**3. G-quad mimicking peroxidase activity in colorimetric-based sensors**

There are several forms of readouts that G-quad structures are capable of producing. A major readout format is by absorbance value where G-quad structures are able to form DNAzymes that exhibit catalytic activity [19]. In nature, DNAzymes are initially known as catalytic enzymes or DNA enzymes because of its ability to catalyze many reactions such as ligation, DNA modification [20, 21], cleavage of DNA or RNA [21, 22] and also methylation of porphyrin rings [9,23]. One of the most important features of G-quad DNAzyme activity is the peroxidase mimicking activity when hemin is bound to the G-quad structure. The hemin–quadruplex complex will catalyze the peroxide-mediated oxidation of the 2,2'-azino-bis(3-ethylbenzthia‐ zoline-6-sulphonic acid) diammonium salt (ABTS) to generate a coloured product [24-26]. This colorimetric change has allowed the development of assays detecting metal ions, aptamer– substrate complexes and even proteins. The conventional method to generate such change in colour is based on enzymatic reactions involving enzymes such as horseradish peroxidase and alkaline phosphatase. These enzymes are usually active in a narrow temperature range and denatures at high temperatures. DNAzymes are stable in a broad temperature range and even at very high temperatures, making it an interesting alternative to enzymes. Another advantage of DNAzymes is the ease in preparation by chemical synthesis or by PCR, whereas protein enzymes require tedious preparation and purification processes. A key advantage of G-quad DNAzyme is the ability to carry out signal amplification by conventional DNA amplification methods, which is impossible with normal protein set-ups. The attractiveness of G-quad DNAzyme to mimic peroxidase-like activity has allowed it to be exploited for the development of biosensors [27-30].

An assay to detect silver ions (Ag+ ) was developed using G-quad as the reporter system. Ag+ ions are able to stabilize cytosine–cytosine (C–C) mismatches by forming the C–Ag+ –C base pairs. Therefore, in the absence of Ag+ , the G-rich sequence will form an intramolecular duplex. The addition of Ag+ in the mixture will allow the G-rich sequence to fold into a quadruplex structure and readily bind to hemin to form a DNAzyme. This will allow the G-quad to exhibit the peroxidase-like activity resulting in a change of the colourless ABTS to a coloured product in the presence of Ag+ ions. The application of G-quad is useful to detect other molecules other than metal ions [31].

Our group has shown the application of G-quad as a sensitive reporter system for the detection of antibody–antigen interaction. The system was based on a pre-formed reporter system whereby a probe was pre-formed by conjugating streptavidin gold nanoparticles with biotinylated antigen and biotinylated daunomycin aptamer to exhibit the hemin-dependent peroxidase-like activity (Figure 1). Thus, the pre-assay generation of such reporter probes allows for rapid one-step incubation in a one-pot synthesis by exploiting the simple yet strong streptavidin–biotin interaction. This helps to eliminate multiple tedious steps of incubation and wash of conventional immunoassay systems. In this direct antibody–antigen assay, antibodies against the target antigen was coated to the microtiter plates and incubated with the probe. The wells were then developed with ABTS solution. The assay was able to generate sensitive readouts for both competitive and direct assays [32].

**Figure 1.** Schematic diagram of the immunoassay that is based on the preformed-reporter system.

Commonly, the peroxidase-like activity of DNAzymes is based on the oxidation of ABTS solution mediated by hydrogen peroxide (H2O2). This mechanism can be manipulated to initiate different sensing outcomes based on the same principles. This was evident in a detection assay for cholesterol by substituting the H2O2 with the catalysed cholesterol oxidase reaction of cholesterol and oxygen to produce the required H2O2 (Figure 2). Therefore, the Gquad DNAzyme will be able to exhibit the peroxidase-like activity after the binding of hemin to catalyze the oxidation of colourless ABTS mediated by the cholesterol oxidase reacted production of H2O2. Therefore, only with the presence of cholesterol, the changes in colour of ABTS will occur. Amplification of the DNA sequences to produce more DNAzyme was carried out to further improve the signal readout [33].

**Figure 2.** Schematic diagram of the G-quad-based colorimetric sensor for cholesterol.

methods, which is impossible with normal protein set-ups. The attractiveness of G-quad DNAzyme to mimic peroxidase-like activity has allowed it to be exploited for the development

structure and readily bind to hemin to form a DNAzyme. This will allow the G-quad to exhibit the peroxidase-like activity resulting in a change of the colourless ABTS to a coloured product

Our group has shown the application of G-quad as a sensitive reporter system for the detection of antibody–antigen interaction. The system was based on a pre-formed reporter system whereby a probe was pre-formed by conjugating streptavidin gold nanoparticles with biotinylated antigen and biotinylated daunomycin aptamer to exhibit the hemin-dependent peroxidase-like activity (Figure 1). Thus, the pre-assay generation of such reporter probes allows for rapid one-step incubation in a one-pot synthesis by exploiting the simple yet strong streptavidin–biotin interaction. This helps to eliminate multiple tedious steps of incubation and wash of conventional immunoassay systems. In this direct antibody–antigen assay, antibodies against the target antigen was coated to the microtiter plates and incubated with the probe. The wells were then developed with ABTS solution. The assay was able to generate

sensitive readouts for both competitive and direct assays [32].

**Figure 1.** Schematic diagram of the immunoassay that is based on the preformed-reporter system.

ions are able to stabilize cytosine–cytosine (C–C) mismatches by forming the C–Ag+

) was developed using G-quad as the reporter system. Ag+

in the mixture will allow the G-rich sequence to fold into a quadruplex

ions. The application of G-quad is useful to detect other molecules other

, the G-rich sequence will form an intramolecular duplex.

–C base

of biosensors [27-30].

The addition of Ag+

in the presence of Ag+

than metal ions [31].

An assay to detect silver ions (Ag+

pairs. Therefore, in the absence of Ag+

124 Nucleic Acids - From Basic Aspects to Laboratory Tools

A new strategy was reported by Tang et al. for nucleic acid detection of the viral load of Hepatitis B virus (HBV) that utilizes G-quad DNAzyme as the probe and involves polymerase chain reaction (PCR). The DNA probe was designed to form a hairpin structure at room temperature. During the denaturation step of PCR, the dsDNA HBV templates will denature, and the DNA probe will be linearized and allowed to hybridize with the target HBV ssDNA. The loop of the DNA probes will anneal to the conserved region of the HBV genome. The DNA probes can be amplified and cleaved so that they can form G-quad DNAzyme. The stem part of the probe is used to prohibit DNAzyme sequence to fold into G-quad at room temperature in order to reduce the background reading. With the involvement of PCR, the sensitivity and specificity of the HBV DNA assay was improved [34].

For further improvement of the DNA probe amplification, single temperature amplification (isothermal amplification) was carried out instead of going through the conventional temper‐ ature cycling with the PCR. As reported by Liu et al., an aptamer-modified microchip that combines rolling circle amplification with the peroxidase mimicking activity by G-quad DNAzyme was developed for thrombin detection (Figure 3). The assay involved an aptamermodified microchip and a reporter aptamer consisting of the thrombin aptamer sequence and a primer with G-quad circular template. When the sample was introduced into the microchip, thrombin was captured by the chip immobilized thrombin aptamer and the reporter aptamer will act as primer for the RCA amplification process. This will allow the amplification process to generate increased amounts of G-quad to form DNAzyme upon binding of hemin. The 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 DNAzyme sensor detection.

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

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 mentioned earlier [36].
