**5. G-quad in luminescence-based sensors**

Although applied widely, fluorescent labelling for sensor development has some limitations. Labelling the oligonucleotide covalently can reduce the binding affinity or selectivity of the oligonucleotide, which in turn hampers the efficiency of the assay. Besides, fluorescent labelling is costly and time consuming. Therefore, the use of luminescent probes in sensor development was an attractive solution. Unlike fluorescent probes, luminescent probes are not covalently attached to the nucleic acid; instead, it can bind with DNA through end stacking or electrostatic interactions, intercalation and groove binding. Such interactions will not affect the functionality of the DNA [43].

A label-free G-quad assay for metal detection was developed by Ma et al. This assay was based on the transition of a DNA structure, which involved specially designed oligonucleotide that is rich in guanine and cytosine. The transition of DNA structure would be from a quadruplex to duplex conformation induced by silver (Ag+ ) ions. In this assay, platinum (II) metallo intercalator was used for the switch on/ switch off response towards the Ag+ ions. Initially, a low luminescence background was generated due to the weak interaction of platinum (II) metallo intercalator and the G-quad structure without the Ag+ ions. However, after adding the Ag+ ions, cystosine-Ag+ -cytosine mismatched base pairing occurred, causing the G-quad structure to revert to a duplex structure. This allowed the intercalation of the platinum(II) metallo intercalator to the duplex DNA structure and generates a luminescent signal [44].

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

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).

Although applied widely, fluorescent labelling for sensor development has some limitations. Labelling the oligonucleotide covalently can reduce the binding affinity or selectivity of the oligonucleotide, which in turn hampers the efficiency of the assay. Besides, fluorescent labelling is costly and time consuming. Therefore, the use of luminescent probes in sensor development was an attractive solution. Unlike fluorescent probes, luminescent probes are not covalently attached to the nucleic acid; instead, it can bind with DNA through end stacking or electrostatic interactions, intercalation and groove binding. Such interactions will not affect

duplex with the complementary strand again [41].

128 Nucleic Acids - From Basic Aspects to Laboratory Tools

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

**5. G-quad in luminescence-based sensors**

the functionality of the DNA [43].

Besides metal detection assays, the same group also developed an assay for the detection of gene deletion (Figure 6). They designed a split G-quad assay whereby it consists of two short oligonucleotides, namely P1 and P2. Both of these strands have complementary regions that can recognize the deletion site of the chemokine receptor gene CCR5 and also contain guaninerich overhangs. These guanine-rich overhangs of both strands can form a split G-quad in close proximity. CCR5 was used as the target because it was reported to be a co-receptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strain and it allows HIV-1 to enter the CD4+ T cells. Mutation on CCR5 gene caused no expression of functional protein, which gives rise to the resistance against HIV infection. In this assay, P1 and P2 strands were able to hybridize with the wild-type CCR5 DNA strand. Due to the large spatial separation between both G-rich overhangs of P1 and P2, a split G-quad structure was not formed, causing the generation of a lower luminescent signal of the iridium(III) complex. However, mutant of the CCR5 DNA sequence that is shorter in length can cause the two G-quad-forming sequences of P1 and P2 to close proximity upon hybridizing with the shorter mutant sequence. This resulted in the formation of the split G-quad, generating a switch-on luminescence effect of the iridium (III) complex [45].

**Figure 6.** Split G-quad luminescent turn-on detection for the detection of gene deletion of HIV DNA sequence.
