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Enhanced Resolution of DNA Separation Using Agarose Gel Electrophoresis Doped with Graphene Oxide

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Abstract

In this work, a novel agarose gel electrophoresis strategy has been developed for separation of DNA fragments by doping graphene oxide (GO) into agarose gel. The results show that the addition of GO into agarose gel significantly improved the separation resolution of DNA fragments by increasing the shift distances of both the single DNA fragments and the adjacent DNA fragments and completely eliminating the background noise derived from the diffusion of the excessive ethidium bromide (EB) dye in the gel after electrophoresis. The improved resolution of DNA fragments in GO-doped agarose gel could be attributed to the successive adsorption-desorption processes between DNA fragments and GO sheets, while the elimination of the background noise could be attributed to the adsorption of the excessive EB dye on the surface of GO sheets and high fluorescence quenching efficiency of GO. These results provide promising potential for graphene and its derivate utilized in various electrophoresis techniques for separation and detection of DAN fragments and other biomolecules.

Background

Graphene has attracted considerable attention in biomedical fields due to its exceptional electronic, thermal, and mechanical properties, as well as extremely large specific surface area [1]. It is of great interest that graphene displays promising potential in DNA analysis and detecting [24]. The theoretical calculations indicate that DNA-graphene hybrids display significant base-dependent features in the electronic local density of states derived from the different interaction energies between DNA bases and graphene, providing an alternative route to DNA sequencing [510]. Studies showed that DNA fragments were quickly adsorbed on the surface of graphene oxide (GO) at room temperature due to the high affinity between GO and DNA nucleobases, while the adsorption and release of the double-stranded DNA from GO were relatively slow [3, 4]. A recent study showed that GO nanoplatelets were successfully utilized for extracting both DNA and RNA from eukaryotic and prokaryotic cells [11]. On the other hand, graphene and its derivates were reported as the super-quenchers with the long-range nanoscale energy transfer property [1214]. Therefore, GO could bind and quench a dye-labeled single-stranded DNA probe and subsequently release the fluorescent probe when it formed a duplex with its target [15]. So far, various GO-based biosensors have also been extensively developed for DNA analysis with improved sensitivity and speed [9, 1618]. For instance, a series of electrochemical biosensors with ultra-high resolution have been developed by depositing GO on the surface of graphite electrode for detection of DNA fragments at single-nucleotide base level and early diagnosis of leukemia (single abnormal cell in approximately 109 normal cells) [1921].

Agarose gel electrophoresis is one of the most important and routine techniques for DNA analysis. Combining with an organic dye (ethidium bromide (EB)), DNA fragments could be well separated according to the nucleobase amount and expediently observed under a UV light. The resolution of agarose gel electrophoresis for DNA separation is mainly dominated by the concentration of agarose gel and working voltage of electrophoresis. In most cases, dispersed and tailed DNA bands were obtained after electrophoresis, accompanying with serious background signals derived from EB dye. Therefore, it will be highly fascinating to develop a novel strategy to improve the electrophoresis resolution of DNA fractions with low-noise background.

Herein, we report a novel electrophoresis strategy for DNA separation by adding GO into agarose gel. Compared with the routine agarose gel electrophoresis, successive adsorption-desorption processes between DNA fragments and the surfaces of GO nanosheets dispersed in the gel net significantly improved the separation of DNA fragments with different nucleobase amounts (Scheme 1). Meanwhile, the background noise derived from the diffusion of EB dye in the gel was completely eliminated because the excessive dye was adsorbed on the surface of GO nanosheets.

Scheme 1
scheme1

Illustration of GO-doped agarose gel electrophoresis for separation of DNA fragments

Methods

Reagents

Natural graphite powder, agarose, EB, and other chemicals were purchased from Sinopharm Chemical Reagent Corp. and used without further purification. Deionized water (16 MΩ cm) was obtained from a Nanopure Water Systems UV (Thomas Scientific, Swedesboro, NJ).

Electrophoresis Separation of DNA Fragment

GO was prepared using natural graphite powder according to the modified Hummer’s method. In a typical DNA electrophoresis experiment, the as-prepared GO was dispersed in deionized water by ultrasonication (KQ2200E system, Kunshan Ultrasonic Instruments Co., Ltd, 40 KHz, 80 W) for 3 h. Then, the dispersed GO solution was added into agarose solution at designed concentrations and heated under microwave irradiation. DNA fragments containing standard DNA markers were separated using a DYCP-32A agarose horizontal electrophoresis system (Beijing Six One Instrument Corp.) at designed voltages and observed under a UV light. The shift distances and width of DNA bands were measured.

