**4. G-quad as fluorescence probes**

The photophysical properties of G-quad structures are highly dependent on the bound metal ions at the location of nucleobase electrons of the structure due to the coordination of ions in the center of the quadruplex. Thus, G-quad is able to exhibit two- to ten-fold higher quantum fluorescence yields. This is due to the structure serving as energy donors to energy acceptors in close proximity to yield fluorescence resonance energy transfer (FRET) systems. G-quad can be utilized as a fluorescent probe in which the nucleobases in the structure are labelled or attached with fluorescent dyes. Besides, the enhancement of the fluorescence of a small dye can be exhibited based on the interaction of the G-quad with the fluorescence ligand [7,37,38]. Some of the ligands such as thiazole orange were reported to yield higher fluorescence yield with the aid of the G-quad structure [39]. On the other hand, the G-quad could also cause fluorescence quenching phenomena. For instance, the classic intercalating agent, ethidium bromide (EtBr), binds to double-stranded DNA and exhibit fluorescence intensity up to 30 fold. However, in the presence of G-quad, the fluorescence intensity of the EtBr can be quenched upon binding to the structure [7].

generated hemin–DNAzyme complex will catalyse the oxidation of ABTS to produce a coloured product [35]. Isothermal DNA amplification strategies can be employed easily for

Besides RCA, G-quad structures can also be amplified using other isothermal strategies like quadruplex priming amplification (QPA) as reported by Kankia et al. QPA allows for efficient generation of G-quad structures by isothermal amplification with the additional ability to selfdissociate for continuous amplification. During the QPA elongation process, the 5' end of the product will fold into intramolecular quadruplex and self-dissociates from the primer-binding site of the template, allowing the template to be accessible to the incoming primer for the next priming cycle. Thus, more quadruplexes were formed and eventually leading to increased signal readout. Our group incorporated the QPA system for the development of a sandwich immunoassay, called immuno-QPA (IQPA). The system exploits the peroxidase mimicking function of G-quad DNAzyme and the quadruplex amplification by QPA. A recombinant scFv was coated on the well and the biotinylated antigen was added and left to bind with the antibody. Streptavidin was introduced as a bridge between the biotinylated antigen and biotinylated QPA template. QPA was then carried out to amplify the QPA template with its specific primer to generate self-dissociating G-quad structures. These self-dissociating G-quad structures will bind to hemin to generate the similar colour change readout with ABTS as

The photophysical properties of G-quad structures are highly dependent on the bound metal ions at the location of nucleobase electrons of the structure due to the coordination of ions in

DNAzyme sensor detection.

126 Nucleic Acids - From Basic Aspects to Laboratory Tools

mentioned earlier [36].

**4. G-quad as fluorescence probes**

**Figure 3.** Ultrasensitive detection of thrombin by RCA and G-quad DNAzyme.

One of the label-free G-quad DNA-based fluorescence biosensors reported was designed to detect cisplatin, which is an anticancer drug widely used in chemotherapy. However, cisplatin overdose can cause neurotic cell death that makes it dangerous for administration without proper monitoring. Therefore, Zhou et al. developed a simple label-free fluorescence biosensor by utilizing the interaction of G-quad and cisplatin as cisplatin was found to bind to G-quad naturally (Figure 4). In the context of the assay, the absence of cisplatin will allow the formed G-quad to bind to N-methyl mesoporphyrin IX (NMM), resulting in an increase in fluorescence intensity. However, when cisplatin was added into the assay, it will bind to the G-quad structure and disintegrate the structure. This will then cause a drastic decrease in fluorescence intensity due to the collapse of the G-quad structure [40].

**Figure 4.** Label-free turn on and turn off fluorescence DNA G-quad-based sensor for the detection of cisplatin.

Wang et al. had proposed a label-free fluorescence biosensor based on G-quad formation in order to detect the lead (II) ion (Pb2+). This biosensor consists of a G-rich DNA strand with its partially complementary strand. The heme-oxygenase-1 inhibitor, zinc protoporphyrin (ZnPPIX) has been utilized as a fluorescence probe, whereby it can interact with the Pb2+/G– quad complex, producing fluorescence readout. In the absence of the Pb2+ ions, both DNA strands will form a DNA duplex. The addition of Pb2+ will unwind the DNA duplex to allow the G-rich strand to fold into the G-quad structure. The Pb2+/G–quad complex will then interact with ZnPPIX to enhance its fluorescence intensity, which is not possible with the DNA duplex. This biosensor could overcome the cumbersome step in which most of the fluorescence-based biosensors would require fluorescence dyes to be probed on the DNA. In addition, this biosensor can be reset easily to the original state by dissociating the G-quad structure. In order to dissociate the G-quad structure, the strong Pb2+ chelator DOTA (1,4,7,10-tetraazacyclodo‐ decane-1,4,7,10-tetraacetic acid) was used as it has high binding capacity against Pb2+ to remove Pb2+ from the G-quad structure, leaving the G-quad structure to dissociate and form a DNA duplex with the complementary strand again [41].

As reported by Kankia and co-workers, a further improvement to their original QPA method was reported [42]. The improved method involves the use of two linear processes that are the QPA and linear nicking amplification, which is based on the study done by Galas and coworkers. The probe DNA was hybridized with the target DNA and polymerase was used to extend the target DNA strand and form QPA-PBS (primer-binding site). Then, the nicking enzyme Nt.BSTNBI was introduced to nick the target strand and release the QPA-primerbinding strand. The QPA-primer-binding strand will bind to the QPA primer and the poly‐ merase will function to extend the strand further. The primer will then dissociate to emit fluorescence signals. The following priming step of QPA will be initiated again when the next primer binds with the QPA-primer binding strand. This method was reported to be able to reduce the background activity to allow sufficient sensitivity of the assay (Figure 5).

**Figure 5.** Principle of the QPA integrating nicking enzyme for diagnostics.
