**6. G-quad integrates with electrochemistry**

Apart from the applications that exploit the properties of the G-quad structures, new strategies were established by integrating G-quad with electrochemistry. Recent studies developed different electrochemical G-quad sensors based on magnetic particles, nanoparticles labelled with metal tags, nanotubes and other nanomaterials. Wang et al. developed a thrombin detection assay using amplified electrochemical signal. This assay consists of an aptamer modified gold electrode and another aptamer modified with Cds hollow nanosphere. These two aptamers are complementary in most part except for the middle bases that are not complementary. This design will allow the aptamer to form a hairpin. Thus, in the presence of thrombin, the conformation of the aptamer will change from a hairpin to a G-quad structure. The nanospheres will allow the electron transfer between the gold electrode and K3[Fe(CN)6], producing an electrocatalytic response [46].

Besides the protein detection sensor, electrochemical methods have also been exploited in the development of biosensor for cancer monitoring or impedimetric biosensor that measures the swelling behaviour of different cancer cells. An electrochemical-based G-quad sensor to detect cancer cells was developed by Qu et al. The biosensor consists of a graphene-modified electrode where aptamer, AS1411 and its complementary strand were used. In the presence of the cations, the aptamer, AS1411 forms G-quad and binds specifically to nucleolins that are expressed on the cancer cell surface with high binding affinity. Thus, only the cancer cells can be captured and it is capable to differentiate cancer cells from normal cells. The cyclic voltam‐ metry (CV) signal of the K3[Fe(CN)6]/K4[Fe(CN)6] decreased over time as the anchored aptamer folded to form a G-quad. However, after the binding of the cancer cell to the G-quad, there was little to no observable CV signal of the K3[Fe(CN)6]/K4[Fe(CN)6]. The sensor is then regenerated using the AS1411 complementary strand to allow it to be reused for the next round of cancer cell detection [47].

Many studies also incorporated electrochemical methods in the DNA detection sensors. Recently, Yao and co-workers integrated the isothermal exponential amplification (EXPAR) with hybridization chain reaction (HCR) of DNAzyme in addition to the merits of electro‐ chemical method for the development of an ultrasensitive DNA sensor for avian flu strain H7N9 (Figure 7). A single-stranded DNA derived from the hemagglutinin (HA)-encoding sequences from avian influenza A (H7N9) was used as the target gene in their sensor devel‐ opment. The molecular beacons (MBs) contained the G-quad that cannot be opened up or assembled together without the target. A duplex probe was anchored to the surface of the electrode. Once the target DNA hybridized with one of the sequence of the probe, the ampli‐ fication through toe-hole-mediated strand displacement (TMSDR) was initiated. The duplex with the target gene was released to the solution and initiated the EXPAR with a primer. On the other hand, the other bound strand of the probe on the electrode was then hybridized with the MBs. The hairpin structure of the MBs was opened through TMSDR and resulted in the formation of G-quad nanowires. Hemin was bound to the G-quad nanowires and formed DNAzyme that catalysed the oxidation of TMB, generating an increase in electrochemical signal in reduction current to be measured. This DNA sensor for avian flu (H7N9) was ultrasensitive with the limit of detection at femtomolar levels [48].

Applying electrochemical methods, the detection limits of biosensors can be improved to allow remarkably low levels to be detected. In addition to the reduced cost of the biosensor with rapid response, the ability to miniaturize the assay allows it to be considered for point-of-care applications. Such remarkable advantages have drawn many researchers to the development

**Figure 7.** Schematic diagram of the DNA detection of H7N9 integrating EXPAR and HCR for electrochemical assay.

of biosensor systems based on electrochemistry for the detection of protein, DNA and other analytes.