Results and Discussion

Figure 1 shows TEM and AFM images of the as-synthesized GO nanosheets. Numerous wrinkles were observed in the plane of the GO nanosheets (Fig. 1a–c). The AFM images show that inhomogeneous GO nanosheets were obtained in single- and multi-layers. The size of the GO nanosheets was calculated by measuring the area of the nanosheets and assuming it as a circle. The inset in Fig. 1f shows the lateral size distribution of the as-synthesized GO nanosheets, and the average size of the GO nanosheets was approximately 4.1 ± 1.3 μm.

Fig. 1
figure1

ae TEM and AFM images of the as-synthesized GO nanosheets. The inset in the AFM image f shows the size distribution of the as-synthesized GO nanosheets

Figure 2 shows Raman and XPS spectra of the as-synthesized GO nanosheets. The characteristic peak at approximately 1580 cm−1 in the Raman spectrum (Fig. 2a) was assigned to the G band derived from the in-plane vibration of symmetric sp2 C–C bonds, while the peak at approximately 1330 cm−1 was derived from the first-order zone boundary phonons (D band). The chemical state of C element was analyzed through C 1s XPS spectrum (Fig. 2b). Four different carbon-bonding states were identified according to the peak fitting. The peaks at approximately 284.5, 286.6, 287.7, and 289 eV could be attributed to the C–C, C (epoxy)/C–OH, C=O, and O–C=O bonding configurations, respectively. In addition, the C/O atomic ratio calculated from the XPS spectrum was approximately 1.90, similar to that reported elsewhere [22].

Fig. 2
figure2

Raman and XPS spectra of the as-synthesized GO nanosheets

Figure 3 shows the agarose gel electrophoresis image of a DNA sample with and without adding GO. In the presence of GO (lanes I–III), a clean gel was observed without EB-derived background noise. The elimination of EB-derived background noise in agarose gel could be attributed to the adsorption of EB dye on GO sheets. Moreover, the shift distances between different DNA bands were significantly enlarged, especially the shift between band 2 and band 3. In comparison, in the absence of GO (lanes IV–VI), the broad DNA bands in agarose gel were observed, accompanied with serious background noise throughout the gel due to the diffusion of EB dye in the gel. In particular, the shift distance between band 2 and band 3 was rather small.

Fig. 3
figure3

Agarose gel electrophoresis images of DNA fragments in the presence and absence of GO. Lane I–III photo image of agarose gel in the presence of GO, Lane IV–VI photo image of agarose gel in the absence of GO

It is generally known that the shift of DNA fragments in agarose gel was primarily depended on the nucleobase amount of DNA fragment and the voltage of electrophoresis. The short DNA fragments shift faster than the long fragments, and the shift rate of DNA fragments is promoted by increasing the voltage of electrophoresis. The influences of GO on the shift of DNA fragments in agarose gel were investigated by adjusting the concentration of GO in agarose gel. As shown in Fig. 4a, the shift distances of DNA fragments were negatively related with the length of DNA fragments regardless of adding GO in agarose gel. The addition of GO in agarose gel significantly increased the shift distance of DNA fragments compared with that in the absence of GO. The largest shift distance of DNA fragments was observed at the GO concentration of 12.5 μg/mL. The increased shift distance of DNA could be attributed to excellent conductivity of GO, promoting the electrophoresis rate of DNA fragments in agarose gel. However, the further increase of the GO concentration did not continually increase the shift distances of the DNA fragment. It is possible that the shift of DNA fragments slowed down in agarose gel due to the frequent adsorption-desorption processes between the DNA fragments and GO sheets when the concentration of GO exceeded 25 μg/mL. Figure 4b shows the influence of the GO concentrations on the shift distance between the two adjacent DNA fragments. With the increase of the GO concentration in agarose gel, the shift distances between the two adjacent DNA fragments were significantly increased, implying the better separation of DNA fragments. However, when the GO concentration in agarose gel increased up to 50 μg/mL, the shift distances between the two adjacent DNA fragments obviously decreased, indicating that the high concentration of GO in agarose gel hindered the separation of DNA fragments.

Fig. 4
figure4

a, b Shift distances of DNA fragments after gel electrophoresis run at different concentrations of GO

Figure 5 shows the influence of electrophoresis voltages on the shift distances of DNA fragments in agarose gel at the GO concentration of 12.5 μg/mL. As shown in Fig. 5a, the shift distance of DNA fragments in agarose gel was positively related with the increase of electrophoresis voltages, indicating that the increase of electrophoresis voltages resulted in the increase of shift distances of DNA fragments. Figure 5b shows that the shift distances between the two adjacent DNA fragments increased with the enhancement of electrophoresis voltages, implying the better separation of DNA fragments.

