A hybrid composite of gold and graphene oxide as a PCR enhancer

Abstract

A hybrid composite of gold-decorated graphene oxide (GO) was synthesized and applied as a PCR enhancer. The analysis of the results of PCR demonstrates that the gold and graphene oxide (Au/GO) hybrid composite improves PCR performance. The chemical interaction of the hybrid composite and the PCR components and the potential mechanism behind the hybrid composite-assisted PCR were also investigated and discussed.

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The highly sensitive and selective detection of specific DNA has been extensively developed because of its important applications such as clinical research and diagnosis, genetically modified food monitoring, and detection of environmental contamination.^1–4 However, a low amount of DNA becomes a major bottle neck to quantitatively and qualitatively analyze genetic information. In this regard, different kinds of DNA amplification methods have been developed, of which, the polymerase chain reaction (PCR) has become the best way to amplify DNA since its discovery in the 1980s.^5–7 PCR is performed under in vitro conditions, and it is composed of three main steps, denaturation, annealing and extension. In spite of its wide utilization, PCR is a delicate and temperature-sensitive technique for amplifying DNA. Therefore, the efficiency and specificity of PCR become a major challenge.⁸

Recently, to address such issues, nanomaterial-assisted PCR especially using materials including metals, carbon-based nanomaterials, semiconductor quantum dots (QDs), and silicon nanowires has been proven as a method to enhance the efficiency and specificity of PCR.^9–15 This nanomaterial-assisted PCR is referred to as nano-PCR.¹⁶ The nanomaterials provide exceptional physical and chemical properties like a large surface area to volume ratio, a good heat transfer rate, and potential binding sites for biological materials.^17–21 Among the various types of nanomaterials, GO has been considered as one of the most exciting materials because of its intrinsic properties and potential applications. GO, a two-dimensional material, is composed of sp²-bonded carbon atoms and is enriched with oxygen functional groups such as carboxyl, hydroxyl, and epoxy groups,²² rendering it water dispersible and providing good building blocks for constructing hybrid nanomaterials.^23–25 In particular, GO exhibits a capability for anchoring biomolecules without any surface modification or coupling reagents due to weak van der Waals and π–π interactions.^26–28 In spite of the merits and capabilities of GO, a serious level of agglomeration leads to a limited surface area, which may impede PCR performance.^29,30

To overcome such challenges, gold nanoparticle (Au NP)-based hybrid nanomaterials have been considered as an alternative choice to meet these requirements. Hybrid nanomaterials maintain only the beneficial features of both precursor materials, but can also provide advantages unique to the hybrid material through the combination of functional components.³¹ GO plays a role as a template and building block for developing graphene based hybrid materials.³² GO also prevents self-aggregation of Au NPs by serving as a substrate.³³ The Au NPs on individual GO sheets can be also exploited to maintain the GO sheets at a certain distance from each other. Furthermore, Au NPs commonly exhibit biocompatibility, chemical stability, and simplicity in preparation and surface modification for immobilization of biomolecules.^34–37 These advantages provide more adhesion layers that can be immobilized together in the same location as the PCR components, thereby significantly increasing the possibility of the amplification reaction.^38–40 Therefore, Au and GO are suitable to increase the efficiency and the fidelity of PCR. However, the mechanism of Au and GO hybrids during PCR still remains a mystery.

Herein, we synthesized a Au NP and GO hybrid composite and applied it as a PCR enhancer. The GO serves as a substrate and provides numerous binding sites to realize the hybrid composite-based PCR. We further investigate the interaction between PCR reagents including DNA, primer, and polymerase and Au NPs, GO and the Au/GO hybrid composite. The potential mechanism behind the hybrid composite-assisted PCR is also proposed and investigated as discussed below. Moreover, the effectiveness of the hybrid composite was confirmed using conventional PCR and real-time quantitative PCR with two different types of DNA.

