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Super-sensitive detection of Alzheimer’s disease biomarkers in plasma by an innovative droplet split-and-stack approach

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Alzheimer’s disease diagnosis through a routine blood test

Early and timely disease detection may save lives. A new sensing device pushes the detection limits of disease biomarkers in routine clinical practice.

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Disease diagnosis relies on measurable indicators in body fluids known as biomarkers. These are traditionally analysed using biochemistry based techniques such as the enzyme-linked immunosorbent assay (ELISA). However, the low concentration of biomarkers coupled with the limited analytical capacity of conventional methods impede detection and hence disease diagnosis. In Alzheimer’s disease (AD) – the most common type of neurodegeneration – beta amyloid and tau biomarkers levels in cerebrospinal fluid increase before the onset of symptoms. Although these and other potential biomarkers can be employed for prompt AD diagnosis, cerebrospinal fluid collection requires lumbar puncture, which is an invasive procedure and requires hospitalisation.

Sample concentration technology

Over the years, scientists have followed different techniques to improve biomarker detection, including the amplification of the target molecules. The EU-funded SensApp project addresses this demand for sensitivity increase in diagnostic assays through preconcentration of target molecules by solvent removal. “Our approach drastically increases the biomarker concentration by reducing the reaction volume, hence achieving an amplification of the signal output,” explains project coordinator Simonetta Grilli. The team developed a device that does not differ in size from other benchtop equipment encountered in diagnostic laboratories. It is based on a new technology known as droplet split-and-stack (DSS) that can generate and accumulate tiny droplets of the liquid sample (plasma, urine, saliva) onto a solid support such as a glass slide.

How does droplet split-and-stack work?

DSS allows the dispensation of sample droplets without spreading, making the few molecules present in a low abundant sample to be more concentrated than in the case of a standard dispensing modality. In this way, the operator can successfully detect those few molecules by a standard detection procedure, such as immunofluorescence, reaching final high sensitivity. The innovation behind this new technology is the ability to generate those tiny droplets by the electric field applied directly to the liquid sample through the pyroelectric effect that achieves a change in polarisation due to temperature change. According to Grilli: “This is the first time this approach is used for this kind of application.” An additional achievement is the combination with a readout module that can detect the fluorescence signal from these scarce biomarkers.

Improved Alzheimer’s disease biomarker detection

As proof of principle, the SensApp device was validated on model samples, demonstrating the ability to detect AD biomarkers down to sub-picogram levels. This was a significant improvement of the standard level of sensitivity of commercial ELISA kits. Although testing in a clinical setting is pending, the SensApp device offers a superior detection approach for fast and sensitive diagnosis of AD. Importantly, biomarker detection can be achieved through a simple blood test, setting new standards for AD screening in the population. Moreover, it is expected to improve patient compliance and follow-up during therapy. It is noteworthy that the application of the SensApp device is not restricted to AD but can be implemented for the detection of other low-in-abundance disease-related biomarkers.

Keywords

SensApp, biomarker, AD, Alzheimer’s disease, biomarker detection, droplet split-and-stack, DSS, pyroelectric effect, immunofluorescence

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