Fig. 5
figure5

a, b Shift distances of DNA fragments after gel electrophoresis run at a GO concentration of 12.5 μg/mL under different voltages

Graphene and its derivate have attracted much attention for applications in DNA detection and sequencing due to its unique electronic property and single atom-layer thickness. The theoretical and experimental investigations demonstrated that single-nucleobase resolution of DNA sequencing could be realized by measuring the nucleobase-dependent transverse conductance derived from the translocation of DNA strands through the nanopores in the graphene plane [59]. Various biosensors for detection of DNA fragments and proteins have been developed based on either the extraordinarily high quenching efficiency of GO or the fluorescence resonance energy transfer between quantum dots and GO [1218]. In addition, various biomolecules, including DNA, proteins, and peptides, were ready to be adsorbed on the surfaces of GO due to intramolecular interaction [10, 23]. The theoretical calculations and experimental measurement by isothermal titration calorimetry have demonstrated that the interaction energy of the DNA nucleobases with graphene was nucleobase-dependent with an order of guanine > adenine > thymine > cytosine, which was also affected by pH value of the solution [24, 25]. In the present study, DNA fragments were adsorbed onto the surfaces of GO nanosheets dispersed in agarose gel net by intramolecular interaction. The oxygen-containing functional groups in GO nanosheets could play a crucial role in the improvement of hydrogen-bonding interaction between DNA nucleobases and GO nanosheets, which is favorable to the adsorption of DNA fragments onto the surfaces of GO nanosheets. Subsequently, DNA fragments were desorbed from the surfaces of GO nanosheets under electrophoresis condition, which could be influenced by the charges carried on both the DNA fragments and GO nanosheets. Therefore, the successive adsorption-desorption processes between DNA fragments and GO nanosheets significantly improved the separation resolution of DNA fragments by increasing the shift distances between the adjacent DNA fragments with different nucleobase amounts. It is noticeable that reduced GO (rGO) nanosheets displayed size- and concentration-dependent cytotoxicity and genotoxicity in human mesenchymal stem cells, which was attributed to rGO-induced oxidative stress, cell membrane damage, DNA fragmentations, and chromosomal aberrations [26, 27]. However, in the present studies, there is no significant increase in both the amount and width of electrophoresis bands, confirming the absence of new DNA fragments derived from GO-induced fragmentations. Meanwhile, the background noise of the gel derived from the diffusion of the excessive EB dye in the gel disappeared because of the adsorption of EB dye molecules on the surfaces of GO nanosheets through the π-π interaction and the high fluorescence quenching efficiency of GO.

Conclusions

In summary, a novel electrophoresis strategy has been developed for separation of DNA fragments using GO-doped agarose gel. The doping of GO in agarose gel resulted in the significant increase of the shift distances of both the single DNA fragment and the adjacent DNA fragments. The increased shift distance of DNA fragments could be attributed to excellent conductivity of GO, promoting the electrophoresis rate of DNA fragments in agarose gel. While the improved separation resolution for DNA fragments could be attributed to the successive adsorption-desorption processes between the surfaces of GO nanosheets dispersed in the gel net and DNA fragments with different nucleobase amounts, the background noise derived from the diffusion of EB dye in gel was completely vanished after electrophoresis due to the adsorption of the excessive EB dye by GO nanosheets. These results provide promising potential for graphene and its derivates utilized in various electrophoresis techniques for separation and detection of DNA fragments and other biomolecules.

References

  1. 1.

    Geim AK, Novoselov KS (2007) The rise of graphene. Nat Mater 6:183–191

  2. 2.

    Kim HS, Kim YH (2015) Recent progress in atomistic simulation of electrical current DNA sequencing. Biosensors Bioelectron 69:186–198

  3. 3.

    Lu CH, Yang HH, Zhu CL, Chen X, Chen GN (2009) A graphene platform for sensing biomolecules. Angew Chem Int Ed 121:4879–4881

  4. 4.

    Tang LH, Wang Y, Li JH (2015) The graphene/nucleic acid nanobiointerface. Chem Soc Rev 44:6954–6980

  5. 5.

    Heerema SJ, Dekker C (2016) Graphene nanodevices for DNA sequencing. Nat Nanotechnol 11:127–136

  6. 6.

    Ahmed T, Kilina S, Das T, Haraldsen JT, Rehr JJ, Balatsky AV (2012) Electronic fingerprints of DNA bases on graphene. Nano Lett 12:927–931

  7. 7.

    Lin J, Teweldebrhan D, Ashraf K, Liu GX, Jing XY, Yan Z, Li R, Ozkan M, Lake RK, Balandin AA, Ozkan CS (2010) Gating of single-layer graphene with single-stranded deoxyribonucleic acids. Small 6:1150–1155

  8. 8.

    Postma HWC (2010) Rapid sequencing of individual DNA molecules in graphene nanogaps. Nano Lett 10:420–425

  9. 9.

    He YH, Scheicher RH, Grigoriev A, Ahuja R, Long SB, Huo ZL, Liu M (2011) Enhanced DNA sequencing performance through edge-hydrogenation of graphene electrodes. Adv Funct Mater 21:2674–2679

  10. 10.