Results and discussion

Characterization of the Au/GO hybrid composite

The GO sheets were prepared from graphite powders using a modified Hummer’s method.^41,42 Initially, graphite powders were placed in concentrated acid in the presence of an oxidizing agent (H₂SO₄). Through the processes, graphene was decorated with oxygen functional groups on both sides of the plane and around the edges. After dispersion of GO in the solution, the Au precursor HAuCl₄ was added and reduced to form Au NPs over the GO surface using a thermal reduction method. The overall chemical reactions are illustrated in Fig. 1. Moreover, after each synthesis step to prepare GO itself and the Au/GO hybrid composite the materials were analysed by UV/vis absorption spectroscopy (Fig. 1 and S1 in ESI†). The strong absorption peak at 230 nm and a shoulder at 300 nm in the GO spectrum correspond to the π–π* transition of the C=C bonds and the n–π* transition of the C=O bonds of the GO, respectively.⁴³ Then, the Au/GO hybrid composite is prepared by redox reaction of the gold ions in situ on the partial GO surface. Additionally, the unique peak at 520 nm corresponds to the Au NPs on the GO sheets.⁴⁴

Fig. 1 Overall synthesis procedure for preparation of the Au/GO hybrid composite and UV-vis absorption spectra of GO and the Au/GO hybrid composite (inset).

The typical morphology of the GO and Au/GO hybrid composite sheets was investigated using transmission electron microscopy (TEM) and atomic force microscopy (AFM) as shown in Fig. 2. In the TEM image of the GO sheets (Fig. 2a), no feature distinct from the stacked, fragmented GO sheets is observed. In the TEM image of the Au/GO hybrid composite sheets (Fig. 2b), however, the randomly distributed Au NPs with an interplanar spacing of 0.23 nm are observed only on the GO sheets. This finding indicates that the Au nuclei are randomly formed only on the GO sheets and subsequently grown for a given thermal reduction time. In particular, the average size of the Au NPs with a spherical shape was 15.2 nm in diameter. In the AFM images (Fig. 2c and d), the height profile along the dashed line is also indicated with a solid line. The entire area of the individual fragmented GO sheets exhibits a uniform thickness, as demonstrated by the AFM image (Fig. 2c). In particular, the average thickness of these GO sheets is nearly 1.05 nm, indicating that such individual GO sheets consist of a single GO layer. It is also demonstrated from the AFM image of the individual Au/GO hybrid composite sheets (Fig. 2d) that some parts of these sheets are of an equivalent thickness to that of the individual GO sheets. Unlike the GO sheets, however, randomly distributed bumps are only exhibited on the individual Au/GO hybrid composite sheets. In particular, the height of these bumps is in the range between 6 and 10 nm which is a similar size to that of the Au NPs from the TEM images. These small particles are attributed to the formation of the Au NPs on the individual GO sheets, indicating that single GO layers can be provided as substrates to load a number of Au NPs.

Fig. 2 Representative TEM images of (a) GO sheets and (b) the Au/GO hybrid composite. AFM images and the height profiles of (c) GO sheets and (d) the Au/GO hybrid composite.

Effect of the Au/GO hybrid composite on PCR

To confirm PCR improvement using the Au/GO hybrid composite, we selected two types of DNA, genomic DNA of Listeria monocyte (200 bp) and Scomber japonicas (aquatic fish, 800 bp) as realistic models. Furthermore, the pristine GO and Au NPs were also employed as control samples to confirm their effectiveness during PCR. After performing the PCR using GO, Au NPs, and the Au/GO hybrid composite, the obtained DNA results were further analysed using agarose gel electrophoresis. The PCR results corresponding to a positive control (lane 1), Au NPs (lane 2), GO (lane 3), and the Au/GO hybrid composite (lane 4) were investigated using agarose gel electrophoresis as shown in Fig. 3a and b. The amounts of PCR products were increased upon introducing Au NPs, GO, and the Au/GO hybrid composite as compared to those of the positive controls regardless of the size of the DNA. It should be noted that both the intensity and band thickness of the DNA of L. monocyte (Fig. 3a) and S. japonicas (Fig. 3b) increase. The Au/GO hybrid composite produced the strongest band thickness and intensity compared to the other materials. In addition, this result indicates the best PCR performance compared to other nanomaterials. For clarity, the individual band intensities of the L. monocyte and aquatic fish DNA were calculated and compared with each other as shown in Fig. 3c and d. In particular, the concentrations of the PCR products of the L. monocyte genomic DNA with Au NPs, GO, and the Au/GO hybrid composite were about 1.3, 1.5, and 2.4 times higher than that of the PCR product from the positive control, respectively (Fig. 3c). In addition, the concentrations of the PCR products of the aquatic fish genomic DNA with Au NPs, GO, and the Au/GO hybrid composite were also about 1.2, 1.3, and 1.5 times higher than that of the PCR product of the positive control, respectively (Fig. 3d). These results exhibited that both the yield and the efficiency of the PCR reaction with the Au/GO hybrid composite are much higher than those of the PCR reaction with Au NPs or GO. From these results, it can be suggested that the Au/GO hybrid composite enhances PCR performance in both short and long DNA.

