title: Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules.
abstract: Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.
authors.affiliation: Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, SE-751 05 Upssala, Sweden.|Science for Life Laboratory, Department of Biophysics and Biochemistry, Stockholm University, SE- 171 65 Solna, Sweden.
authors.forename: Malte
authors.initials: M
authors.lastname: Kühnemund
authors.affiliation: Science for Life Laboratory, Department of Biophysics and Biochemistry, Stockholm University, SE- 171 65 Solna, Sweden.
authors.forename: Iván
authors.initials: I
authors.lastname: Hernández-Neuta
authors.affiliation: Science for Life Laboratory, Department of Biophysics and Biochemistry, Stockholm University, SE- 171 65 Solna, Sweden.
authors.forename: Mohd Istiaq
authors.initials: MI
authors.lastname: Sharif
authors.affiliation: Laboratory of Microsystems, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
authors.forename: Matteo
authors.initials: M
authors.lastname: Cornaglia
authors.affiliation: Laboratory of Microsystems, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
authors.forename: Martin A M
authors.initials: MAM
authors.lastname: Gijs
authors.affiliation: Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, SE-751 05 Upssala, Sweden.|Science for Life Laboratory, Department of Biophysics and Biochemistry, Stockholm University, SE- 171 65 Solna, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Nilsson
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015374:Biosensing Techniques; D015780:Carbapenems / Q000494:pharmacology; D002232:Carbocyanines / Q000737:chemistry; D015342:DNA Probes / Q000378:metabolism; D004270:DNA, Circular / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D056656:Lab-On-A-Chip Devices; D008712:Methicillin / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D002232:Carbocyanines; D015342:DNA Probes; D004270:DNA, Circular; D004129:Dimethylpolysiloxanes; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; C089187:cyanine dye 3; C013830:baysilon; D001618:beta-Lactamases; C000601047:beta-lactamase OXA-48, E coli; D016650:Fluorescein-5-isothiocyanate; D008712:Methicillin