Aptamer-based quantification of small molecules using qPCR (Apta-qPCR)

Small molecules remain hard to measure. The established tools are hyphenated gas chromatography (GC) and high-performance liquid chromatography (HPLC), read by UV or fluorescence detection. These methods are sensitive, but they need costly equipment and highly trained staff, and the extraction and clean-up steps they depend on lose sample and add time and cost. Simple label-free alternatives are scarce, and most work for only one target analyte. We present a different approach. Apta-qPCR is a magnetic bead-based, aptamer-assisted real-time PCR assay for measuring small molecules. It is label-free, and we characterised it using adenosine triphosphate (ATP) as a model analyte.

The assay works by target-induced dissociation (TID). Deoxythymidine (d(T)25) was immobilised on carboxyl-coated magnetic beads by one-step EDC coupling, which gives dT-beads. A short oligonucleotide carrying d(A)25, complementary to the ATP-binding region of the aptamer, links the aptamer to these beads. ATP then disrupts that link. The aptamer is released into the supernatant, where qPCR measures how much came off. We tested several settings. Complementary sequences of 11, 14, 17, 20 and 25 nt were compared, and detection was optimal with the 14 nt sequence.

We also optimised the density of aptamer on the beads and the TID incubation time. Specificity was checked against GTP, CTP and TTP at ten-fold excess over ATP. Apta-qPCR detected ATP with a limit of detection of 17 nM, over a wide dynamic range from 50 nM to 5 mM in biological samples. One determination used 6.25 μL of sample and took 2.5 h. In cell lysate the assay recovered 90–110 % of added ATP between 0.5 and 10 μM, matching the CellTiter-Glo® reference assay. It also detected 0.1 μM ATP, which CellTiter-Glo® could not. The platform then transferred directly to a second, unrelated analyte. Ochratoxin A (OTA) was detected with a sensitivity of 0.2 nM over a broad dynamic range from 0.5 nM to 10 μM, including in red wine and peanut oil. The method depends only on an aptamer and a complementary oligonucleotide, so it transfers readily to a broad range of aptamer–target pairs.