburg, Germany; Lindemann T et al., 2004).

The BioSpot provided the easiest way to combine all LOC modules due to the 70 cm long slide apparatus, included with the BioSpot, and furthermore it will provide the important interface for enabling automation of the total LOC system. As a modular unit of the lab-on-a- chip system, the BioSpot was used as a dispenser for various liquids needed for the molecular biological analysis executed on the LOC chip surface. The BioSpot, as a computer-controlled dispensing platform, was capable of unloading reagents at any destined domain on the LOC chip surface. The z-axis, where the PipeJets were attached to, allowed movements of up to 40 cm, while y-positions of the PipeJets needed to be adjusted manually. The distance between the three PipeJets provided enough space that each PipeJet could reach the chip surface.

The software gave access to four single active control windows for operating the whole BioSpot device, namely (1) Axis Control and Axis Movement, (2) PipeJet Control, (3) Valve Control and Pump Control and (4) Batch Mode. A total operating procedure of the BioSpot started with dispensing e.g. 1 µl of master mix to reaction center B on the LOC chip

surface for sample uptake. For that, the designated PipeJet was approached to a reservoir flask (figure 10 A and B), was driven to aspirate a distinct amount of fluid and was moved to the chip surface for unloading (figure 10 C). Subsequently, the chip-holder was moved to the microscope, waiting for sample uptake after microdissection and SPATS transfer. Sample material, released into predispensed fluid, was immediately covered by mineral oil. For that, the chip-holder was moved back to the designated PJ3 for applying Sealing Solution either directly by dropping onto the liquid droplet or by dispensing onto the LOC chip surface (figure 10 D), while droplet fusion was achieved by surface acoustic wave actuation.

Figure 10. Workflow of the BioSpot applied on the lab-on-a-chip system. A) Via the slide rail the flasks-

holding reservoir device was centered to the PipeJets. B) A distinct amount of fluid was aspirated by one of the PipeJets. C) The designated PipeJet was approached to the LOC chip surface and centered to reaction center A for liquid dispension. D) Via the piston actuation a small volume of 1 µl of liquid was dispensed onto the chip surface (red arrow), which was going to be covered with 5 µl of pre-dispensed mineral oil (green arrow) for forming a virtual reaction chamber PCR droplet arrangement.

Pipetting workflows like this could either be performed manually by operating the various active control windows of the software or automatically by executing pre-programmed operations using the “Batch mode” setting of the software.