    Varghese N, Mogera U, Govindaraj A, Das A, Maiti PK, Sood AK, Rao CNR (2009) Binding of DNA nucleobases and nucleosides with graphene. ChemPhysChem 10:206–210

  11. 11.

    Hashemi E, Akhavan O, Shamsara M, Valimehr S, Rahighi R (2014) DNA and RNA extractions from eukaryotic and prokaryotic cells by graphene nanoplatelets. RSC Adv 4:60720–60728

  12. 12.

    Sthör RJ, Kolesov R, Xia KW, Reuter R, Meijier J, Logvenov G, Wrachtrup J (2012) Super-resolution fluorescence quenching microscopy of graphene. ACS Nano 10:9175–9181

  13. 13.

    Chang HX, Tang LH, Wang Y, Jiang JH, Li JH (2010) Graphene fluorescence resonance energy transfer aptasensor for the thrombin detection. Anal Chem 82:2341–2346

  14. 14.

    Li SH, Aphale AN, Macwan IG, Patra PK, Gonzalez WG, Miksovska J, Leblanc RM (2012) Graphene oxide as a quencher for fluorescent assay of amino acids, peptides, and proteins. ACS Appl Mater Interfaces 4:7069–7075

  15. 15.

    He SJ, Song B, Li D, Zhu CF, Qi WP, Wen YQ, Wang LH, Song SP, Fang HP, Fan CH (2010) A graphene nanoprobe for rapid, sensitive, and multicolor fluorescent DNA analysis. Adv Funct Mater 20:453–459

  16. 16.

    Liu X, Wang JY, Mao XB, Ning Y, Zhang GJ (2015) Single-shot analytical assay based on graphene-oxide-modified surface acoustic wave biosensor for detection of single-nucleotide polymorphisms. Anal Chem 87:9352–9359

  17. 17.

    Huang YX, Yang HY, Ai Y (2015) DNA single-base mismatch study using graphene oxide nanosheets-based fluorometric biosensors. Anal Chem 87:9132–9136

  18. 18.

    Dong HF, Gao WC, Yan F, Ji HX, Ju HX (2010) Fluorescence resonance energy transfer between quantum dots and graphene oxide for sensing biomolecules. Anal Chem 82:5511–5517

  19. 19.

    Akhavan O, Ghaderi E, Rahighi R (2012) Toward singly-DNA electrochemical biosensing by graphene nanowalls. ACS Nano 4:2904–2916

  20. 20.

    Akhavan O, Ghaderi E, Rahighi R, Abdolahad M (2014) Spongy graphene electrode in electrochemical detection of leukemia at single-cell levels. Carbon 79:654–663

  21. 21.

    Akhavan O, Ghaderi E, Hashemi E, Rahighi R (2014) Ultra-sensitive detection of leukemia by graphene. Nanoscale 6:14810–14819

  22. 22.

    Chen D, Zhao X, Chen SS, Li HF, Fu XN, Wu QZ, Li SP, Li Y, Su BL, Ruoff RS (2014) One-pot fabrication of FePt/reduced graphene oxide composites as highly active and stable electrocatalysts for oxygen reduction reaction. Carbon 68:755–762

  23. 23.

    Ou LC, Luo Y, Wei GH (2011) Atomic-level study of adsorption, conformational change, and dimerization of an α-hilical peptide at graphene surface. J Phys Chem B 115:9813–9822

  24. 24.

    Gowtham S, Scheicher RH, Ahuja R, Pandey R, Karna SP (2007) Physisorption of nucleobases on graphene: density-functional calculations. Phys Rev B 76:033401

  25. 25.

    Antony J, Grimme S (2008) Structures and interaction energies of stacked graphene-nucleobase complexes. Phys Chem Chem Phys 10:2722–2729

  26. 26.

    Akhavan O, Ghaderi E, Akhavan A (2012) Size-dependent genotoxicity of graphene nanoplatelets in human stem cells. Biomaterials 33:8017–8025

  27. 27.

    Akhavan O, Ghderi E, Emamy H, Akhavan F (2013) Genotoxicity of graphene nanoribbons in human mesenchymal stem cells. Carbon 54:419–431

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Acknowledgements

This work was financially supported by the Natural Science Foundation of China (Nos. 30800256 and 31300791) and the basic research project of Wuhan Science and Technology Bureau (No. 2014060101010041).

Authors’ Contributions

JL, YY, ZM, and WH carried out the synthesis and characterization of GO and DNA separation by agarose gel electrophoresis. JL, TQ, and QW designed the whole work and drafted the manuscript. All authors read and approved the final manuscript.

Competing Interests

The authors declare that they have no competing interests.

Author information

Correspondence to Tong Qiu or Qingzhi Wu.

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Keywords

  • DNA fragments
  • Agarose gel electrophoresis
  • Graphene oxide