Proximity hybridization-induced competitive rolling circle amplification to construct fluorescent dual-sensor for simultaneous evaluation of glycated and total hemoglobin

• Published in: Biosensors & bioelectronics 2022-04, Vol.202, p.113998-113998, Article 113998
• Main Authors: Chen, Zhao-Peng, Su, Ming-Li, Chen, Hao-Ran, Yuan, Ruo, Li, Yan, Liang, Wen-Bin
• Format: Article
• Language: English
• Subjects: Biosensing Techniques - methods; DNA; DNA Probes; Glycated hemoglobin; Glycated Hemoglobin A - analysis; Hemoglobin; Nucleic Acid Amplification Techniques - methods; Nucleic Acid Hybridization; Proximity hybridization; Rolling circle amplification
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2022.113998

The glycated hemoglobin (HbA1c) level, defined as the ratio of HbA1c to total hemoglobin (tHb), has been considered as a standard index for diabetes mellitus diagnosis, avoiding any short-term fluctuation of the blood glucose level. However, it is still a key challenge that some complicated separation procedures are required to detect HbA1c and tHb in a single experiment. Herein, we developed a dual-sensing fluorescent assay for HbA1c and tHb based on proximity hybridization-induced rolling circle amplification (RCA) and T7 exonuclease aided signal cycle to calculate the HbAlc level in one single experiment. Typically, four DNA-tagged probes were used for immunoreactions of anti-HbA1c antibodies (Ab1) and anti-Hb antibodies (Ab2) with HbA1c and Hb, respectively. The RCA1 and RCA2 were induced by the complexes of assistant DNAs and the products of proximity hybridization with anti-HbA1c-DNA1 (2) and anti-Hb-DNA3 (4), respectively. Meanwhile, the tHb in solution could prevent the existing RCA2 based on its competing with Hb-DNA3 for Ab2-DNA4. Thus, RCA1 and RCA2 routes create “signal-on” and “signal-off” readouts, respectively. After the hybridization of signal probes and RCA products, the quenched fluorescence was recovered by the digestion of T7 exonuclease. Under optimized conditions, the method displayed high sensitivity with a limit of detection 2.17 ng/mL and 33.4 ng/mL for HbA1c and tHb, respectively, and the real sample analysis results were found to match well with the theoretical data, holding feasibility for rapid, homogeneous, and easy HbA1c level evaluation and great promise as a potential alternative tool to analyze HbA1c level clinically. •Using HbA1c as a target biomarker to avoid any short-term fluctuations in blood glucose levels for diabetes diagnosis.••The assay was constructed for simultaneous and distinguishable detection of HbA1c and tHb without separation procedures.•The obtained HbA1c and tHb concentrations could be used for further glycated level evaluation in a single experiment.•The proximity hybridization assay could be easily extended to various protein analyses by employing different antibody.