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Posted: Sep 20, 2011

Nanotechnology sensor could provide a much earlier warning signal for lung cancer

(Nanowerk Spotlight) Every aspect of cellular activities, including cell proliferation, differentiation, metabolism and apoptosis, can be regulated by a class of tiny but very important nucleic acids fragments called microRNAs (miRNAs). They bind to specific messenger RNAs and cause messenger RNA degradation or inhibit translation, thereby regulate gene expression at the post-translational level. In cancer cells, the homeostasis of these normal biological processes is disrupted, partially by dysregulated miRNAs, therefore the level of microRNAs is an indicator to the disease development, and miRNAs in cancer tissues or biofluids, either whole spectrum or a set of miRNAs, can be utilized as a diagnostic biomarker for cancer detection.
Now, researchers report a miRNAs-based discovery that could provide a much earlier warning signal for lung cancer. The new technique is based on a nanopore sensor and can detect a specific molecule type in the bloodstream when lung cancer is present.
"We have designed a nanopore-based microRNA sensor that uses a programmable oligonucleotide probe to generate a signature electrical signal for the direct and label-free detection of target microRNA in a fluctuating background, such as plasma RNA extracts from clinical samples," Liqun Gu, an associate professor in biological engineering Dalton Cardiovascular Research Center and at the University of Missouri, explains to Nanowerk.
Led by Gu and Michael Wang, assistant professor of pathology and anatomical sciences at Ellis Fishel Cancer Center and MU, the team reports their findings in the September 4, 2011 online edition of Nature Nanotechnology ("Nanopore-based detection of circulating microRNAs in lung cancer patients"). The work in this paper is to develop a novel single-molecule technology that demonstrates a great potential for ultrasensitive and specific detection of microRNA levels in plasma of cancer patients.
For years, nanopore technology has been developed as a biosensor at the single-molecule resolution to detect an array of biomedical molecules, such as DNA, RNA, protein, bio-toxin (see for instance "For the first time single proteins were detected with nanopores" or "Novel, fast DNA nanopore detector features integrated tunneling electrodes"), and various nanopore projects have been funded to develop the next generation of DNA sequencing technology.
Liqun Gu is a biophysicist working in the interdisciplinary field of nanobiotechnology. One of the long-term goals of Gu's lab is to explore the nanopore application in solving life science problems and medical diagnostics. In particular the latter task has a high impact and needs a collaborative effort. Motivated by this goal, Gu made the first step collaborating with Michael Wang and his lab, who are interested in cancer detection using various genetic and epigenetic biomarkers including microRNAs. The integration of nanopore and microRNA detection may potentially provide a non-invasive method for diagnostics and prognostics of diseases including cancer.
During the course of diseases such as cancer, miRNAs are released from the primary tumor site into the bloodstream. Circulating microRNAs are very stable and can be utilized as biomarkers for cancer detection. However, detection of highly diluted miRNA molecules in blood is a technical challenge. Real-time reverse transcription polymerase chain reaction (RT-PCR) is the principle method for this purpose.
Wang notes that to distinguish similar sequences, or single-nucleotide polymorphisms, and accurately measure the levels of the specific miRNAs by quantitative RT-PCR (qRT-PCR) is problematic. "Compared with qRT-PCR, the nanopore method we developed can directly detect microRNA without the need of enzymatic reaction, labeling and amplification, while it is also simpler to operate at a low cost. Our nanopore method measures miRNA at single molecular level with very high sensitivity and specificity," say Gu and Wang.
The core of this nanosensor comprises a very tiny protein pore assembled by the toxin alpha-hemolysin. This nanopore is only 2 nm wide and 10 nm long. It is unique because when each type of microRNAs in hybrid with its probe interacts with the pore lumen, it can specifically modulate the ionic current through the pore to generate a signature signal, which acts as a fingerprint allowing us to recognize individual microRNA molecules one by one.
The key component of the nanopore sensor is the DNA probe, the sequence of which has been specifically designed and optimized to achieve high specificity and sensitivity.
"We found that the nanopore can distinguish sequence-similar microRNAs with a single nucleotide difference" says Gu. "This is important because single nucleotide change – also known as single nucleotide polymorphism or SNP – in its target sequences or miRNA itself has significant impact in diseases. For instance, a let-7 microRNA-binding site polymorphism in the KRAS 3'-untranslated region (known as KRAS-LCS6) is associated with poor prognosis in lung cancer, colorectal cancer and oral cancer patients. KRAS is a classic oncogene and its expression is regulated by let-7 microRNA family."
The researchers expect that the probe composition will not be limited to the four nucleotides. "The incorporation of unnatural compounds such as locked nucleic acids and peptide nucleotide acids into the probe sequences may enhance selectivity because of the strengthened hybridization between probe and target. The probe can also be engineered with a specific barcode through chemical modification, so that multiple microRNAs can be simultaneously detected using distinct probes."
This work may have potential applications in early diagnostics and monitoring of disease, especially in lung cancer. It can also be used in discovery of new biomarkers for other diseases.
The nanopore sensor is not limited to the detection microRNA. In principle, it can be used to detect any pathogenic DNA or RNA fragments, and detect a single nucleotide polymorphism. For instance, detection of circulating mutant DNA fragments in cancer patient blood has many clinical applications including early diagnosis, prediction of metastasis, monitoring of response to therapy, and detection of minimal residual disease.
"Following this work, we are planning to validate our method using a large set of lung cancer patient blood samples which includes comparison of lung cancer at different stages and various tissue types, as well as in high risk populations," Gu and Wang outline the team's next steps.
"We also are focused on overcoming two challenges that we currently are faced with: Firstly, the existing sensor prototype is not small enough. A smaller device not only consumes minimal amount of clinical sample, but greatly increases the sensitivity and decreases the measuring time. We need to develop a kind of miniature, durable and portable device that is integrated with the sensitive nanopore so that it can be conveniently usable by physicians and researchers."
"Secondly, another issue is the multiplex measurement" he continues. "The current sensor configuration can only detect one microRNA species per nanopore per test. In clinical settings, we need to test several miRNAs as a panel. One of our current efforts is the exploration of a nanopore array that allows simultaneous screening of multiple microRNAs for clinical diagnosis."
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