PCR‑ALERA introduces a new way to detect DNA without antibodies. The method links modified nucleotides used in PCR to a special peptide via click chemistry. This peptide activates the enzyme papain in solution. Papain cuts a fluorescent substrate, so the signal is visible in a microplate well. The whole process, from PCR to readout, takes about four hours.

The workflow starts with PCR that incorporates commercially available modified nucleotides into the target DNA. A selenocysteine‑containing peptide is then attached to these nucleotides by click chemistry. The DNA‑peptide hybrid is immobilised on a streptavidin‑coated microplate. Latent papain is added; it is rapidly reactivated by the peptide. The reactivated papain processes a fluorogenic substrate, producing a bright fluorescent signal. The signal’s intensity reaches more than a 200‑fold difference between positive and negative controls.

PCR‑ALERA removes the need to handle costly, time‑consuming antibodies. The assay relies on the highly efficient activation of papain by a selenolate‑triggered disulfide cleavage, giving it high catalytic turnover. A major limitation is unspecific binding of the activator peptide or the DNA‑peptide hybrid to the plate surface. This reduces sensitivity and requires careful purification or washing steps to achieve optimal performance.