Alzheimer’s disease– a progressive neurological disorder that leads to brain shrinkage (atrophy) and neuronal loss– is the most common cause of dementia worldwide. It affects about 6.5 million people aged 65 or above in the United States, with the number projected to grow to around 13.8 million by 2060. It is also currently the seventh leading cause of death in the US (1).
Besides the lack of a cure for Alzheimer’s, the condition is also notoriously challenging to diagnose before the onset of clinical symptoms such as memory loss and decline of other cognitive skills (2). Early detection, however, would enable patients and caregivers to adopt better management strategies– from lifestyle changes to symptomatic treatments– and help improve their quality of life. Additionally, it would allow future treatments to be able to target the disease in its earliest stages, before significant and irreversible brain damage has occurred. Early detection of Alzheimers disease could also help reduce healthcare costs– a recent study by the Alzheimer's Association found that diagnosis of Alzheimers in the early stages could save an estimated $7 trillion dollars associated with medical and long-term care costs for patients with unmanaged dementia (1).
Alzheimer's disease is characterized by the aberrant processing and misfolding of soluble neuronal proteins which leads to plaque and tangle formation in the brain. Two of these main proteins that misfold and form aggregates are amyloid beta (Aβ1–42) and Tau protein. They are therefore also the primary biomarkers of Alzheimers disease. Multiple studies have confirmed that Alzheimers patients have elevated levels of Aβ1–42 and Tau in their cerebrospinal fluid and blood(3).
In our iGEM project, we have designed a non-invasive aptamer-based detection technique that can detect two major biomarkers associated with Alzheimer’s disease– amyloid beta and tau– from blood samples with high sensitivity and specificity.
We have built a portable, plasmonic biosensor-based device using the self-assembly of gold nanoparticles (AuNP) that produces wavelength extinction shifts via a localized surface plasmon resonance (LSPR) signal. Localized surface plasmon resonance (LSPR) is a widely used label-free and highly sensitive method of detection used in biosensors and can even be used for real-time sensing. Conventional SPR sensors involve a thin gold film and exploits the phenomenon of surface plasmons which refers to the collective oscillation of conduction electrons at the interface between a metal and dielectric caused by electromagnetic radiation (4). Resonance with surface plasmons cause the local electromagnetic wave to be selectively magnified, resulting in the increase of many optical phenomena, such as absorption and transmission, at the surface plasmon frequency. In our chip, the AuNPs attach to a glass slide with the help of an amine group linker, APTES. Negatively charged AuNPs are distributed uniformly in a monolayer on the amine functional group on the glass substrate. The LSPR chip is then functionalized with amyloid beta and tau specific aptamers under optimized conditions. The aptamers are then able to specifically bind to our peptides of interest– amyloid beta and tau. Reading taken from a spectrophotometer are then used to obtain the absorbance signals and detect our peptides of interest.
Aptamers are short (length: usually 15-60 nucleotides; size: < 20 kDa, ~2nm), single-stranded DNA or RNA oligonucleotides that bind to numerous molecules, proteins, nucleic acids, and even live cells with high affinity (KD = 10 pM to 10 nM) and selectivity. The term aptamer stems from the combination of the Latin word aptus, “to fit,” and the Greek word meros, meaning “part” or “region.” Aptamers bind to their targets not via Watson-Crick base pairing but their stable tertiary structure, which is comparable to how antibodies bind to antigens. A simplified schematic is shown in Figure 1. Their complex 3D structures result from the combination of their secondary structures, which include loops, stems, hairpins, pseudoknots, bulges, or G-quadruplexes (5). Aptamers possess complementary ligand-binding sites for the target of interest and binding occurs via induced fit, van der Waals, hydrogen bonding, and electrostatic interactions (6). For our project, we shortlisted aptamers specific to beta amyloid and tau (a complete list can be found in the parts page). We ended up using the apatmer TSO508 isolated by Tsukakoshi et. al. complementary to beta amyloid oligomers (7).
Our sensing device works better than other similar diagnostic methods that utilize antibodies to detect amyloid beta and tau proteins from blood in terms of its reduced cost and time of detection as well as an improved sensitivity. Aptamers are advantageous over antibodies due to their long-term stability, low cost, and recognition of a wide variety of targets with high sensitivity. In addition, the development process of aptamers is simpler and takes less time than antibodies: monoclonal antibodies take about 4-6 months to be produced and requires eliciting an immune response in an animal model, whereas aptamers can be developed by enrichment of an oligonucleotide library through SELEX process and takes around 1-3 months only. Aptamers are also required in lower concentrations than antibodies. Finally, antibodies can typically be conjugated with only one type of binding molecule, whereas aptamers can be modified at both 5’ and 3’ ends which increases their scope of utility (4).
Our study, in addition to providing direction for the early detection of Alzheimer’s, suggests the potential of using aptamers as a recognition probe in the field of diagnostics.
