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Posted: Sep 13, 2010

Detecting signs of early-stage cancer with graphene oxide and carbon nanotubes

(Nanowerk Spotlight) Biomarkers are of increasing importance in modern medicine for the purpose of early detection and diagnosis of a disease, for instance cancer. Biomarkers are mostly protein molecules that can be measured in blood, other body fluids, and tissues to assess the presence or state of a disease. For example, the presence of an antibody may indicate an infection or an antigen, such as PSA, might indicate the presence of prostate-specific cancer cells. Although protein-based approaches to early detection and diagnosis of cancer have a clear advantage over other, more invasive, methods, protein detection is a challenging problem owing to the structural diversity and complexity of the target analytes.
Current protein detection approaches are mainly dominated by heterogeneous immunological (or separation) assay methods. These assays are usually low-throughput and frequently require multiple steps including multiple incubation and careful washing of a surface onto which the labeled reagent has bound.
In contrast, homogenous immunoassays can overcome these problems. In these assays, the signal is affected by binding and can often be run without a separation step. Such assays can frequently be carried out simply by mixing the reagents and sample and making a physical measurement.
Researchers in China and Japan have now developed a graphene oxide based fluorescence assay for fast, ultra-sensitive, and selective detection of protein and demonstrated its use for detection of a prognostic indicator in early-stage cancer, cyclin A2.
Cyclin A2, as a member of the cyclin family, is critical for the initiation of DNA replication, transcription and cell cycle regulation through the association of cyclin-dependent kinases (CDK). Cyclin A2 is over-expressed in many types of cancers and has been a prognostic indicator in early-stage cancer and a promising anti-cancer target by targeting the cyclin A2 substrate recruitment element called the cyclin binding groove (CBG). Therefore, developing a simple, ultra-sensitive and selective assay of cyclin A2 is important for both clinical diagnosis of cancer in the early stage and the treatment.
"At present, homogenous protein assays are mainly focused on enzymes – protease, kinase, etc – and model proteins such as thrombin," Xiaogang Qu, a professor of chemistry at Changchun Institute of Applied Chemistry, explains to Nanowerk. "Few reported research efforts deal with the proteins that have no enzyme activity and no binding aptamer, which is most likely due to the lack of general protein sensing strategies. Protein-protein interaction provides a general molecular recognition for sensing. However, it is not easy to transform protein-protein interaction into an easily output signal."
In a paper in the August 30, 2010, online issue of Advanced Functional Materials ("Ultrasensitive and Selective Detection of a Prognostic Indicator in Early-Stage Cancer Using Graphene Oxide and Carbon Nanotubes"), Qu, together with his team at the Laboratory of Chemical Biology, and collaborators from the Frontier Institute for Biomolecular Engineering Research (FIBER) at Konan University, led by Naoki Sugimoto, describes the use of single-walled carbon nanotubes (SWCNT) and graphene oxide to selectively read out peptide-protein binding events.
In developing their assay, the researchers took advantage of graphene and single-walled carbon nanotube as highly efficient fluorescence quenchers for organic fluorescent molecules and it appears that graphene oxide is even superior to the SWCNTs for cyclin A2 detection. The team's assays show little background interference and high signal to background ratio (∼21 at 200 nm cyclin A2).
According to Qu, the direct detection limit using graphene oxide is 0.5 nm, 10-fold better than using SWCNTs, and 1200-fold better than the latest reported value of 0.6 µm using terbium-chelating macrocycle modified peptides (see "Cyclin A Probes by Means of Intermolecular Sensitization of Terbium-Chelating Peptides").
This compares very favorably with the few currently reported methods – such as peptide beacon (PB) and hairpin peptide beacon (HPB) based bio-sensing architectures – that require the difficult synthesis and optimization of peptide probes and show only limited signal increase (less than 10-fold, most 2-4 fold at saturated concentration) upon target proteins binding.
"Our results demonstrate the ability of carbon nanotubes and graphene oxide to read out peptide probe-target protein binding events" he says.
Although the researchers have managed to detect cyclin A2 in their lab set-up with high sensitivity and specificity, this method still can not be applied to detect cyclin A2 in clinical samples due to the trace amount of cyclin A2 in the cell total protein.
"We are trying to incorporate a signal amplification step to increase the output signal and detection sensitivity to solve this problem" says Qu.
He notes that several studies have demonstrated that cyclin A2 can serve as a promising anticancer target for the development of tumor therapeutic agents. One of the most effective approaches for identification of new cyclin A2-CDK complex inhibitors is targeting a cyclin A2 substrate recruitment motif named cyclin binding groove (CBG).
"These efforts may considerably benefit from the existence of a cyclin A2 assay using collagen-binding motif (CBM) peptide, which can be conveniently converted to screen CBG inhibitors by a competitive format" Qu points out.
This method might also be applicable to other non-protease protein detection by employing the corresponding peptide probes.
The research team therefore believes that their novel method presents a promising technique to screen new therapeutic drugs and to monitor protein - small molecule interactions in the laboratory, where relatively pure samples are the norm. Although at present this method cannot be used for homogeneous evaluation of the expression level of cyclin A2 in clinical samples, it represents a big step toward this goal.
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