Phage‐Derived and Aberrant HaloTag Peptides Immobilized on Magnetic Microbeads for Amperometric Biosensing of Serum Autoantibodies and Alzheimer's Disease Diagnosis

Abstract

An electrochemical biosensing platform for serum autoantibodies (AAbs) detection is reported in this work, exploiting for the first time six Alzheimer's disease (AD)-specific phage-derived and frameshift aberrant HaloTag peptides as receptors, immobilized on magnetic microbeads (MBs) surface and captured on disposable electrodes to perform amperometric detection. Operational analytical characteristics and clinical diagnostic ability of the bioplatform were probed in optimized key experimental conditions by analysing serum AAbs of AD patients and healthy subjects. The value of 100 % obtained for AUC, sensitivity, and selectivity from the all peptides combined ROC curve, indicate full AD-diagnostic capability of the methodology, which was further implemented, as proof of concept, in a POC multiplexing platform to detect the signature in a single test over clinically actionable times (1 h 15 min), opening great promise for the type of diagnosis and AD patients' monitoring follow-up currently pursued.Alzheimer's disease (AD) is the most common form of dementia in elderly age groups worldwide, with an estimated prevalence of 10-30 % and an average duration of about 10 years since the first clinical symptoms. [1,2] However, AD has long preclinical and prodromal stages which extend the disease for up to 20 years.

Electrochemical Cell‐Free Biosensors for Antibody Detection

“…Our technology can be in principle adapted for the detection of different classes of antibodies (e.g., IgG, IgA, or IgM) by designing a specific gene circuit that can be activated only upon recognition of a specific antibody class. Phage‐derived and HaloTag peptides can also be easily employed as recognition elements to widen the range of possible target antibodies [28, 29] . The approach we propose here is convenient as it requires only inexpensive synthetic nucleic acid‐modified strands and commercially available transcription kits.…”

“…Moreover, electrochemical sensors can be mass‐produced, require low sample volumes, are reusable, less prone to interference and, in conclusion, better suited for point‐of‐care applications than optical approaches [27] . Several electrochemical platforms have been reported to date for the rapid detection of antibodies showing high sensitivity, reproducibility, and multiplexing ability [28–32] . Despite these advantages, only a few reports have so far described the adaptation of cell‐free biosensors with electrochemical detection [2, 15] .…”

“…[27] Several electrochemical platforms have been reported to date for the rapid detection of antibodies showing high sensitivity, reproducibility, and multiplexing ability. [28][29][30][31][32] Despite these advantages, only a few reports have so far described the adaptation of cell-free biosensors with electrochemical detection. [2,15] In one clever demonstration, Pardee, Kelley and co-workers designed cell-free transcription/translation responsive gene circuits that, upon recognizing a specific RNA sequence, trigger the expression of a restriction enzyme that liberates a redox reporter-conjugated strand.…”

Multiomics Profiling of Alzheimer’s Disease Serum for the Identification of Autoantibody Biomarkers

“…Remarkably, this panel should be further validated using larger cohorts of patients and controls to verify their diagnostic ability. In this sense, the analysis of independent patient cohorts and larger number of samples, including blinded samples, is assured not only to establish their utility for the diagnosis of AD but also for implementing these novel AD biomarkers in combination with previously reported AD autoantigens in multiplexed bio­(sensing) platforms (i.e., protein microarrays, biosensors or Luminex) to reach enough sensitivity, specificity, and AUC to get the blood-based clinical diagnosis of the disease. ,,, …”

Empowering Electrochemical Biosensing through Nanostructured or Multifunctional Nucleic Acid or Peptide Biomaterials

“…The bioplatform involved the use of MBs modified with the identified four phage‐derived and two aberrant HaloTag monomeric peptides to capture the corresponding serum autoantibodies efficiently and selectively. [ 69 ] The diagnostic capability of the bioplatforms was assessed with a cohort of 20 serum samples (10 from healthy subjects and 10 from AD patients). The excellent values of area under curve, sensitivity, and specificity, all above 90%, and 100% combining all peptides, obtained by receiver operating characteristic curve analysis, proved the high diagnostic ability of the developed bioplatforms for discriminating AD patients in ≈2 h, in a simple, affordable, and point‐of‐care manner.…”