**7. G-quad integrates with quantum dots**

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],

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

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

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

ultrasensitive with the limit of detection at femtomolar levels [48].

producing an electrocatalytic response [46].

130 Nucleic Acids - From Basic Aspects to Laboratory Tools

of cancer cell detection [47].

The emergence of quantum dots (QD) in molecular sensing methods has attracted a lot of attentions from many researchers. QD have been extensively used as optical labels, probes for FRET and also as energy acceptors from metal complexes or from energy generated by luminescence or photoelectrochemical for the detection of DNA or formation of aptamer– substrate complexes [49]. Wilner and co-workers utilized the G-quad forming DNAzyme, which is conjugated to semiconductor QDs. It functions as a light source that promotes chemiluminescence resonance energy transfer (CRET) to QDs.

In this assay, anti-thrombin aptamer was used where it was also previously used in a colori‐ metric sensor for thrombin whereby hemin/G-quad forming DNAzyme was used to catalyse the oxidation of ABTS in the presence of H2O2. Meanwhile, in this assay, the hemin/G-quad forming DNAzyme aptamer–thrombin complex was used to generate chemiluminescence in the presence of H2O2/luminol. Then, in close proximity, this complex was able to excite the CdSe/ZnS QDs, resulting in CRET to the QDs triggering the luminescence of QDs. The glutathione-modified CdSe/ZnS QDs were attached with the anti-thrombin aptamer. A low CRET and chemiluminescence signal was observed even if it was without thrombin due to the diffusional hemin. Upon adding thrombin into the assay, the chemiluminescence signal and the CRET-stimulated luminescence of the QDs were greatly increased. The increase in CRET signals was proportional to the concentration of thrombin detected [50].

Besides the detection of aptamer–substrate complex, the detection of metal ions can also be carried out by exploiting the hemin/G-quad forming DNAzyme complex. The same group also developed a G-quad-based sensor to detect mercury ions (Hg2+) (Figure 8). This assay comprised two strands of DNA whereby each of the strand has G-rich sequence forming DNAzyme (1) and (2) and a T-containing site that functions as the recognition site of Hg2+, (3) and (4). As the T-containing sites of (3) and (4) were partially complementary, (1) and (2) could not assemble to form a G-quadruplex. However, in the presence of Hg2+, the T-Hg2+-T complexes formed between the T-containing sites of (3) and (4) and formed a duplex structure. Therefore, this led to the formation of the hemin/G-quad complex between (1) and (2). The hemin/G-quad complex catalyzed the oxidation of luminol in the presence of H2O2, generating chemiluminescence signal. Then, this signal acted as an internal light source that can excite CdSe/ZnS QDs, resulting in CRET to the QDs. Then, this stimulat‐ ed the luminescence signal from QDs [51].

**Figure 8.** Detection of Hg2+ through CRET from luminol that is oxidized by DNAzyme to QD.

### **8. Conclusion**

In comparison with conventional reporter systems, G-quad-based systems provide many advantages in terms of cost, thermostability and ease of synthesis. With all these advantages, it makes them very useful for the development of sensing probes for diagnosis, applicable to DNA, protein and metal detection. The vast application of G-quad structures to generate various forms of readouts ranging from colorimetric to electrochemical-based readouts puts it at the forefront of sensing reporters. G-quad-based assays are flexible as they can be adapted to many different types of diagnostic platforms. This makes G-quad an attractive alternative for the development of sensitive reporter systems for the sensing of various samples ranging from small drug molecules, chemical compounds to large biomolecules.