Fig. 3 Agarose gel electrophoresis for analyzing the concentration of the PCR products obtained from two different templates, the genomic DNA of (a) L. monocyte (size: 280 bp) and (b) S. japonicas (aquatic fish, size: 800 bp), (c and d) the DNA band intensities of the gel images from (a) the L. monocyte genomic DNA and (b) the aquatic fish genomic DNA quantified with the Quantity One program from Bio-Rad. Lane M: DNA marker, lane 1: positive control, lane 2: Au NP-assisted PCR, lane 3: GO-assisted PCR, lane 4: Au/GO hybrid composite-assisted PCR.

The effects of Au NPs, GO, and the Au/GO hybrid composite on the PCR were also investigated by employing real-time quantitative PCR with SYBR Green I (Fig. 4), to simultaneously quantify the changes in fluorescence intensity during amplification of the target DNA. In this case, relative fluorescence intensity is directly associated with the yield of the PCR products in response to the nanomaterials. Compared to the control, the PCR efficiencies of both the L. monocyte and aquatic fish genomic DNA were enhanced in the presence of the nanomaterials. Among them, the Au/GO hybrid composite also produced the best efficiency of the real-time PCR compared with the others. In particular, the efficiency produced by the Au/GO hybrid composite was about 1.8 and 1.7 times higher than that of the positive control (Fig. 4c and d). These results demonstrated that the Au/GO hybrid composite results in the most efficient PCR process. Moreover, both the conventional PCR and real-time PCR exhibited a similar enhancement efficiency, and these results also proved that the Au/GO hybrid composite nanomaterial can not only reduce the PCR cycle but also increase the PCR products.

Fig. 4 The efficiency of PCR amplification using L. monocyte genomic DNA (a) and aquatic fish genomic DNA as templates (b). The fluorescence intensity of real-time PCR assays which corresponded to (c) L. monocyte genomic DNA and (d) aquatic fish genomic DNA.

The annealing temperature is a critical parameter in the PCR step, and thus the inspection of a broad range of annealing temperatures is highly important to enhance both the efficiency and accuracy of PCR. To examine the effects of the annealing temperature on nanomaterial-assisted PCR performance, the PCR performances of Au NPs, GO, and the Au/GO hybrid composite were separately tested with only the genomic DNA of aquatic fish in the range of annealing temperatures from 40 °C to 55 °C. The final PCR products were analyzed using gel-electrophoresis (ESI, Fig. S2†). The Au/GO hybrid composite produced a yield improvement at a broad range of annealing temperatures (40–55 °C) in comparison with the Au NPs and GO. The results indicated that Au/GO hybrid composite-assisted PCR guarantees an improvement in PCR performance at a broad range of annealing temperatures.

Mechanism of the Au/GO hybrid composite during PCR

To study the mechanism of the Au/GO hybrid composite in the enhancement of PCR, we firstly hypothesized that such nanomaterials including Au NPs, GO, and the Au/GO hybrid composite might be interacting with PCR components such as DNA, primer, and polymerase. In order to investigate and understand the role of Au NPs, GO, and the Au/GO hybrid composite during PCR, fluorescent dye (FAM)-labelled ssDNA was used as a primer model.

