A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva

Abstract

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

Fig. 1. The design of our cocaine-binding CBSA. Two split aptamer pairs were derived from 38-GC. Stem 1 of one split aptamer (A) was merged with stem 2 of the other split aptamer (B) to form an engineered CBSA (C) comprising a short fragment (SF) and a long fragment (LF).

Fig. 2. ATMND as a fluorescence reporter of target-induced CBSA assembly. (A) We modified the core CBSA sequence (left) by replacing the adenosine (at position 10 from 5′) between the two binding domains of the short fragment with a C3 spacer abasic site (marked as X) to yield the final CBSA construct (middle). Upon binding cocaine, the CBSA undergoes target-induced assembly, forming a dinucleotide bulge within each three-way junction (right). The cocaine–CBSA-5325 complex features four complementary double-stranded regions (A–D) and a duplexed AP site. (B) In the absence of cocaine, LF and SF remain separated and the unbound ATMND in solution generates a strong fluorescence signal. Cocaine induces CBSA assembly, forming the duplexed AP site that binds ATMND and thereby quenches its fluorescence. (C) Time-course of ATMND quenching by cocaine-induced split aptamer assembly. (D) Sequence of the parent split aptamer (PSA) with incorporated AP site.

Fig. 3. Analyzing the dual binding domains of CBSA for target-induced assembly. (A) We generated multiple derivatives of CBSA-5325, including split aptamers with a single binding pocket (LSA) and a pair of point-mutants (CBSA-M1 and CBSA-M2) with sequence alterations that disrupt either of the two binding domains (pink circle). (B) ATMND quenching in the presence or absence of 250 μM cocaine. Quenching was calculated by (F _A – F)/F _A × 100%, where F _A is the fluorescence of 200 nM ATMND in 1× binding buffer alone and F is the fluorescence of the ATMND–CBSA mixture with 250 μM cocaine or without cocaine, respectively.

Fig. 4. Effect of target-binding affinity (K _1/2) on CBSA cooperativity. (A) The working principle of the CBSA-based fluorophore/quencher assay. (B) Calibration curves for the assay at cocaine concentrations ranging from 0 to 2500 μM (left). Right panel shows a linear response at 0–10 μM. Reactions were performed with 1 μM CBSA long fragment, 1 μM fluorophore/quencher-modified CBSA short fragment, and different concentrations of cocaine in 10 mM Tris–HCl, 100 μM MgCl₂ (pH 7.4) at room temperature.

Fig. 5. Effect of the intrinsic affinity of the PSA on CBSA target-binding affinity (K _1/2). (A) We generated CBSA-5335-GT by replacing a G–C base-pair (highlighted) in the 5′ binding domain of CBSA-5335 with a G–T wobble pair. (B) Calibration curves for the assay at cocaine concentrations ranging from 0 to 1000 μM after a 15 min incubation (left), with a linear response at 0–10 μM (right). Experiments were performed with 1 μM CBSA long fragment, 1 μM fluorophore/quencher-modified CBSA short fragment, and different concentrations of cocaine in 10 mM Tris–HCl, 100 μM MgCl₂ (pH 7.4) at room temperature.

Fig. 6. Successful detection of cocaine in saliva samples with fluorophore/quencher-modified CBSA-5335. (A) Dilution effects on cocaine detection in saliva. Saliva samples spiked with cocaine were tested with CBSA-5335 at 50% or 10% dilutions. Calibration curves were constructed based on signal gain at each concentration of cocaine in the pre-dilution samples. (B) Time course of CBSA-5335 in the presence of 1, 5 and 10 μM cocaine in 10% pooled samples.

Fig. 7. High sensitivity and specificity of our CBSA-5335-based fluorophore/quencher assay for cocaine detection in saliva. (A) Calibration curve for the assay in buffer and 10% saliva. (B) Signal gains from the CBSA assay in the presence of 50 μM (left) and 5 μM (right) cocaine (COC) or potential interferents including cocaethylene (EC), benzoylecgonine (BZE), anhydroecgonine methyl ester (MEG) and nicotine (NIC) in 10% saliva. Inset shows the interferent structures. Error bars show standard deviation of signal gain from three measurements at each concentration.