Abstract
Graphene oxide (GO) is a promising material for the development of cost-effective detection systems. In this work, we have devised a simple and rapid GO-based method for the sequence-specific identification of DNA molecules generated by PCR amplification. The csp genes of Escherichia coli, which share a high degree of sequence identity, were selected as paradigm DNA templates. All tested csp genes were amplified with unlabelled primers, which can be rapidly removed at the end of the PCR taking advantage of the preferential binding to GO of single-stranded versus duplex DNA molecules. The amplified DNAs (targets) were heat-denatured and hybridized to a fluorescently-labelled single strand oligonucleotide (probe), which recognizes a region of the target DNAs displaying sequence variability. This interaction is extremely specific, taking place with high efficiency only when target and probe show perfect or near perfect matching. Upon GO addition, the unbound fraction of the probe was captured and its fluorescence quenched by the GO’s molecular properties. On the other hand, the probe-target complexes remained in solution and emitted a fluorescent signal whose intensity was related to their degree of complementarity.
Highlights
The capacity of protecting ourselves from infectious diseases depends, to large extent, on our ability to precisely detect the pathogen in the environment and rapidly activate safeguarding actions and adequate treatments
The Graphene oxide (GO) Raman spectrum with signature-like spectral feature of the D and G peaks at 1330 and 1600 cm-1, respectively, is shown in S1 Fig. The position, width and relative intensity of these peaks are in agreement with those reported in literature [20]
The chemical composition of the GO used in the experiments was investigated by means of X-ray photoemission spectroscopy (XPS) (S2 Fig)
Summary
The capacity of protecting ourselves from infectious diseases depends, to large extent, on our ability to precisely detect the pathogen in the environment and rapidly activate safeguarding actions and adequate treatments. The detection step often relies on biomolecular approaches that exploit the unique features of the DNA molecule [1]. These DNA-based methodologies are crucial for the identification of the human genetic variations known to be associated with diseases or that may otherwise predispose to pathological conditions [2]. Nanomaterials have proven helpful for the development of new DNA-based assays that can speed up the procedures and reduce the costs for the detection of pathogens or for the early diagnosis of unfavourable genetic conditions [3,4].
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