Initially, as-prepared Au NPs, GO, and Au/GO hybrid composite were separately reacted with the FAM-labelled ssDNA. After the interaction of the Au NPs, GO, or Au/GO hybrid composite with the ssDNA, the fluorescence intensity of the FAM was dramatically quenched by 39, 51, and 89% which corresponded to the Au NPs, GO, and Au/GO hybrid composite, respectively (Fig. 5a). Among them, the Au/GO hybrid composite exhibited a strong fluorescence quenching of more than 89% compared to its original signal. These phenomena are mainly attributed to adsorption through interaction between the ssDNA and the Au NPs, GO, or Au/GO hybrid composite. These phenomena inevitably led to fluorescence quenching of the FAM dyes on the ssDNA due to the energy transfer from the dye to the nanomaterials.²⁷ To avoid a potential misunderstanding between the experimental procedures of the quenching and real-time PCR experiments, a schematic illustration is also shown in Fig. S3.†

Fig. 5 (a) Fluorescence spectra of (1) pristine FAM-ssDNA and after interaction of FAM-ssDNA with (2) Au NPs, (3) GO, and (4) the Au/GO hybrid composite. (b) UV/vis absorbance changes after the interaction of polymerase and primer with Au NPs, GO, and the Au/GO hybrid composite.

In the case of the interaction of primers and polymerase with the nanomaterials, we analyzed the UV/vis absorbance change before and after interaction of the nanomaterials with primer or polymerase (Fig. 5b). To investigate the effects, the Au NPs, GO, and the Au/GO hybrid composite were separately mixed and reacted with the primers or polymerase. After centrifugation, the remaining PCR components in the supernatants were measured using UV/vis spectroscopy. After the reaction between the polymerase and the Au NPs, GO, or the Au/GO hybrid composite, the absorbance signals were decreased by about 12, 7, and 11%, respectively, as shown in Fig. 5b. In addition, the absorbances were also gradually decreased by about 18, 23 and 37% even after the interaction between the primers and the Au NPs, GO, and Au/GO hybrid composite, respectively (Fig. 5b). Therefore, the obtained results of strong fluorescence quenching and UV/vis absorbance changes indicated that the Au/GO hybrid composite exhibited a relatively strong affinity for DNA, primer and polymerase compared with the Au NPs and GO. Moreover, as previously reported, the interaction of ssDNA, primer, and polymerase with graphene-based materials is mainly attributed to π–π stacking and electrostatic attraction.^27,28 These interactions improve the stability of the PCR components including DNA, polymerase and primer, and it allows us to use the Au/GO hybrid composite as a PCR enhancer.

Conclusions

In summary, we have reported a simple, biocompatible way to enhance PCR efficiency using a gold-coated GO hybrid nanomaterial as a bio-adhesive and carbon source. We directly observed the general effects of the Au/GO hybrid composite in PCR. The Au/GO hybrid composite enhanced PCR performance by interacting with the PCR components. These observations suggested that the Au/GO hybrid composite enables the creation of harmonious conditions which influence the PCR reagents and contribute to PCR enhancement. The different characteristics of the nano-composites facilitate a unique environment and diverse conditions to interact with PCR reagents. Therefore, these findings could provide a new insight for the understanding of the PCR effects of composite materials and the study of their mechanism. Moreover, these materials will be valuable for future use in biomedical, diagnostic, and biosensing applications.

Acknowledgements

This work was supported by the IT R&D program of MoTIE/MISP/KEIT (10044580, Korea) and the Public Welfare & Safety research program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korea (NRF-2013M3A2A1073991). This work was also supported by BioNano Health-Guard Research Center funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea as Global Frontier Project (Grant Number H-GUARD2013M3A6B2078945).

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Footnote

† Electronic supplementary information (ESI) available: The experimental details and the effects of annealing temperature. See DOI: 10.1039/c5ra12932j

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