DNA-based biosensors have been widely used for its stable chemical properties, cost-effectiveness, and easy modification. G-quadruplex (G4) DNA has attracted growing interest since it can be used for both target recognition and signal transduction. Much of G4 DNA's relevance in biosensors is related to its ability to bind specific metal ions, dyes and porphyrins, and these properties are first reviewed. Then, the role of G4 for molecular recognition is discussed including the sensing of metal ions, proteins and enzymes. Subsequently, G-quadruplex for signaling transduction is reviewed. In this part, G4 binds some substrate to enhance fluorescence, and binds hemin to show peroxidase-like activity. Finally, some sensors used G4 DNA for both target recognition and signaling. This review mainly contains specific examples of biosensors published in the last five years. Finally, some challenges in the field are discussed and a few future research opportunities are proposed.

G-quadruplex DNA for construction of biosensors

G-quadruplex: Flexible conformational changes by cations, pH, crowding and its applications to biosensing.

Review of recent progress on DNA-based biosensors for Pb2+ detection

Sensitive detection of the SARS-CoV-2 protein remains a great research interest in clinical screening and diagnosis owing to the coronavirus epidemic. Here, an ultrasensitive chemiluminescence (CL) imaging strategy was developed through proximity hybridization to trigger the formation of a rolling circle-amplified G-quadruplex/hemin DNAzyme for the detection of the SARS-CoV-2 protein. The target protein was first recognized by a pair of DNA-antibody conjugates, Ab-1 and Ab-2, to form a proximity-ligated complex, Ab-1/SARS-CoV-2/Ab-2, which contained a DNA sequence complemental to block DNA and thus induced a strand displacement reaction to release the primer from a block/primer complex. The released primer then triggered a rolling circle amplification to form abundant DNAzyme units in the presence of hemin, which produced a strong chemiluminescent signal for the detection of the target protein by catalyzing the oxidation of luminol by hydrogen peroxide. The proposed assay showed a detectable concentration range over 5 orders of magnitude with the detection limit down to 6.46 fg/mL. The excellent selectivity, simple procedure, acceptable accuracy, and intrinsic high throughput of the imaging technique for analysis of serum samples demonstrated the potential applicability of the proposed detection method in clinical screening and diagnosis.

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

Selection of a metal ligand modified DNAzyme for detecting Ni2.

Metalloporphyrins play important roles in biology, such as magnesium porphyrin for photosynthesis and iron porphyrin for carrying and transferring oxygen They are also powerful molecules for the development of biosensors, phototherapy, photocatalysis, photodegradation, light harvesting, and water splitting However, the porphyrin metalation reaction is difficult to achieve at room temperature due to a high kinetic barrier Inspired by the pioneering work in catalytic antibodies, ribozymes, DNAzymes and nanozymes have been developed as enzyme mimics to accelerate this reaction This review summarizes the progress in DNAzymes and nanozymes due to their excellent stability and low cost We first introduce the structure and property of common porphyrins and metalloporphyrins DNAzymes for porphyrin metalation are then reviewed, including early work, recent work using Pb2+ as a cofactor, and non-G-quadruplex DNAzymes The catalytic mechanisms of DNAzymes are also discussed, especially the role of metal ions Subsequently, nanozymes for porphyrin metalation based on graphene and a few other nanomaterials are reviewed In this part, the interactions between the nanozymes and porphyrins are elucidated to describe the catalytic effect In addition, beta-cyclodextrin and some surfactants that can form micelles in water were also found to have catalytic activity Finally, we review the applications of porphyrin metalation reactions for the detection of various metal ions, improving photocatalytic activity, and removing heavy metal ions in water

Porphyrin metalation catalyzed by DNAzymes and nanozymes

We herein report a DNAzyme named T30695 (sequence: (G3 T)4 ) that can catalyze Zn2+ insertion into three different porphyrins in the presence of Pb2+ as a cofactor. Meanwhile, T30695 with Pb2+ alone was found to cause a shift in both the fluorescence and UV-vis spectra of protoporphyrin IX (PPIX), thus suggesting that metalation of Pb2+ was also achieved at room temperature. From kinetic measurements, the reaction required two Pb2+ ions; this is consistent with one being a cofactor and the other being a substrate. No previous reports inserted Pb2+ into porphyrins by using DNAzymes or protein-based enzymes. This reaction was most significantly inhibited in the presence of K+ followed by Na+ and Li+ , suggesting the importance of the Pb2+ -stabilized G-quadruplex. When Pb2+ is inserted into PPIX, its emission blue shifts from 635 to 590 nm, thus allowing simple ratiometric fluorescent sensing with a detection limit of 1.2 nM Pb2+ .

Yu Zhou

Papers

G-quadruplex DNA for construction of biosensors

Porphyrin metalation catalyzed by DNAzymes and nanozymes

Pb2+ as a Substrate and a Cofactor of a Porphyrin Metalation DNAzyme