Preparation and functionalization of graphene nanocomposites for biomedical applications

Journal name:
Nature Protocols
Year published:
Published online


Functionalized nano-graphene– and graphene-based nanocomposites have gained tremendous attention in the area of biomedicine in recent years owing to their biocompatibility, the ease with which they can be functionalized and their properties such as thermal and electrical conductivity. Potential applications for functionalized nanoparticles range from drug delivery and multimodal imaging to exploitation of the electrical properties of graphene toward the preparation of biosensing devices. This protocol covers the preparation, functionalization and bioconjugation of various graphene derivatives and nanocomposites. Starting from graphite, the preparations of graphene oxide (GO), reduced GO (RGO) and magnetic GO–based nanocomposite, as well as how to functionalize them with biocompatible polymers such as polyethylene glycol (PEG), are described in detail. We also provide procedures for 125I radiolabeling of PEGylated GO and the preparation of GO-based gene carriers; other bioconjugation approaches including drug loading, antibody conjugation and fluorescent labeling are similar to those described previously and used for bioconjugation of PEGylated carbon nanotubes. We hope this article will help researchers in this field to fabricate graphene-based bioconjugates with high reproducibility for various applications in biomedicine. The sample preparation procedures take various times ranging from 1 to 2 d.

At a glance


  1. A schematic of Step 1 that includes preparation and surface functionalization of GO derivatives including GO, RGO, nRGO and RGO-IONP, as well as nGO-PEG, RGO-PEG, nRGO-PEG and RGO-IONP-PEG.
    Figure 1: A schematic of Step 1 that includes preparation and surface functionalization of GO derivatives including GO, RGO, nRGO and RGO-IONP, as well as nGO-PEG, RGO-PEG, nRGO-PEG and RGO-IONP-PEG.
  2. A schematic to show further bioconjugation of PEGylated nano-GO (Step 3).
    Figure 2: A schematic to show further bioconjugation of PEGylated nano-GO (Step 3).

    Step 3A is 125I labeling of nGO-PEG, and Step 3B is the preparation of nGO-PEG-PEI and the subsequent complexing with pDNA for gene transfection. The procedures for antibody conjugation, 64Cu labeling and drug loading to PEGylated nano-GO can be found in the preparation of corresponding bioconjugates based on PEGylated SWNTs, as described in detail in ref. 44.

  3. Characterization of various functionalized GO derivatives.
    Figure 3: Characterization of various functionalized GO derivatives.

    (a) The photos show various functionalized GO derivative in different solutions including DI water, saline and serum. (b) UV-visible NIR spectra of GO, nGO-PEG, RGO-PEG and nRGO-PEG. (c) UV-visible spectra of GO and RGO-IONP-PEG. RGO-PEG, nRGO-PEG and RGO-IONP-PEG exhibit much higher NIR absorbance compared with GO. (d) AFM images of various functionalized GO derivatives. This figure is adapted from refs. 24,25 and reproduced with permission from Elsevier.

  4. 125I-labeled nGO-PEG for the in vivo biodistribution study.
    Figure 4: 125I-labeled nGO-PEG for the in vivo biodistribution study.

    (a) A schematic of 125I-nGO-PEG. (b) The radiolabeling stability of 125I-nGO-PEG in saline, serum and mouse plasma at 37 °C. (c) Biodistribution of 125I-nGO-PEG in mice measured at different time points after i.v. injection. Error bars show s.d., n = 4 or 5 mice per group. This figure is adapted from ref. 33 and reproduced with permission from ACS Publications.

  5. Graphene-based gene transfection.
    Figure 5: Graphene-based gene transfection.

    (a) An AFM image of nGO-PEG-PEI (Step 3B). (b) Photos of nGO-PEG-PEI in saline and serum-containing cell medium. (c) Relative viabilities of cells treated with nGO-PEG-PEI, GO-PEI and free PEI. Error bars show s.d. on the basis of triplicate samples. (d) Confocal images of EGFP-transfected HeLa cells using nGO-PEP-PEI, GO-PEI or free PEI as the transfection agent in the absence and presence of 10% FBS. (e) Relative EGFP transfection efficiencies of nGO-PEP-PEI, GO-PEI and free PEI in the presence of different concentrations of FBS. Error bars show s.d. of at least four parallel measurements. This figure is adapted from our previously published work21 and reproduced with permission from Wiley-VCH.

  6. Biodistribution of free 125I and 125I-nGO-PEG at 6 h post i.v. injection.
    Supplementary Fig. 1: Biodistribution of free 125I and 125I-nGO-PEG at 6 h post i.v. injection.

    Minimal uptake of free 125I was observed in the liver, spleen, as well as most other organs except thyroid and stomach due to the fast renal excretion of small iodine ions33.


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Author information


  1. Institute of Functional Nano and Soft Materials (FUNSOM), Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow Univeristy, Suzhou, Jiangsu, China.

    • Kai Yang,
    • Liangzhu Feng &
    • Zhuang Liu
  2. Department of Radiology, University of Wisconsin, Madison, Wisconsin, USA.

    • Hao Hong &
    • Weibo Cai
  3. Department of Medical Physics, University of Wisconsin, Madison, Wisconsin, USA.

    • Hao Hong &
    • Weibo Cai
  4. University of Wisconsin Carbone Cancer Center, Madison, Wisconsin, USA.

    • Weibo Cai


Z.L. and W.C. designed the experiments and wrote the manuscript; K.Y., L.F. and H.H. performed the experiments, analyzed the results and wrote the manuscript.

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The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Biodistribution of free 125I and 125I-nGO-PEG at 6 h post i.v. injection. (55 KB)

    Minimal uptake of free 125I was observed in the liver, spleen, as well as most other organs except thyroid and stomach due to the fast renal excretion of small iodine ions33.

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  1. Supplementary Figure 1 (151.0 KB)
  2. Supplementary Table 1 (174.0 KB)

    Summary of in vivo toxicity of different polymer-functionalized GO.

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