Publications by authors named "Roberta Menezes"

3 Publications

  • Page 1 of 1

Isothermal digital detection of microRNAs using background-free molecular circuit.

Sci Adv 2020 01 22;6(4):eaay5952. Epub 2020 Jan 22.

Laboratoire Gulliver, CNRS, ESPCI Paris, PSL Research University, 10 rue Vauquelin, 75005 Paris, France.

MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.
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http://dx.doi.org/10.1126/sciadv.aay5952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976291PMC
January 2020

Streamlined digital bioassays with a 3D printed sample changer.

Analyst 2020 Jan 26;145(2):572-581. Epub 2019 Nov 26.

Laboratoire Gulliver, UMR7083 CNRS, ESPCI Paris, PSL Research University, 10 rue Vauquelin, 75005 Paris, France.

Droplet-based microfluidics has permeated many areas of life sciences including biochemistry, biology and medicine. Water-in-oil droplets act as independent femto- to nano-liter reservoirs, enabling the parallelization of (bio)chemical reactions with a minimum sample input. Among the range of applications spanned by droplet microfluidics, digital detection of biomolecules, using Poissonian isolation of single molecules in compartments, has gained considerable attention due to the high accuracy, sensitivity and robustness of these methods. However, while the droplet throughput can be very high, the sample throughput of these methods is poor in comparison to well plate-based assays. This limitation comes from the necessity to convert independently each sample into a monodisperse emulsion. In this paper, we report a versatile device that performs the quick sequential partitioning of up to 15 samples using a single microfluidic chip. A 3D printed sample rotor is loaded with all samples and connected to a pressure source. Simple magnetic actuation is then used to inject the samples in the microfluidic chip without pressure disruption. This procedure generates monodisperse droplets with high sample-to-sample consistency. We also describe a fluorescent barcoding strategy that allows all samples to be collected, incubated, imaged and analyzed simultaneously, thus decreasing significantly the time of the assay. As an example of application, we perform a droplet digital PCR assay for the quantification of a DNA amplicon from 8 samples in less than 2 hours. We further validate our approach demonstrating the parallel quantification of 11 microRNAs from a human sample using an isothermal nucleic acid amplification chemistry. As an off-chip device, the sample changer can be connected to a variety of microfluidic geometries and therefore, used for a wide range of applications.
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http://dx.doi.org/10.1039/c9an01744eDOI Listing
January 2020

Emerging isothermal amplification technologies for microRNA biosensing: Applications to liquid biopsies.

Mol Aspects Med 2020 04 23;72:100832. Epub 2019 Nov 23.

Centre de Recherche des Cordeliers, INSERM, CNRS, Sorbonne. Université, USPC, Université Paris Descartes, Université Paris Diderot, F-75006, Paris, France; INSERM UMR-S1147, CNRS SNC5014, Paris Descartes University, 45 rue des Saints-Pères, Paris, 75006, France; Equipe labellisée Ligue Nationale contre le cancer, France. Electronic address:

The potential of microRNAs (miRNAs) as biomarker candidates in clinical practice for diagnosis, prognosis and treatment response prediction, especially in liquid biopsies, has led to a tremendous demand for techniques that can detect these molecules rapidly and accurately. Hence, numerous achievements have been reported recently in miRNA research. In this review, we discuss the challenges associated with the emerging field of miRNA detection, which are linked to the intrinsic properties of miRNAs, advantages and drawbacks of the currently available technologies and their potential applications in clinical research. We summarize the most promising nucleic acid amplification techniques applied to the in vitro detection of miRNAs, with a particular emphasis on the state of the art for isothermal alternatives to RT-qPCR. We detail the sensitivity, specificity and quantitativity of these approaches, as well as their potential for multiplexing. We also review the different detection formats to which these chemistries have been adapted, including analog readouts such as real-time monitoring, digital counting based on single-molecule amplification in compartments, and surface-based strategies.
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http://dx.doi.org/10.1016/j.mam.2019.11.002DOI Listing
April 2020
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