Fabrication of Microdroplet Arrays applied to Single Molecule Counting for the Detection of Alzheimer’s Disease Blood-Based Biomarkers

Alzheimer’s disease (AD) is a neurodegenerative disease that affects more than 25 million people worldwide, and this number is projected to triple within the next 30 years. Despite intensive efforts, no disease-modifying treatments or preventive therapies are available. This is mainly due to the lack of specific, sensitive, and minimally invasive diagnostic tools to identify individuals in early-stage AD.

The analysis of biomarkers in blood would be desirable as a first line screening tool in the diagnostic procedure to identify individuals at risk of developing AD, as a blood test is easy to perform in most health-care facilities, and on large populations. However, the current obstacle limiting the access to a robust blood test for AD diagnostics is that the levels of the relevant biomarkers in blood are extremely low.

To overcome this problem, ultrasensitive approaches such as digital ELISA for the detection of proteins have been developed, which have shown a 1000-fold improved detection limit compared to traditional ELISA methods. The aim of this work was to develop an ultrasensitive tool for the detection of blood-based AD biomarkers at low concentrations based on digital counting.

This was realized using a recently described microdroplet array platform, where hydrophilic spots are surrounded by a hydrophobic background and by contacting the array with an aqueous solution, droplets are formed spontaneously when the aqueous solution is displaced by air. In particular, two new fabrication methods for producing this type of hydrophilic-inhydrophobic microdroplet arrays (HiH-MDAs) were successfully developed.

One of the processes enabled moving the microfabrication from cleanroom facilities to a normal chemical lab. With this fast and straightforward method, it has been possible to produce robust and reproducible MDA substrates with feature diameters down to 4 μm, with a 2 μm to 3 μm accuracy and 3 % to 5 % spot-to-spot variance.

The second method involved the fabrication of HiH-microwell arrays using FluorAcryl 3298, a UV cross-linkable fluoropolymer. With this approach, arrays of wells with 10 μm diameter and various depths, ranging from 300 nm to 1 μm, were fabricated and applied to single molecule counting. Regardless of the fabrication method, the MDA substrates were assembled in modular 3D printed microfluidic systems enabling multiplexing or highly parallel assay formats.

To demonstrate the versatility of the fabricated MDA substrates, digital ELISA assays and digital hybridization assays were conducted. Furthermore, the effect of static and dynamic incubation of the target on the assay quality was investigated. Finally, digital ELISA assays with detection limits in the attomolar range were achieved for the detection of total Tau protein, and Amyloid Precursor Protein, both proteins believed to be involved in the development of AD.

The fabrication of FluorAcryl well arrays allowed capturing elements other than biomolecules due to their three-dimensional structure. On these MDAs, an innovative method for trapping and selecting specific bacteria was established as a proof of principle for single-cell isolation and phenotype-based selection for high-throughput screening.

In conclusion, this work has demonstrated the possibility for the future development of a cost-effective, robust, and ultrasensitive analysis tool, which can be easily miniaturized and matured into a portable, fully automated device suitable for point of care testing, e.g., at the general practitioners’ office, where digital assays can have the greatest impact in routine patients’ screening and early-stage disease diagnosis.