A Modular, DNA‐Based Beacon for Single‐Step Fluorescence Detection of Antibodies and Other Proteins

Abstract

A versatile platform for the one-step fluorescence detection of both monovalent and multivalent proteins has been developed. This system is based on a conformationswitching stem-loop DNA scaffold that presents a smallmolecule, polypeptide, or nucleic-acid recognition element on each of its two stem strands. The steric strain associated with the binding of one (multivalent) or two (monovalent) target molecules to these elements opens the stem, enhancing the emission of an attached fluorophore/quencher pair. The sensors respond rapidly (< 10 min) and selectively, enabling the facile detection of specific proteins even in complex samples, such as blood serum. The versatility of the platform was demonstrated by detecting five bivalent proteins (four antibodies and the chemokine platelet-derived growth factor) and two monovalent proteins (a Fab fragment and the transcription factor TBP) with low nanomolar detection limits and no detectable cross-reactivity.Recent years have seen a significant increase in the number of well-characterized biomarkers, proteins present in the blood or on cells that are diagnostic of disease. [1][2][3] Unfortunately, however, current methods for the detection of such markers are based on either multistep, wash-, or reagentintensive processes and require sophisticated, laboratorybased measurements (e.g., ELISA or Western blot assays) or are at best only semi-quantitative (e.g., immunochemical dipsticks).[4-6] These drawbacks limit the accessibility of quantitative molecular diagnostics, resulting in delayed treatment, reduced compliance, and poorer outcomes. [1,2, 7] In contrast to the cumbersome, multistep nature of current detection schemes, however, biomolecular receptors present in organisms respond to changes in the concentration of their targets in a quantitative fashion and without needing the addition of reagents or wash steps. [8,9] Indeed, these receptors detect thousands of distinct molecules in real time even in the complex in vivo environment.[10] Building artificial biosystems of similar simplicity, convenience, and selectivity represents a major bioengineering goal.One of the strategies used by naturally occurring "sensors" [11] is based on conformational switching in which a receptor undergoes a large-scale, binding-induced conformational change in the presence of its target. [8,9] As their signaling is linked to a specific, binding-induced event, such "switches" are highly selective and enable detection even in highly complex sample matrices.[10] Moreover, such conformational changes can also be harnessed in artificial systems, where they can be used to generate an optical or electrochemical signal without the addition of exogenous reagents or coupling to exogenous biochemical reactions. [12,13] Motivated by these considerations, we have developed a single-step method for the quantitative detection of specific proteins that is rapid, inexpensive, and highly selective. We drew inspiration from DNA molecular beacons, which are synthetic nucleic acid switches for t...

A DNA nanoswitch-controlled reversible nanosensor

“…In the past decades, DNA nanosensors have attracted much attention for their wide applications in environmental monitoring ( 1 ) and biological diagnosis ( 2 ), including the specific and sensitive detections of metal ions ( 3 , 4 ), proteins ( 5 , 6 ), nucleic acids ( 7 ), small molecules ( 8 , 9 ), etc. Recently, several DNA nanodevices have been built upon DNA nanosensors such as DNA logic sensors ( 10 ), DNA sensing dendrimers ( 11 ) and DNA sensing nanomachines ( 12 ).…”

Specific Biosensing Using DNA Aptamers and Nanopores

“…Our two‐step assay has built a bridge between the nanopore‐based single molecule method and DNA‐based circuitry designs. Apart from the aptamer‐based conformation changes used in this work, direct binding of antibodies and other proteins can also facilitate successful signal translation into output strands . Compared to the traditional fluorescence assays, our method avoids fluorophores conjugation and spectra overlap.…”

Robotic DNA Nanostructures

“… 96 Omitting often complex and large DNA nanostructures, they have designed sensors that consist of only several functionalized DNA sequences and realized sensitive antibody probing based on the structural transition of their devices. 97 , 98 Other than sensors, it is worth noting that with the help of antibodies the group has also produced DNA robotic vectors for controlled release of molecular payloads. 99 , 100 …”