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Capillary systems for advanced point-of-care diagnostics

Periodic Reporting for period 2 - CAPSYS (Capillary systems for advanced point-of-care diagnostics)

Reporting period: 2020-01-01 to 2021-12-31

In 2017, the global in vitro diagnostic (IVD) market was valued at $64 billions with an estimated annual growth rate of 4.8% from 2018 to 2025. Point-of-care (POC) diagnostic tests represent at least 36% of the IVD market and are performed by medical professionals or directly by the patients themselves. Even though there are already many POC diagnostic tests available on the market, several important needs for portable diagnostics are still unmet: POC tests still require a relatively large volumes of samples, often lack sensitivity and accuracy and cannot be readily multiplexed. In addition, there are opportunities to adapt POC diagnostics for better connectivity in the context of mobile health and to make them wearable.

CAPSYS will have a double impact on society. On the one hand the results obtained from this synergetic research work will contribute to push the current state of the art of POC technologies by combining advanced capillary-driven microfluidic chips with innovative bioassay architectures (Fig. 1). On the other hand, CAPSYS provided a unique multidisciplinary education to two Early Stage Researchers (ESRs). In addition to high quality technical work, the ESRs were exposed to the process of IP generation and discussion with companies and potential partners. This real-world research environment trained and inspired the ESRs, who acquired skills beyond academic research and have now competences for becoming the next generation of entrepreneurs in Europe.

The overall objective of CAPSYS is to push the state-of-the-art technology in POC diagnostics by taking advantage of the physics of liquids at the microscale. Two distinct but complementary projects were led by two PhD students: ESR1 and ESR2. In these projects ESR1 and ESR2 designed and fabricated capillary-driven microfluidic chips in order to perform bioassays with very small volumes of sample and reagents and with controlled assay kinetics. In particular, capillary-driven microfluidic chips allow to program the flow rate of liquids by design, integrate and resuspend reagents precisely on chip, and perform a sequence of steps automatically. This multidisciplinary industrial and academic project will contribute to improve rapid testing and will impact the field of diagnostics, medicine and technology.
The work packages described in this report cover the implementation of complex DNA reactions on chip, the in situ formation of hydrogels in microchannels and the use of microfluidics for performing a precise sample dilution step on chip.

ESR1 – Complex DNA reactions on chip

ESR1 designed, fabricated and characterized a programmable hydraulic resistor array for silicon microfluidic chips using electrogate arrays. Taking advantage of an electrogate array the flow rate inside microfluidic chips can be programmed easily without using external and bulky equipment. As an illustration she demonstrated the possibility to use such an array to create laminar flows and “tune” an enzymatic assay in a capillary-driven microfluidic chip. The signal of these enzymatic assays was localized on receptor-coated microbeads trapped in microfluidic structures.
ESR1 also worked on the implementation of complex nucleic acid reactions inside capillary-driven microfluidic chips taking advantage of a self-coalescence module (SCM) and spotting of reagents inside the chip. An SCM allows the reconstitution of spotted reagents inside the chip in a precise manner. In particular, two isothermal reactions, a molecular beacon and a more complex clamped hybridization chain reaction (C-HCR), have been implemented in chip. For both reactions, the concentration of an initiator DNA sequence was measured using a fluorescence measurement. The formation of fluorescent DNA polymers was observed inside the chip as a result of a C-HCR (Fig. 2). Such results demonstrated the possibility to implement and study complex reaction systems inside microfluidic chips.
Moreover, ESR1 worked on a module allowing the filtration of red blood cells. Using a 3D printed device, she filtered successfully red blood cells from a few microliters of a whole blood sample, which allowed the filling of capillary-driven microfluidic chips with plasma only.
In summary, it was elaborated that the various techniques developed by ESR 1 can be combined to pave the way for rapid, accurate, and amplified DNA-based assays in silicon microfluidic chips.

ESR2 – Sequential assays

ESR2 developed an innovative method for forming hydrogels with precise geometries in a sealed microfluidic chip. This method can be used to create in situ compartments on chip, filter samples, and localize bioassays. In particular, ERS2 identified a robust two-component chemical system for performing a fast and reliable polymerization reaction for forming PEG-based hydrogels. The polymerization reaction is based on thiol-maleimide coupling and can be performed in physiological conditions. ERS2 designed a specific capillary-driven microfluidic chip for shaping the interface of the two precursor solutions. This allows to form hydrogels with a well-defined geometry in a sealed microfluidic chip by interfacial polymerization within 2 minutes. The molecular weight cut off (MWCO) of the hydrogel was characterized by monitoring the diffusion of fluorescent molecules with different molecular weights through the hydrogel. Moreover, ERS2 was able to change the MWCO of the hydrogel easily, by tuning the concentration of the precursor solutions and by modifying the length of the PEG chains of the precursors. ERS2 demonstrated that receptors such as antibodies could be easily immobilized in the hydrogel and would retain their functionality. This allowed to perform a novel format of heterogeneous competitive assay on chip, which does not require a rinsing step.
Additionally, ERS2 also implemented a microfluidic module for the precise metering of two liquids by performing volumetric mixing. Such a module can be used for example for diluting a sample solution with a precise volume of buffer/reagents independently on their viscosity. Sample dilution is a critical step in rapid tests, which is often performed by the user and is prone to errors. Therefore, it could be useful to integrate such a dilution module in rapid tests to automate sample dilution and make tests more reliable.
The objectives and the corresponding scientific work of this project are in general beyond the state of the art and resulted in five publications in top journals and three patents filed. Moreover, ERS1 and ERS2 presented their work at several international conferences and were awarded with multiple prices.
We expect that the results achieved and published within the project CAPSYS will have a direct impact in the field of POC diagnostics. In particular, the promising results in immunoPCR, enzymatic assays, and DNA assays will benefit the European research community working on high sensitivity assays. Moreover, the outcome of this project will contribute to the research in microtechnology and microfluidics for POC diagnostics applications by reducing the volumes of sample and reagents required and by replacing bulky equipment for active pumping with capillary-driven microfluidic circuits. Furthermore, this microfluidic technology has the potential to be scaled up and fabricated on polymeric chips by hot embossing or injection molding, which will considerably reduce the costs of fabrication.
Confocal microscopy images of DNA polymers formed inside a microfluidic chip
Microfluidic chips filled with blood by capillary forces