Nanomechanical Detection of DNA Melting on Microcantilever Surfaces

Biswal, Sibani Lisa; Raorane, Digvijay; Chaiken, A.; Birecki, H.; Majumdar, Arun

doi:10.1021/ac052171y

Cited by 72 publications

(54 citation statements)

References 42 publications

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“…A similar effect was discussed by Majumdar et al, who applied nanomechanical detection of DNA melting on microcantilever surfaces and also observed lower melting temperatures for the surface confined duplexes [33]. Using covalent ferrocene labels, Hartwich et al also found similar melting behavior [34].…”

Section: Resultssupporting

confidence: 66%

Electrochemical Detection of DNA Melting Curves by Means of Heated Biosensors

Surkus¹,

Flechsig²

2009

Electroanalysis

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This article reports about the detection of DNA melting curves at heated electrochemical biosensors. Osmium tetroxide-bipyridine-labeled target oligonucleotides are hybridized with probe oligonucleotides immobilized on gold electrodes. Then, the gold electrode is successively heated in order to measure a complete melting curve consisting of alternating current voltammetric signals. Melting temperatures W m , determined at various ionic strengths and in dependence on different numbers of base pair mismatches, have been compared with those obtained by means of UV spectrophotometry. The proposed method holds great promise for the fast and easy parallel detection of nucleic acids sequences on selectively heated electrode arrays. A stringent hybridization temperature can be easily adjusted in order to discriminate base pair mismatches.

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Section: Resultssupporting

confidence: 66%

Electrochemical Detection of DNA Melting Curves by Means of Heated Biosensors

Surkus¹,

Flechsig²

2009

Electroanalysis

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“…In recent years a versatile platform for biodetection has been developed based on microfabricated arrays of silicon cantilevers 14,15,16,17,18,19,20,21 , each coated with a sensitive layer for molecular recognition, e.g. a gene specific oligonucleotide.…”

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confidence: 99%

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

et al. 2013

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Malignant melanoma, the deadliest form of skin cancer, is characterised by a predominant mutation in the BRAF gene. Drugs that target tumours carrying this mutation have recently entered the clinic. Therefore patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real time polymerase chain reaction (PCR) and sequencing methods. Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild type cells using as little as 20 ng/µl of RNA material, without prior PCR amplification and use of labels.The identification of alterations in specific signalling pathways and recurrent oncogenic mutations in particular types of cancers has led in the past decade to the explosion of targeted therapy approaches. In cutaneous melanoma, a significant improvement in overall survival has been achieved by vemurafenib and similar drugs that selectively inhibit tumours carrying a mutated BRAF 2 gene 1,2 . Additional drugs for combination therapies with higher efficacies and fewer side effects are in clinical trials 3 . BRAF is one of three RAF genes (rapidly accelerated fibrosarcoma A, B and C) encoding cytoplasmic protein serine/threonine kinases belonging to the mitogen-activated protein kinase (MAPK) signal transduction cascade, a pathway controlling various cellular processes such as proliferation, migration and survival 4,5 . BRAF somatic mutations are present in half of cutaneous melanomas. Over 90% of the mutations are a single T to A transversion at position 1799 in the BRAF coding sequence (cT1799A), which converts a valine amino acid residue at position 600 in the protein to a glutamic acid (V600E). This mutation renders the protein constitutively active, resulting in a deregulated MAPK pathway 6 and thus uncontrolled cell growth and cancer. BRAF mutations are also present in other neoplasms, including hairy cell leukemias, thyroid and colon carcinomas 7,8,9 . As the presence of the cT1799A/V600E BRAF (hereafter BRAF V600E ) mutation determines eligibility to BRAF inhibitor treatment, molecular screening of tumour biopsies is now carried out routinely. with sensitivity comparable to COBAS TM , silicon nanowire field-effect transistors 12 and a threedimensional gold nanowire platform 13 . The latter technologies have only been shown to work using synthetic oligonucleotide targets, larger gene fragments or still rely on an initial PCR amplification. In particular, they have not been applied to the direct identification of a mutated messenger RNA (mRNA) sequence in total RNA (constituted primarily by ribosomal RNA and containing all mRNAs transcribed from gen...

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“…3c) ). Such nanomechanical biosensors can be used for applications in ultrasensitive detection of: DNA hybridization (Biswal et al 2006;Fritz et al 2000), biomarker transcripts in human RNA , and viruses (Gupta et al 2006). In addition, cantilever-based force spectroscopy has emerged as a powerful tool for characterizing spatiotemporal dynamics of the interaction between individual ligands and receptors, either on isolated molecules or on cellular surfaces (Hinterdorfer and Dufrene 2006), such as VEGFR2 (Lee et al 2007b) or integrin a2b1 (Taubenberger et al 2007).…”

Section: Cantilever-based Biosensorsmentioning

confidence: 99%

Applications, techniques, and microfluidic interfacing for nanoscale biosensing

Jungkyu

Junkin

Kim

et al. 2009

Microfluid Nanofluid

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Biosensors based on nanotechnology are rapidly developing and are becoming widespread in the biomedical field and analytical chemistry. For these nanobiosensors to reach their potential, they must be integrated with appropriate packaging techniques, which are usually based on nano/microfluidics. In this review we provide a summary of the latest developments in nanobiosensors with a focus on label-based (fluorescence and nanoparticle) and label-free methods (surface plasmon resonance, micro/nanocantilever, nanowires, and nanopores). An overview on how these sensors interface with nano/microfluidics is then presented and the latest papers in the area summarized.

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Nanomechanical Detection of DNA Melting on Microcantilever Surfaces

Cited by 72 publications

References 42 publications

Electrochemical Detection of DNA Melting Curves by Means of Heated Biosensors

Electrochemical Detection of DNA Melting Curves by Means of Heated Biosensors

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

Applications, techniques, and microfluidic interfacing for nanoscale biosensing

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