Target-induced cyclic DNAzyme formation for colorimetric and chemiluminescence imaging assay of protein biomarkers

Abstract

A target-induced cyclic strategy for DNAzyme formation was proposed to achieve simple, sensitive and universal detection of protein biomarkers with convenient colorimetric or chemiluminescence imaging readout. In the assay, the target protein was recognized by a pair of DNA-labeled antibodies (Ab1-DNA1 and Ab2-DNA2) to form a proximate complex, which could hybridize with the conjugate DNA3/DNA4 to release the guanine-rich DNA4 and thus formed G-quadruplex/hemin horseradish peroxidase-mimicking DNAzyme. The process could be further recycled with Exonuclease III by cleaving DNA3 to free the proximate complex, resulting in the cyclic formation of DNAzyme. The G-quadruplex/hemin DNAzyme could catalyze the HO-mediated oxidation of 3,3,5,5-tetramethylbenzidine to produce the color change from colorless to blue or enhance the chemiluminescence of a luminol-HO system. Thus the signal could be read out with the naked eye, and by colorimetry and chemiluminescence imaging. Using a carcinoembryonic antigen as a model target, the proposed assay showed a detection range of 4 orders of magnitude along with detection limits of 170 and 16 pg mL for colorimetric and chemiluminescence imaging assays respectively. This assay had the advantages of easy operation, sensitive detection, target flexibility and diversified signal readout, providing a great opportunity for commercial application.

A Rolling Circle-Amplified G-Quadruplex/Hemin DNAzyme for Chemiluminescence Immunoassay of the SARS-CoV-2 Protein

“…Synthesis of DNA−Antibody Conjugates. According to previous studies, 33,34 DNA−antibody conjugates were pre-pared using MBS as a biofunctional connector. Antibody 1 (Ab1) (1 mg/mL) and antibody 2 (Ab2) (1 mg/mL) were first treated with excess 40-fold M MBS in 20 μL of 10 mM PBS (pH 7.2) for 2 h under low-frequency vibration, and excess MBS was removed by ultrafiltration (50 kDa Millipore, 10,000 rpm) to obtain Ab1-MBS and Ab2-MBS, respectively.…”

Nucleic-Acid Driven Cooperative Bioassays Using Probe Proximity or Split-Probe Techniques

“…In this section, we highlight some recent examples of new proximity binding modes as well as some clever adaptations of previously reported modes. The proximity binding concept has also been widely used to trigger amplification methods like the original proximity ligation assay (PLA), ,,, catalytic hairpin assembly (CHA), , circular strand displacement, , nicking enzyme amplification, , and others. ,− Some examples of assays coupled with these amplification methods will be discussed in this review, although we will focus more on the proximity binding concepts, on recent advancements in understanding the binding mechanisms, and on general guidelines for assay design. For a better understanding of the amplification techniques, we direct the readers to the cited articles or to a recent review focused on the subject …”

“…In this way, any available means of detecting DNA hybridization can now be used to report the quantity of the protein. Indeed, proximity assays have been designed using fluorescence resonance energy transfer (FRET), ,, electrochemistry, − chemiluminescence, , quantitative real-time polymerase chain reaction (qPCR), , DNAzyme assembly, − along with other analytical techniques.…”

Thermofluorimetric Analysis (TFA) using Probes with Flexible Spacers: Application to Direct Antibody Sensing and to Antibody-Oligonucleotide (AbO) Conjugate Valency Monitoring

“…A key advantage of this technique is its simple mix-and-read format, which can be adopted to either a homogenous or surface based assay 4,12,22,[25][26][27] . This proximity dependent annealing can be converted into a signal readout by coupling to fluorescent, electrochemical, or colorimetric detection platforms 5,16,21,28,29 .…”