Molecular-electromechanical system for unamplified detection of trace analytes in biofluids

Abstract

Biological research and diagnostic applications normally require analysis of trace analytes in biofluids. Although considerable advancements have been made in developing precise molecular assays, the trade-off between sensitivity and ability to resist non-specific adsorption remains a challenge. Here, we describe the implementation of a testing platform based on a molecular-electromechanical system (MolEMS) immobilized on graphene field-effect transistors. A MolEMS is a self-assembled DNA nanostructure, containing a stiff tetrahedral base and a flexible single-stranded DNA cantilever. Electromechanical actuation of the cantilever modulates sensing events close to the transistor channel, improving signal-transduction efficiency, while the stiff base prevents non-specific adsorption of background molecules present in biofluids. A MolEMS realizes unamplified detection of proteins, ions, small molecules and nucleic acids within minutes and has a limit of detection of several copies in 100 μl of testing solution, offering an assay methodology with wide-ranging applications. In this protocol, we provide step-by-step procedures for MolEMS design and assemblage, sensor manufacture and operation of a MolEMS in several applications. We also describe adaptations to construct a portable detection platform. It takes ~18 h to construct the device and ~4 min to finish the testing from sample addition to result.

Detection of miR-155 Using Peptide Nucleic Acid at Physiological-like Conditions by Surface Plasmon Resonance and Bio-Field Effect Transistor

“…In this regard, the injections were started when an average current variation of less than 0.01 µA over 200 s was detected. The current value after stabilization was normalized to the current variation according to the formula (I ds − I 0 )/I 0 = ∆I/I 0 , where I ds stands for the real-time recorded current, while I 0 is the current value obtained just after stabilization [56]. Furthermore, five single injections of working buffer were performed on active electrodes to obtain the blank signal required for the calculation of the LOD of our system.…”

“…Furthermore, five single injections of working buffer were performed on active electrodes to obtain the blank signal required for the calculation of the LOD of our system. LOD, defined by IUPAC as the smallest measure that can be reasonably detected for a given analytical procedure [56], was calculated from the calibration curve, using five times the standard deviation of the blank, according to the procedure in refs. [57,58] (see also ref.…”

Sensitivity-Enhancing Strategies of Graphene Field-Effect Transistor Biosensors for Biomarker Detection

“…123−126 Their reduced size compared to antibodies lessens the impact of the Debye screening effect by decreasing the distance to the graphene sensing interface, thus enhancing biomolecule detection. 22,127 Additionally, aptamers' small and flexible nature allows binding to smaller targets or domains inaccessible to antibodies, enabling GFET biosensors equipped with aptamers to achieve highly sensitive and selective biomarker detection.…”

“…Nucleic acid aptamers, derived via systematic evolution of ligands by exponential enrichment (SELEX), are stem-loop structured probes that offer small size, high designability, and strong affinity. , Aptamers can recognize a wide range of targets, including proteins, viruses, bacteria, cells, and heavy metals, making them promising for GFET biosensor development for biomarker detection. − Their reduced size compared to antibodies lessens the impact of the Debye screening effect by decreasing the distance to the graphene sensing interface, thus enhancing biomolecule detection. , Additionally, aptamers’ small and flexible nature allows binding to smaller targets or domains inaccessible to antibodies, enabling GFET biosensors equipped with aptamers to achieve highly sensitive and selective biomarker detection.…”