Point-of-care device based on KETs for diagnosis of food allergies

Periodic Reporting for period 3 - AllerScreening (Point-of-care device based on KETs for diagnosis of food allergies)

Période du rapport: 2021-01-01 au 2022-03-31

The most relevant achievements of Allerscreening project are:
1. Development of disposable highly multiplexed diagnostic KITs capable of measuring 65 determinations. That means 20 diferent allergens in triplicate in a single Diagnostic KIT.
2. Diagnostic KIT scalable at wafer fabrication and compatible with semiconductor industry
3. Improvement of the PoC device capable of reading out 65 determinations of the diagnostic KIT in 12 minutes.
4. Easy and scalable biofunctionalization process thanks to the use of optical interferometers based on an epoxy resist sensing surface.
5. The standardisation of an immunoassay using bio-nanoparticles for the specific detection of IgE, without any labelled secondary antibodies, conforming the reagent set.
6. Easy to used PoC system and management of the disposable Diagnostic KITs and reagent SETs
At the beginning of the project, the technology was established at TLR2, being now at TRL6 , that is, the AllerScreening in-vitro system has been proved in a real scenario.

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.

5. Clinical trial
The clinical trial was carried out using Bio-SiO2 by the 5 clinical partners.
The clinical partners that have participated in the trial pointed out some aspects to improve in the Allescreening Diagnostic system:
• The current size of the instrument still makes its use in a medical office overly complicated.
• In-vitro diagnostic assay should be simplified to avoid lab expertise.
• The assay time consume long time for obtaining the results and must be reduced.
• Regarding results obtained in the experiments performed on sera from the patients recruited for the study and their comparison with the results obtained by the so-called “gold standard” (Thermo Fisher ImmunoCAP singleplex) the AllerScreening system has shown excellent specificity and good sensitivity, although the latter was less impressive with certain specific allergens (e.g. LTP) possibly due to the intrinsic features of these proteins.

6. Allerscreening as a business opportunity
The level of satisfaction of the system has reached the scored enough to be proposed as an improved in-vitro system. thus, the exploitation plan was designed as a live document planned to be updated every new milestone will be reached.
The evolving from the TRL6 to a TRL9 is planned and several actions have been taken to achieve this goal, being the most important one the certification of the system to obtain the CE mark. This first step is critical for the two lines of commercialization: offering allergy screening services and commercialization of the In-vitro system (PoC, Diagnostic KITs and reagent SETs). Moreover, is remarkable that the protection has been extended to Europe, UK, Canada, Japan and USA.
The cease of activities of BIOD has partially truncated the immediate commercialization of the device. However, the effort of the UPM in warrantying the exploitation of the project's results has been implemented in several individual actions. In fact, a large PHARMA company has shown its interest in the technology developed and protected by the UPM related to the patents applications already granted. This company is committed to give access right in fair and reasonable conditions according to the grant agreement for the sector of food allergies.

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Periodic Reporting for period 3 - AllerScreening (Point-of-care device based on KETs for diagnosis of food allergies)

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