Abstract
Sensitive quantification of protein biomarkers is highly desired for clinical diagnosis and treatment. Yet, unlike DNA/RNA which can be greatly amplified by PCR/RT-PCR, the amplification and detection of trace amount of proteins remain a great challenge. Here, we combined allosteric probe (AP) with magnetic bead (MB) for assembling an on-bead DNA synthesis system (named as APMB) to amplify protein signals. The AP is designed and conjugated onto the MB, enabling the protein biomarker to be separated and enriched. Once recognizing the biomarker, the AP alters its conformation to initiate DNA synthesis on beads for primary signal amplification. During the DNA synthesis, biotin-dATPs are incorporated into the newly synthesized DNA strands. Then, the biotin-labeled DNA specifically captures streptavidin (STR)-conjugated horseradish peroxidase (HRP), which is used to catalyze a colorimetric reaction for secondary signal amplification. By using carcinoembryonic antigen (CEA) as a protein model, the APMB can quantify protein biomarkers of as low as 0.01 ng/mL. The response values measured by APMB are linearly related to the protein concentrations in the range 0.05 to 20 ng/mL. Clinical examination demonstrated good practicability of the APMB in quantifying serum protein biomarker. The on-bead DNA synthesis could be exploited to improve protein signal amplification, thus facilitating protein biomarker detection of low abundance for early diagnosis.