Monday, 6 November 2017

The future possibilities of graphene for microbiology


Graphene is the most widely researched new material. It is an allotrope of carbon and due to special properties the material is being tested out within the fields of consumer and medicinal electronics. While these applications have received considerable attention developments relating to microbiology are taking place. This short review article considers bacterial staining and anti-bacterial activity, as two of the most promising future developments.

An essay by Tim Sandle

Graphene is a material derived from carbon and it has unique physicochemical properties. Graphene is formed where graphite is taken and atom thick layers are sliced away. The resultant structure is a single-layer of carbon atoms linked in a hexagonal chicken-wire pattern. Within the structure each of the atoms share a cloud of electrons moving freely about the surface. The material is light, transparent, strong and very conductive (Allen et al, 2010). Compared to other carbon allotrope, such as fullerenes, carbon nanotubes and graphite, graphene exhibits many exceptional physical and chemical properties. Graphene related materials are of great interest in the field of biomedicines and applications are underway in biosensing and drug delivery.

Graphene as a Gram-stain alternative

New research, from the University of Illinois at Chicago, suggests that graphene can be used for performing Gram-stains, as part of microbial identification. A review of the properties of graphene shows it can detect variances to cell vibration when a cell comes into contact with the material. So far the tests undertaken with graphene have related to cancer. Here atomic vibration differs depending upon whether the cell is a cancer cell or a normal cell. This happens because the cancer cell’s hyperactivity leads to a higher negative charge, and this causes a higher level of protons to be released. This difference can be detected, helping medical technologists to identify cancerous growth.

Assessing the variances in vibration is possible using an established laboratory method called Raman spectroscopy (a spectroscopic technique used to observe vibrational, rotational, and other low-frequency modes in a system).

According to lead researcher Vikas Berry, who is the associate professor and head of chemical engineering, who led the research along with Ankit Mehta, assistant professor of clinical neurosurgery in the UIC College of Medicine: “We may be able to use it with bacteria to quickly see if the strain is Gram-positive or Gram-negative…We may be able to use it to detect sickle cells.” Berry made this remark to Controlled Environments magazine (Anon, 2017).

In a parallel development, one research group have created a graphene sensor for Escherichia coli. This involved fabricating a flexible substrate onto which a sensor device with O-ring is fitted. Once contact takes place with the suspected organism, Raman spectra is used to indicate the presence (Basu et al, 2014).

As all microbiologists know, the Gram-stain is the key test for distinguishing between two groups of bacteria based on cell wall morphologies (Sandle, 2014). The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decolouring process (Sandle, 2004).


While the Gram-stain technique is well described it is sometimes prone to error. This can relate to the types of organisms, the age of the cultures, or due to errors made by the person performing the test (such as over decoloursation). A method based on graphene would error proof. Whether such a method becomes commercially available will depend on development costs.

Graphene as an antibacterial agent

As well as using graphene as a potential diagnostic tool, graphene can also be used as an anti-bacterial measure (where liposome-embedded graphene reduces the growth capability of bacteria). Research by Zappacosta and colleagues showed that graphene aqueous dispersion is stable for several days and demonstrates significant antibacterial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) strains, with a reduction in the growth of S. aureus and E. coli as high as 60 and 78%, respectively.

In a similar application, researchers have looked at graphene-iodine nano-composites, formed via electrostatic interactions between positively charged graphene derivatives and triiodide anions, as anti-bacterial agents (Some et al, 2015). With this, the antibacterial potential of these graphene-iodine composites against Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus mirobilis, Staphylococcus aureus, and Escherichia coli has been demonstrated. The success against the organisms relates to the inherent cytotoxicity of the nanocomposite, specifically through electron transfer interaction from microbial membrane to graphene.


Furthermore, scientists are studying graphene oxide with the aim of creating bacteria-killing catheters and medical devices. Here coating surgical tools with this carbon-based compound could kill bacteria, reducing the need for antibiotics, decreasing the rates of post-operative infections and speeding recovery times. This is with graphene oxide, which is a form of graphene with molecular oxygen incorporated into it. This compound protects against infection by destroying bacteria before it gets inside the body. In terms of the process the graphene oxide wraps around the bacteria, puncturing its membrane. A broken membrane prevents the bacteria from growing and often kills it.

Studies conducted at the Universit√† Cattolica del Sacro Cuore in Rome indicate that the compound is most effective when paired with salt. Getting the salt balance correct is important. With too little salt and then the graphene oxide is unable to wrap around the bacteria; and with  too much salt and the graphene aggregates, failing to puncture the bacteria's membrane. In order to destroy both Gram positive and Gram negative bacteria a 300 nanometer sheet of graphene oxide solution must be mixed with low molarity (<10 mM) calcium chloride is required (Anon, 2015).

Summary

These two related research strands (for differential microbiology and as an antibacterial agent) signal that graphene, the so-called ‘wonder material’ of our age, is set to make a significant impact upon microbiology. As with the development of any novel method, progress will be slow. However, the research results reported to date suggest that graphene is set to make a major contribution to microbiology.

References

Allen, M. J., Tung, V.C. and R. B. Kaner, R.B. Honeycomb carbon: a review of graphene, Chem. Rev., 2010, 110, 132

Anon. Biophysical Society report “Towards a “green” antimicrobial therapy: Study of graphene nanosheets interaction with human pathogens”, 2015 (http://tinyurl.com/zzgsofu)

Anon. First Use of Graphene to Find Cancer Cells, Controlled Environments, 2017 (http://www.cemag.us/news/2017/02/first-use-graphene-find-cancer-cells)

Basu, P.K., Indukuri, D., Keshavan, S., Bhat, N. (2014) Graphene based E. coli sensor on flexible acetate sheet, Sensors and Actuators B Chemical 190:342-347 (https://www.researchgate.net/publication/256926101_Graphene_based_E_coli_sensor_on_flexible_acetate_sheet)

Sandle, T. (2004) Gram’s Stain: History and Explanation of the Fundamental Technique of Determinative Bacteriology’, IST Science and Technology, No. 54, pp3-4

Sandle, T. (2014). ‘Microbial Identification: Laboratory Techniques and Methods. In Chesca, A. (Ed.) Methods for Diseases: Diagnostic with Applicability in Practice, Lambert Academic Publishing, Germany, pp15-26

Some, S., Sohm, J., Kim, J. et al Graphene-Iodine Nanocomposites: Highly Potent Bacterial Inhibitors that are Bio-compatible with Human Cells, Scientific Reports 6, Article number: 20015 (2016). doi:10.1038/srep20015

Zappacosta, R., Di Giulio, M., Ettorre, V. et al Liposome-induced exfoliation of graphite to few-layer graphene dispersion with antibacterial activity, J. Mater. Chem. B, 2015, 3, 6520-6527 (http://pubs.rsc.org/en/content/articlehtml/2015/tb/c5tb00798d)

by Dr. Tim Sandle