Aptamer Biosensor Based on Fluorescence Resonance Energy Transfer from Upconverting Phosphors to Carbon Nanoparticles for Thrombin Detection in Human Plasma

Abstract

We presented a new aptamer biosensor for thrombin in this work, which was based on fluorescence resonance energy transfer (FRET) from upconverting phosphors (UCPs) to carbon nanoparticles (CNPs). The poly(acrylic acid) (PAA) functionalized UCPs were covalently tagged with a thrombin aptamer (5'-NH(2)- GGTTGGTGTGGTTGG-3'), which bound to the surface of CNPs through π-π stacking interaction. As a result, the energy donor and acceptor were taken into close proximity, leading to the quenching of fluorescence of UCPs. A maximum fluorescence quenching rate of 89% was acquired under optimized conditions. In the presence of thrombin, which induced the aptamer to form quadruplex structure, the π-π interaction was weakened, and thus, the acceptor was separated from the donor blocking the FRET process. The fluorescence of UCPs was therefore restored in a thrombin concentration-dependent manner, which built the foundation of thrombin quantification. The sensor provided a linear range from 0.5 to 20 nM for thrombin with a detection limit of 0.18 nM in an aqueous buffer. The same linear range was obtained in spiked human serum samples with a slightly higher detection limit (0.25 nM), demonstrating high robustness of the sensor in a complex biological sample matrix. As a practical application, the sensor was used to monitor thrombin level in human plasma with satisfactory results obtained. This is the first time that UCPs and CNPs were employed as a donor-acceptor pair to construct FRET-based biosensors, which utilized both the photophysical merits of UCPs and the superquenching ability of CNPs and thus afforded favorable analytical performances. This work also opened the opportunity to develop biosensors for other targets using this UCPs-CNPs system.

Inorganic lanthanide nanoprobes for background-free luminescent bioassays

“…The commonly used acceptors are those molecules or nanomaterials with a large absorption band overlapped with the emission bands of UCNPs. For instance, based on different acceptor entities such as organic dyes, Au NPs, MnO 2 nanosheets, carbon NPs, graphene and graphene oxide (GO), Liu and coworkers [192][193][194][195][196][197][198][199][200] proposed a series of UC-FRET pairs for the detection of diverse biomolecules including glucose, thrombin, DNA, matrix metalloproteinase-2 (MMP-2), CEA, and Kanamycin, etc. For point-of-care testing, they further designed a straightforward paper-based microfluidic device (namely UC-μPAD) for UC-FRET assays based on a normal office printing sheet with a simple plotting method [192].…”

Biosensor Design with Molecular Engineering and Nanotechnology

“…As previously mentioned on a number of occasions in this section, aptamers often undergo significant conformational changes, adopting defined three dimensional structures upon binding to their targets. This conformational coupling property paves the ways for optical biosensors based on changes in fluorescence intensity or polarisation where an organic fluorophore is introduced into the aptamer molecule [32]. In some cases, a pair of fluorescence donor/receptor groups can be incorporated into an aptamer molecule as conformational changes can bring certain sites on the aptamer molecule closer/further from each other modulating a FRET signal [18].…”

Photonen aufkonvertierende Nanopartikel zur optischen Codierung und zum Multiplexing von Zellen, Biomolekülen und Mikrosphären

“…[211][212][213] Für diese Art von Assay ist es notwendig, dass die Emissionsintensität der UCNPs ausschließlich durch den Analyten verändert wird. Dies wird meistens durch LRET erreicht, wobei die UCNPs als Donor und ein organischer Farbstoff, [214] Goldnanopartikel, [149,215,216] Quantenpunkt, [217] Kohlenstoffnanopartikel, [218,219] Graphenoxid, [220] Mangandioxid [221] oder Phycobiliprotein [222] als Akzeptor wirken. Goldnanopartikel haben sich als die effizientesten Akzeptoren für UCNPs herausgestellt (Abbildung 10), doch ihr großer Durchmesser (gegenüber denen organischer Farbstoffe) kann zur sterischen Hinderung während der Analyterkennung führen.…”

“…Da der fluoreszierende Donorfarbstoff durch ein UCNP ersetzt werden kann, ist es unkompliziert, die LRET-Aufkonversionstechnik auf vorhandene FRETSubstrate zu übertragen. Dieses Detektionssystem wurde eingesetzt, um Assays für Endonucleasen, [224] Caspasen, [234] Thrombin [218] oder Metalloproteinasen [219] zu entwickeln.…”