Chemiluminescence Imaging for a Protein Assay via Proximity-Dependent DNAzyme Formation

An array-based chemiluminescence (CL) imaging method is presented for simple and high throughput detection of protein targets via the formation of a proximity-dependent DNAzyme to produce sensitive CL signal. The protein array is prepared by covalently immobilizing single-stranded guanine-rich nucleic acid 1-labeled antibody 1 (GDNA1-Ab1) or GDNA-thrombin aptamer subunit 1 (Apt-P1) as the capture probe on each sensing site on an aldehyde-functionalized disposable glass chip. In the presence of target protein, hemin, and another GDNA2-Ab2 or Apt-P2 probe, a sandwich complex among the protein and two probes can be formed to trigger the proximity assembly of GDNA1, hemin, and GDNA2, which leads to the formation of hemin-G-quadruplex DNAzyme. At different sensing sites, the DNAzyme-induced CL signals are simultaneously collected by a charge-coupled device for imaging readout of the sensing events. As a proof of concept, the proposed array-based CL imaging strategy is applied to detect carcinoembryonic antigen and thrombin and shows wide linear ranges over 4 and 5 orders of magnitude with the detection limits of 0.15 ng mL(-1) and 0.49 pM, respectively. Benefiting from the one-step proximity-dependent DNAzyme formation, the assay method is extremely simple and can be carried out within 40 min. By using different probes, the array can be easily used to detect more protein analytes. The advantages of easy operation, short assay time, good sensitivity, and versatility make it a promising candidate for point-of-care testing and commercial application.