Figure 1 Two wells filled with methyleneblue. The overmoulded well is tight (left), while the well on the right is leaking (see the blue colour under the rim)
Dr. Lutz Staemmler, Greiner Bio-One, examines, joining technology for cell culture labware.
Multiwell plates within cell culture applications are supplied with a variety of properties from coatings applied to the surface to the actual colour/transparency of the resin. Increasingly a transparent base is required which is independent of the main body to enable compatibility with complex microscopy. For products designed for cell culture, the technique to combine two separate components must meet the following conditions:
• The bond must be stable/leak-proof > 14 days at 37 °C and at a rel. humidity of 95 % (incubator conditions).
• The bond must withstand centrifugation at 4800 g.
To follow is an overview of common techniques utilised to join plastics.
Overmoulding
For applications that require enhanced optics, eg cell imaging, a film bottom is attached to the plate producing micro-plates whose wells have a very thin base. This process starts with the thin film being placed into the open injection moulding tool. The material for the body of the plate is then injected onto the film. The temperature of molten resin coupled with the pressure inside the tool melts the film and the plastic together. Greiner’s µClear plates are produced by a patented overmoulding process to produce a coloured microplate with a transparent bottom [1]. This processes guaranties sealed wells.
Gluing
Although many adhesives are available, the principle of the gluing remains constant: An adhesive forms the connection between two components. Hardening of the adhesive is either instantaneous or by a treatment eg UV light.
For bonding in series production, adhesives cured by UV light are most commonly used because curing can be initiated at a predetermined point in time. Heat curing is less popular as most plastics are susceptible to heat.
The entire area that will form the bond must be covered by adhesive. In the case of a microplate the adhesive is dispensed around each well; making gluing a serial and slow process. Furthermore, the viscous adhesive takes a relatively long time to flow from the needle. Equating to a rate of coverage achievable being 10 mm/s. In addition the gluing process must fulfil the following conditions:
- To not be cytotoxic.
- It should not fluoresce
- Fluorescence dyes should not attach to the adhesive or interact with fluorescence measurements.
- Be transparent.
Efficient adhesives should:
- be applied using an automated dispenser
- have a short curing time.
The main requirement is the ability to join two plastic components. Examples of unsuitable plastics are:
- Polyoxymethylene (POM)
- Polytetrafluorethylene (PTFE)
- Polyethylene (PE).
However, specific surface treatments may render a plastic suitable for adhesive bonding [2]. Gluing is the best method when joining two different materials; such as glass to plastic.
Figure 2 Greiner Bio-One SensoPlate: 96 well polystyrene (PS) microplate with a glass bottom
Laser welding
In all welding technologies the bond is formed by the two components themselves where they are melted to form the bond. This process is identical for all welding applications. The difference is in the way the heat is applied. To succeed both welding components must have a similar melting temperature. If not, incompatible states will exist.
The laser beam is absorbed by at least one of the two components and is heated to its melting temperature. Where one component absorbs the energy, the other component is melted indirectly by the heat generated. [3].
A mirror (galvohead) drives the laser beam along the weld line (Figure 3). Again a serial process, but faster than gluing at 500 mm/s.The limiting factor is bonding time.
Figure 3 A moving mirror drives the laser beam along the weld line.
Figure 3 shows the welding zone being between the two sheets of plastic. The laser only penetrates the upper sheet. Therefore the upper sheet has to be a material that is transparent to the incoming beam of light and the lower one opaque. Thus, heat is generated in the lower opaque sheet only.
Ultrasonic welding
The disadvantages of serial welding can be circumvented by the use of ultrasonic welding, as it offers welding in parallel and is, by virtue, a faster process. Welding times of a fraction of a second are standard.
Figure 4 Laser welded wells. The weld seam appears black around the wells.
A piezo crystal oscillates between 20 and 50 kHz and drives a mechanical resonator (sonotrode) to oscillations with an amplitude of microns. The sonotrode is placed on top of the upper surface. It induces friction, creating a heating effect, between the two surfaces, which is sufficient to melt both components forming a welded seam. It is possible to make a weld seam as small as 100 µm wide. Thus, allowing very complex structures to be manufactured, a criterion that is a prerequisite when producing microfluidic structures.
No colour restrictions like in laser welding exist for ultrasonic welding, as it is a mechanical process.
Comparison
Advantages and disadvantages of the aforementioned techniques are shown below:
References
[1] Patent DE 197 12 484 C2: Mikroplatte mit transparentem Boden und Verfahren zu deren Herstellung (1999-07-08). Greiner GmbH
[2] HABENICHT, G.: Applied Adhesive Bonding, Weinheim: Wiley-VCH (2009)
[3] RUSSEK, U. A.: Laserschweißen von Kunststoffen, München: Süddeut- scher Verlag onpact (2009)