Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA

Zhang, J.; Lang, H.P.; Huber, François; Bietsch, Alexander; Grange, Wilfried; Certa, Ulrich; McKendry, Rachel A.; Güntherodt, H.‐J.; Hegner, Martin; Gerber, Ch.

doi:10.1038/nnano.2006.134

Cited by 290 publications

(231 citation statements)

References 35 publications

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

et al. 2013

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“…miRNA is another class of RNA, which is a short ribonucleic acid molecule (18-25 nucleotides), involved in gene silencing. RNA detection has become an important part of current biomedical research, because it is considered a signature for pathogen identification 1 and its expression profile is linked with disease pathogenesis 2 , allowing for biomarker identification 3 . Thus, RNA as a target of interest has attracted a lot of attention from scientific community, because of their great value in medical diagnostics 4 , drug discovery 5 , disease pathophysiology 6 , biochemical pathways 7 and microbial identification 8 .…”

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Cleavage-based signal amplification of RNA

2013

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RNA detection has become an integral part of current biomedical research. Up to now, the reverse transcription-PCR has been the most practical method to detect mRNA targets. However, RNA detection by reverse transcription-PCR requires sophisticated equipment and it is highly sensitive to contamination with genomic DNA. Here we report a new isothermal reaction to simultaneously amplify and detect RNA, based on cleavage by DNAzyme and signal amplification. Cleavage-based signal amplification of RNA cannot be contaminated by genomic DNA and is suitable for the detection of both mRNA and microRNA targets, with high specificity and sensitivity. Moreover, the detection results can be reported in a colorimetric or real-time fluorometric way for different detection purposes.

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“…Unfortunately, typical optical detection schemes (10) require complex instrumentation which may preclude them from many low-cost point-of-care applications. Further, the response of nanomechanical biosensors varies only linearly (5) or logarithmically (6,14,15) with the change in the mass or surface stress of the cantilever, and therefore these sensors may not be sufficiently sensitive to detect target molecules at very low analyte concentrations, unless sophisticated, low-noise setup is used.To overcome the respective limitations of classical electrical and mechanical nanoscale biosensors, we propose the concept of a Flexure-FET biosensor that integrates the key advantages of both technologies but does not suffer from the limitations of either approach. The Flexure-FET consists of a nanoplate channel biased through a thin-film suspended gate (Fig.…”

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Flexure-FET biosensor to break the fundamental sensitivity limits of nanobiosensors using nonlinear electromechanical coupling

Jain

Nair

Alam

2012

Proc. Natl. Acad. Sci. U.S.A.

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In this article, we propose a Flexure-FET (flexure sensitive field effect transistor) ultrasensitive biosensor that utilizes the nonlinear electromechanical coupling to overcome the fundamental sensitivity limits of classical electrical or mechanical nanoscale biosensors. The stiffness of the suspended gate of Flexure-FET changes with the capture of the target biomolecules, and the corresponding change in the gate shape or deflection is reflected in the drain current of FET. The Flexure-FET is configured to operate such that the gate is biased near pull-in instability, and the FET-channel is biased in the subthreshold regime. In this coupled nonlinear operating mode, the sensitivity (S) of Flexure-FET with respect to the captured molecule density (N s ) is shown to be exponentially higher than that of any other electrical or mechanical biosensor.In other words, while S Flexure ∼ e ðγ 1 ffiffiffiffi N s p −γ 2 N s Þ , classical electrical or mechanical biosensors are limited to S classical ∼ γ 3 N S or γ 4 lnðN S Þ, where γ i are sensor-specific constants. In addition, the proposed sensor can detect both charged and charge-neutral biomolecules, without requiring a reference electrode or any sophisticated instrumentation, making it a potential candidate for various low-cost, point-of-care applications.label-free detection | genome sequencing | cantilever | spring-softening | critical-point sensors N anoscale biosensors are widely regarded as a potential candidate for ultrasensitive, label-free detection of biochemical molecules. Among the various technologies, significant research have focused on developing ultrasensitive nanoscale electrical (1) and mechanical (2) biosensors. Despite remarkable progress over the last decade, these technologies have fundamental challenges that limit opportunities for further improvement in their sensitivity ( Fig. 1A) (3-6). For example, the sensitivity of electrical nanobiosensors such as Si-Nanowire (NW) FET (field effect transistor) (Fig. 1B) is severely suppressed by the electrostatic screening due to the presence of other ions/charged biomolecules in the solution (7), which limits its sensitivity to vary linearly (in subthreshold regime) (3, 7) or logarithmically (in accumulation regime) (4,7,8,9) with respect to the captured molecule density N s . Moreover, the miniaturization and stability of the reference electrode have been a persistent problem, especially for lab-onchip applications (1). Finally, it is difficult to detect charge-neutral biological entities such as viruses or proteins using chargebased electrical nanobiosensor schemes.In contrast, nanomechanical biosensors like nanocantilevers (10, 11) (Fig. 1C) do not require biomolecules to be charged for detection. Here, the capture of target molecules on the cantilever surface modulates its mass, stiffness, and/or surface stress (5,11,12). This change in the mechanical properties of the cantilever can then be observed as a change in its resonance frequency (dynamic mode), mechanical deflection, or change in the resist...

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Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA

Cited by 290 publications

References 35 publications

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

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

Cleavage-based signal amplification of RNA

Flexure-FET biosensor to break the fundamental sensitivity limits of nanobiosensors using nonlinear electromechanical coupling

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