Protein detection in blood via a chimeric aptafluorescence assay: toward point-of-care diagnostic devices

Abstract

Paper-based analytics allows building portable and disposable devices for point-of-care (POC) diagnosis. Conventional methods for quantifying proteins exhibit substantial disadvantages related to costs and difficulty of the technique when used in settings where fast and cost-effective assays are needed. We report the successful application of a simple, rapid, easy to use, and label-free aptasensor strategy based on the selective fluorescence of the NMM IX dye. For the probe design, the three-dimensional (3-D) structures of the DNA components were carefully analyzed using software for the 3-D visualization of crystallographic structures. The chimeric aptafluorescence molecule consists of two modules, a detection aptamer and a transduction sequence that induces the specific fluorescence of NMM IX. In the presence of thrombin, a fluorescent spot visible to the naked eye can be observed. The fluorescent response is directly proportional to protein concentration and can be easily quantified colorimetrically using a low-cost microscopy system. The recognition probe design might be adaptable to other relevant biological analytes by changing the sequence of the aptamer. This proof of principle represents a contribution to the development of useful, cheap, reliable, and simple protein quantification assays for POC testing.

Rational Design and Experimental Analysis of Short-Oligonucleotide Substrate Specificity for Targeting Bacterial Nucleases

“… 10 − 12 A key aspect in the development of any diagnostic tool based on oligonucleotide probes is the necessity of designing probes which are both efficient and specific. 13 Efficiency is directly related to the extent to which the target nuclease cleavages the synthetic substrate. In this respect, the oligonucleotide probe needs to be readily cleaved by the target nuclease even when the latter is in very low concentrations, such as in the early stages of infection.…”

“…Nucleic acids have been proven useful recognition molecules for the development of several diagnostic strategies, taking advantage of their flexibility to be adapted to various transduction mechanisms, such as fluorescence, electrochemistry, piezoelectric, and colorimetric mechanisms . In particular, the use of nuclease-activatable oligonucleotide probes (NAOPs) as biosensors has proven its potential as a groundbreaking solution in early diagnosis of serious infectious diseases. , The detection of bacterial infection, − malignant cells in biopsies of breast cancer, − or food contamination by pathogenic bacteria 1 has been previously reported to be feasible on the basis of their nuclease activity profile. − A key aspect in the development of any diagnostic tool based on oligonucleotide probes is the necessity of designing probes which are both efficient and specific . Efficiency is directly related to the extent to which the target nuclease cleavages the synthetic substrate.…”

The Molecular Basis of JAZ-MYC Coupling, a Protein-Protein Interface Essential for Plant Response to Stressors

“…We collected data for a specific protein-protein interaction from the literature and predicted the effect on the whole group of proteins for which minimal experimental data was available. Integrating computational insights to characterize or design complex system increases our understanding and optimizes experimental resources ( Montero-Oleas et al., 2018 ; Ryan et al., 2019 ). By doing so, we can escalate the study of protein-protein interactions to specific families of proteins, as we did with the Arabidopsis TIFY class II family.…”

Nuclease Activity as a Biomarker in Cancer Detection

“…Importantly, oligonucleotides are degradable by nucleases [1]. Hence, a cleaving nuclease activity is an optimal target of such nucleic acid probes, provided that substrate oligos are sufficiently sensitive and specific to report on such an enzymatic activity [132].…”