Over the last two years, the PoCCardio project’s scientific and industrial partners have collaborated closely to develop proteomic and genomic electrochemical sensors, microfluidic cartridges, sample preparation technologies and instrumentation that together constitute the PoCCardio point-of-care (PoC) solution.

Detection of protein biomarkers and genetic variations related to myocardial infarction

Led by Ghent University and the Interfibio Research Group at Universitat Rovira i Virgili, this work aims to create a generic electrochemical platform capable of:

1. simultaneously detecting multiple blood protein biomarkers with different concentration ranges and detection limits and
2. rapidly detecting multiple genetic variations, known as single nucleotide polymorphisms in genomic DNA from a finger-prick blood sample.

For proteomic biomarker detection, the project employs sandwich assays using monoclonal antibodies, aptamers or aptamer-antibody combinations. Aptamers are artificial nucleic acids selected through a process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Where performance requirements are met, the project aims to replace antibodies with aptamers. Aptamers offer several advantages, including lower cost, higher stability and a fully in vitro selection process that eliminates the need for animal hosts.

To date, the project has defined SELEX strategies for the selection of aptamers against biomarkers that meet the performance requirements of the PoCCardio tool, targeting major contributors to cardiovascular disease: inflammation (CRP), heart failure (NT-proBNP), metabolic dysfunction (HbA1c), and myocardial injury (cTnT). In parallel, commercially available antibodies have been screened and evaluated to identify functional pairs targeting these same biomarkers for use in the sandwich immunoassay or in combination with the aptamers. Sensor arrays and multi-electrode proteomic and genomic biosensor designs that operate on patient blood samples are being developed with balanced consideration of cost, ease of manufacturability and performance.

Genetic variants linked to cardiovascular disease 

For the genomic platform, PoCCardio has identified an initial set of eight single-nucleotide polymorphisms (SNPs) associated with cardiovascular disease (CVD). Detection is based on Isothermal solid phase primer extension based on Recombinase Polymerase Amplification (RPA), a DNA amplification technique that copies specific DNA segments at a constant temperature.

The project team has designed and tested short synthetic DNA probes for these eight SNPs, which include genetic variants and mutations associated with inflammation (IL-6, IL-6 receptor D358A mutation, NLRP3), hypercholesterolemia (ApoA, ApoE, PCSK9, PCSK9 R46L mutation), and response to treatments known to improve CVD outcomes, such as statins (SLCO1B1).

Due to the generic nature of the PoCCardio electrochemical platform, the project is also considering the addition of further SNPs. To date, a total of 16 SNPs have been identified that could potentially be implemented.

Handling and preparing the blood samples

To process and prepare the finger-prick blood samples, Fraunhofer IMM is developing a microfluidic manifold that integrates either the proteomic or genomic sensor into a cartridge for multiplexed marker detection, depending on the assay to be performed. The main challenges include reagent storage, sample handling and the integration, fluidic actuation and read-out of the sensor array.

Reagent storage will be achieved using small tablets or lyophilised pellets housed in cavities within the cartridge. The project has successfully demonstrated the transfer of filler material (accounting for 95–99% of the pellet volume) into tablet or pellet form, as well as its subsequent resuspension. The next step is to incorporate the active reagents and conduct tests using the chemical compounds that will ultimately be deployed in the cartridge.

In parallel, the project has been testing essential microfluidic functions required for lab-on-chip assays, including fluid splitting, merging, mixing and bubble removal. This work is ongoing, alongside the continued design and optimisation of the sensors and microfluidic manifold. Additional complexity arises from the requirement that both the proteomic and genomic fluidic cartridges must share the same footprint and interfaces to ensure full compatibility with the PoCCardio instrument.

Reading the measured results

The PoCCardio PoC instrument, which integrates the microfluidic cartridges and provides a readout of the measured results, is being developed by Labman Automation.

At an early stage, the project defined the key instrument requirements, including device operation, required functionalities, graphical user interface, user interaction, environmental operating conditions, physical dimensions and power consumption.

To address these requirements, Labman has adopted a modular strategy whereby each functional element of the instrument e.g. cartridge management, fluidic operations, assay execution and readout, is designed as an independent module, rather than as part of a fully integrated system. This modular design enables the flexible assembly of modules to construct the final instrument, serving as adaptable building blocks.

Such an approach greatly enhances both modification capabilities and serviceability. The former is particularly valuable during the development phase, during which instrument specifications may evolve in parallel with ongoing cartridge development. The latter enhancement facilitates efficient maintenance and servicing once instruments are deployed in the field.

With this methodology, Labman has developed and evaluated ten innovative modules through proof-of-concept prototypes and specialised test rigs. All testing yielded positive outcomes, leading to successful integration of the modules into a unified instrument. The forthcoming stage involves rigorous testing of the integrated instrument using test cartridges, to be followed by optimisation in preparation for the finalised clinical device.