4 STUDY OF PROTEIN INTERACTIONS IN CIRCULATING TUMOR CELLS BY PROXIMITY LIGATION ASSAY . 120
4.2 INTEGRATION OF PROXIMITY LIGATION ASSAY INTO EPHESIA DEVICE FOR THE DETECTION OF
4.2.1 TECHNOLOGICAL DEVELOPMENT OF THE DEVICE
OPTIMIZATION OF MICROFABRICATION OF COC EPHESIA
The first consideration to integrate PLA into the Ephesia system was to adapt the fabrication material to be compatible with the protocol. The PLA protocol contains steps requiring heating up to 37°C, and previous Ephesia chips, made of PDMS, led to potential problems upon heating due to its permeability to gases. This could lead, for instance, to the creation of air bubbles, disrupting magnetic columns, the evaporation of the reagents during the long incubation times. For this reason, it was decided to switch to a thermoplastic material, cyclic olefin copolymer (COC), for microfabrication. The use of COC has been increasing over the years, since it offers several advantages over PDMS: COC chips can be prepared in mass production by hot embossing or injection molding, not requiring delicate clean room techniques. As a material it is inert and highly resistant to hydrolysis, acids and alkalis, and most organic polar solvents such as acetone, methanol, and isopropyl alcohol. Other important properties in the present application are high purity, good biocompatibility, and excellent optical properties, notably high transparency in visible and near ultraviolet61.
A previous Ph.D. student of the team, Karla Perez-Toralla, had developed a protocol for replicating COC Ephesia using a master mold made of NOA (Norland optical adhesive).
For further COC replication, hot embossing parameters were optimized during the course of this PhD. The Ephesia device is made by the bonding of two microstructured parts, a top part comprising the microchannels array, and a bottom part comprising the microwells used for capillary assembly of magnetic beads. The fine structures of the upper part involves microstructures below 10μm in width, impossible to prepare with conventional micromillig instrument An alternative method is to use NOA for making master using a PDMS sacrificial mold for transferring the structure from a SU-8 master. This way provides NOA master molds for hot embossing of COC. They have, however, a limited lifetime of about 10 times usage. In order to avoid this, a nickel master mold has been produced by LIGA process (a method to fabricate microstructures using lithography, electroplating and molding) on a stainless steel disc (AREMAC-Polymer), and the hot embossing parameters were optimized for this new master. A NOA master mold produced from a sacrificial PDMS mold
was also used for the bottom part. A Nickel master mold has been produced for this bottom part, but ultimately it could not be used because the minimum thickness of the disc was not compatible with the rolling embossing machine. Roll embossing is needed for the bottom part, because the high resolution imaging used in the PLA protocol for fluorescent signal detection requires a lower layer with a thickness of no more than 250μM, with micrometer thickness uniformity. In spite of numerous attempts, such uniformity could not be achieved by hot embossing in a press, so finally a NOA master had to be used for the bottom part of the chip.
Hot embossing is based on the stamping the desired structure by softening the substrate above the glass transition temperature (Tg) and applying the pressure. In this thesis TOPAS® 8007S-04 has been used whose Tg is 78 °C with 0.5 mm thickness for upper part and 245μm for bottom part. Bottom part is replicated with a dry film laminator, upper part replication and sealing is made by hot press. The final optimized parameter for COC Ephesia microfabrication is summarized in TABLE 4-2-1 below. The detailed protocol for making NOA master and hot embossing with the other tested parameters is detailed in ANNEX II.
TABLE 4-2-1 OPTIMIZED HOT EMBOSSING PARAMETERS FOR COC EPHESIA MICROFABRICATION
In summary, the fabrication of COC Ephesia is done by press using hot embossing for upper part and roll embossing for the bottom part which is very thin. A nickel master is used for upper part and a NOA master is used for bottom part, which is made by sacrificial PDMS mold. After chip preparation, the fabrication of the final device follows the same protocol as with PDMS fabrication. Capillary assembly is done with a different composition of solution, having less surfactant concentration. This was required to adapt the contact angle of the recessing meniscus, critical for capillary assembly, due to the different hydrophobicity of COC versus PDMS (see details in ANNEX II). The surface treatment of the chip was also different: instead of PDMA-AGE, a solution of Pluronic (1%in H2O-
F127) is incubated for an hour to decrease the non-specific adsorption of beads and cells
on the chip 62.
INTEGRATION OF A TEMPERATURE CONTROL SYSTEM
Another very important requirement for performing the PLA protocol in the chip was to have a temperature control system on the system. A specific difficulty of the Ephesia system is the need to keep the magnetic field on during all treatment and imaging to keep the magnetic columns in place. Thus, the protocol must be performed in situ, and the heating system must preserve the possibility to perform imaging with high magnification, and be compatible with the presence of a magnetic coil around the chip during whole experiment.
Therefore for heating, we have selected an Indium Tin Oxide (ITO) coated glass slide which can be easily manipulated to heat by applying appropriate voltage to reach the desired temperature. Indium Tin Oxide is transparent and electronically conductive63. It has a wide application range in industry such as electronics, smart windows, and thin film photovoltaics, or aircraft windshields for defrosting. It is also used in research, e.g. for electroporation of cells for inserting molecules such as DNA64, for monolayer culture of different cell types like endothelial and neuronal and also as stimulation/recording electrodes for mapping of whole heart preparations65.
For the Ephesia system, ITO glass slide is connected to a custom made module comprising a high accuracy temperature control system for adjusting the temperature. The temperature is read by a thermocouple and a PID (proportional–integral–derivative controller) regulator provides output for reaching or maintaining the temperature66. Implementing ITO into the system required shape matching to the chip and ensuring a homogenous heating over the surface. Due to difficulties in finding commercially or prepare an ITO covered circular 5 cm slide, the heating slide was cut as square having a diagonal length of 5 cm with 3.5 cm edge length to fit into the magnetic coil. Later the electrodes were soldered on a copper sheet that was glued all along the two sides of the slide with silver conductive glue. This allowed having a homogenous current applied on the whole surface. Finally the heating distribution was confirmed by measuring the temperature in the center and in the side of the chip with an external thermocouple. It was observed that the temperature in the center was matching to the one measured by the external thermocouple confirming the accuracy of the control system. However the temperature measured at the side had difference of about 3-6°C. We interpret this difference as a consequence of the limited coverage of the chip by the heating slide, and also of the presence of the surrounding magnetic coil, which has a high heat capacity and must be cooled to avoid internal damage of the coil. This non-uniformity could affect the reliability of the system, in particular regarding the homogeneity of the PLA signals, within the chip due to the sensitivity of the enzyme to the temperature differences. This problem was finally solved by supplying heat by the water cooling system of the magnetic coil: When this cooling water
is heated up to 30°C, the temperature non-uniformity of the chip is reduced to only 1°C.
It is also very crucial to avoid temperature overshooting not to diminish the enzymatic activity by degrading it with high temperatures. So for that, the internal parameters of the temperature controller have been adjusted to increase the temperature slowly after around 30°C to reach to 37°C. Finally, insulation from external air was achieved by an aluminum sheet to keep the heat stable. Details of heating measurement are given in TABLE 4-2-2. In the final protocol, this ITO glass slide is integrated in Ephesia system, under the chip, allowing optical observation during the experiments. This heating element is removed at the end of the PLA protocol for high resolution imaging (60X) by taking out the ITO glass slide under the coil meanwhile the chip is lifted in the magnetic coil without removing it completely.
TABLE 4-2-2 TEMPERATURE CALIBRATION OF COC EPHESIA
T in the center measured by
4.2.2 CELL LINE MODELS FOR STUDYING HER2:HER3