Direct Detection of Adenosine in Undiluted Serum Using a Luminescent Aptamer Sensor Attached to a Terbium Complex

Abstract

Aptamers, single-stranded nucleic acids that can selectively bind to various target molecules, have been widely used for constructing biosensors. A major challenge in this field, however, is direct sensing of analytes in complex biological media such as undiluted serum. While progress has been made in developing inhomogeneous assay by using a pre-separation step to wash away the interferences within serum, a facile strategy for direct detection of targets in homogenous unprocessed serum is highly desired. We herein report a turn-on luminescent aptamer biosensor for the direct detection of adenosine in undiluted and unprocessed serum, by taking advantage of a terbium chelate complex with long luminescence lifetime to achieve time-resolved detection. The sensor exhibits a detection limit of 60 µM adenosine while marinating excellent selectivity that is comparable to those in buffer. The approach demonstrated here can be applied for direct detection and quantification of a broad range of analytes in biological media by using other aptamers.

A general approach for rational design of fluorescent DNA aptazyme sensors based on target-induced unfolding of DNA hairpins

“…Using the aptazyme sensor with 8 bp under the optimized condition, our method was able to detect adenosine in solution with a detection limit of 18 µM based on the definition of 3σ b /slope (inset of Figure 2B). This sensitivity was comparable or even better than some other reported adenosine sensors using the aptamers or aptazymes for adenosine [23,24,40,52,60]. Other two nucleosides, uridine and cytidine, almost did not induce any rate enhancement ( Figure 2D), suggesting the selectivity of the aptamer was well preserved in the aptazyme design.…”

DNAzyme Hybridization, Cleavage, Degradation, and Sensing in Undiluted Human Blood Serum

“…Indeed, we observed target concentration dependent cleavage in serum, with a detection limit of 6 nM ( Figure 5B). The sensitivity can be improved by incorporation of other signaling methods such as lanthanide luminescence, 43 gold nanoparticles or polymerase enzymes. 59 We also observed high specificity; if a non-target DNA was added, no cleavage occurred ( Figure 5C).…”

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

“…LRET using LLCs has been applied in a number of applications, including monitoring protein-protein interactions in cells [356], monitoring orthogonal ligand-dependent protein-peptide binding events [352], high-throughput screening of potential drug candidates [357], and numerous in vitro bioassays [347,353,[358][359][360][361]. Kupstat et al, for example, developed a homogeneous time-resolved immunoassay for prostate-specific antigen (PSA) that was sensitive and quantitative, and could be incorporated into a point-of-care testing (POCT) device [360].…”

“…A similar format, using a Tb donor and five different acceptor dyes, was recently used to detect five different lung cancer tumor markers simultaneously in a 50 ml human serum sample [362]. Li et al developed an adenosine sensor using an aptamer-based sensor design, which functioned by inducing a conformational change that disrupted the LRET (Figure 6.19b) [361]. The sensor was able to detect selectively 60 mM of adenosine in undiluted serum samples.…”