1.Improvement of device, MOX
The adaptation of the read-out platform was developed to fulfil the stablished requirements:
• Adaptation of positioning hardware and control software for the multiplexed measurement of 65 sensing sites per KIT. A Sensing site is a Bio Photonic Sensing Cell (BICELL) per KIT.
• Software control and user interface adaptation easy to use for the final user
• Reduction in the readout time: 65 BICELLs in 12 minutes.
• User interface: novel user interface available focus on a better user experience by using this complex multiplexed Diagnostic KIT was performed.
2.To develop multiplexed Diagnostic KITs
Design and fabrication of cost effective multiplexed diagnostic KITs integrated with 65 Fabry-Perot Interferometers made of SU-8 resist as sensing surface of the BICELLs. This innovative approach not only ensure the scalability at mass fabrication, but also the scalability for the biofunctionalization saving drastically the reagent and allergen amount needed because only 1000 nL of volume per well is needed. Thus, with about 100 ng of allergen a sensing site of the KIT can be biofunctionalized.
Scalability in biofunctionalization: The immobilization of the 20 allergens was performed on the SU-8 resist sensing surface through a O2 plasma activation step of the BICELLs. In the final version of Diagnostic KITs a microfluidic chamber was included in order to be sure the homogeneous of patient sample distribution, as well as the volume of the liquid sample needed.
The stability of the Diagnostic KITs was tested for 3 months after immobilization. KITs were functionalized with allergens and stored at 4 ºC. The functional stability can be maintained for at least 10 weeks.
3.Immunoassays to detect specific IgE
IgE is considered the marker antibody for allergy or sensitization. For the detection of allergen-specific IgE binding, without the use of labeled secondary antibodies, the presence of IgG, whose ratio is 10000 IgG vs 1 IgE, must first be avoided. For this purpose, two strategies were implemented:
a) Fishing strategy with bio-SiO2 NPs (strategy A).
b) Strategy of direct binding of IgE with Bio-AuNPs (strategy B).
4. Transfer of technology to third parties for mass production
Briefly, ten platforms were built in total, 5 for clinicians (Antwerp, Málaga, Milan, Munich, Wien), one for CQS, one for UPM, one for BIOD and the rest for backup, following ISO9001 and ISO 13485. The production of the Diagnostic KITs used for the clinical validation was conducted in different batches (for example a batch of 100 Diagnostic KITs) under international quality standards. The yield and quality of each batch was assessed by an in-line quality control verifying the response at wafer level and a percentage of a number of KITs after fabrication. Each batch of Diagnostic KITs were identified by a code number, in order to ensure traceability. Finally, the biofunctionalization of the KITs was carried out at CQS and UPM facilities automatically and packed for the final users together with the reagent Sets. A reusable clamping device was assembled to end the packing of the Diagnostic Kit, and the production of them were ready before the clinical validation launch.
