Sensitive Microscale Thermophoresis Assay Using Aptamer Thermal Switch

Abstract

Rapid sensitive detection of targets is desired in broad fields, such as environmental monitoring, food safety, and molecular diagnosis. We demonstrated an innovative strategy of constructing aptamer thermal switch for target detection, which affords a large difference in heating-induced conformation changes between the bound and the unbound states. This aptamer thermal switch is obtained by rational engineering aptamer sequence and modulating aptamer conformation stability under heating. We further introduced a conformation-sensitive fluorophore into the aptamers, converting heating-induced conformation changes to heating-induced fluorescence changes. The bound and unbound aptamers showed a large difference in heating-induced fluorescence change. Using these aptamers in microscale thermophoresis (MST), which is a powerful tool for quickly measuring fluorescence responses to heating with precise ratiometric analysis, we achieved detection of a variety of targets (e.g., cadmium ions (Cd 2+ ), small molecule aflatoxin B1, and protein biomarker immunoglobulin E) within seconds with pronounced sensitivity. We envision that this strategy will have wide applications to detection of targets of interest in broad research fields, and it will also be helpful for affinity binding study.

Sensitive microscale thermophoresis assay for rapid ochratoxin A detection with fluorescently labeled engineered aptamer

“…2), which may be due to the insensitivity of FAM at O36 to the conformational change of the aptamer during heating. 11 O33-3′FAM generated a small F norm change of 27.2‰ in response to OTA, and the K d was 3.8 ± 2.1 nM as determined by MST (Fig. 2).…”

“…The results indicate that for the aptamer probes O31-3′FAM, O32-3′FAM, and O33-3′FAM, the OTA-binding-induced MST signal change dramatically decreased with increasing stem length of the aptamer probes. 11 This can be attributed to the fact that aptamers with longer stems ( e.g. , O33-3′FAM) may already have a stable conformation and high stability under heating in the absence of OTA, and the unbound and bound aptamers have a small difference in fluorescence response to heating in MST.…”

“…The F norm signal of the fluorescently labeled molecule is mainly governed by molecular thermophoresis and the temperature-related intensity change (TRIC) property of fluorophores; 9 it is reported that TRIC can make the larger contribution. 10,11 MST has many applications in analyzing diverse molecular interactions, and MST and related techniques also provide a novel tool in the rapid quantification of targets, which usually requires an affinity ligand for specific recognition of targets. 11–19…”

“…10,11 MST has many applications in analyzing diverse molecular interactions, and MST and related techniques also provide a novel tool in the rapid quantification of targets, which usually requires an affinity ligand for specific recognition of targets. 11–19…”

“…11 In MST analysis, a high-affinity aptamer probe showing a large MST signal change upon target binding is favorable for sensitive MST detection of targets. 9,11…”