On-bead DNA synthesis triggered by allosteric probe for detection of carcinoembryonic antigen

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. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00604-022-05404-4.

Bridge-DNA synthesis triggered by an allosteric aptamer for the colorimetric detection of pathogenic bacteria

“…† The Cy3 and Cy5 labeled aptamers were dissolved in 2× TAE buffer, and diluted to 15 mM. The AAP solution was incubated at 60 °C for 3 min and cooled to 25 °C to ensure they were correctly folded into a hairpin structure, as described by Ling et al 22 Next, 30 mL AAP solution was pipetted into 30 mL E. coli O157:H7 solution (10 8 cfu mL −1 ), and mixed well. The mixture was subjected to uorescence spectrophotometry to obtain the emission spectrum at an excitation wavelength of 480-490 nm.…”