A new gravity-driven microfluidic-based electrochemical assay coupled to magnetic beads for nucleic acid detection.
In this work, the characterisation and the optimisation of hybridisation assays based on a novel, rapid and sensitive micro-analytical, gravity-driven, flow device is reported. This device combines a special chip containing eight polymer microchannels, with a portable, computer-controlled instrument. The device is used as a platform for affinity experiments using oligonucleotide-modified paramagnetic particles. In our approach, both hybridisation and labelling events are performed on streptavidin-coated paramagnetic microparticles functionalized with a biotinylated capture probe. Modified particles, introduced in the microchannel inlet of the chip, accumulate near the electrode surface by virtue of a magnetic holder. After hybridisation with the complementary sequence, the hybrid is labelled with an alkaline phosphatase conjugate. The electrochemical substrate for alkaline phosphatase revelation is p-aminophenyl phosphate. Solutions and reagents are sequentially passed through the microchannels, until enzyme substrate is added for in situ signal detection. Upon readout, the magnet array is flipped away, beads are removed by addition of regeneration buffer, and the so-regenerated chip is ready for further analysis. This protocol has been applied to the analytical detection of specific DNA sequences of Legionella pneumophila, with an RSD=8.5% and a detection limit of 0.33 nM.
Dipartimento di Chimica, Università degli Studi di Firenze, Firenze, Italy.
Serena
S
Laschi
Rebeca
R
Miranda-Castro
Eva
E
González-Fernández
Ilaria
I
Palchetti
Frédéric
F
Reymond
Joël S
JS
Rossier
Giovanna
G
Marrazza
D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006112:Gravitation; D055029:Inverted Repeat Sequences; D016952:Legionella pneumophila / Q000235:genetics; D016014:Linear Models; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D008863:Microspheres; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009696:Nucleic Acids / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism
D004269:DNA, Bacterial; D058185:Magnetite Nanoparticles; D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D019809:Streptavidin