Development of a fluid functionalized lipidic matrix applied to direct in situ polynucleotide detection.
This work presents a new approach for direct detection of polyelectrolytes at the air-water interface, based on the investigation of the interfacial properties of an active lipidic matrix especially designed for polynucleotide immobilization. A synthetic lipid with a cationic spermine headgroup, DiOctadecylamidoGlycylSpermine (DOGS), was spread at the interface to form a distortable film able to capture polynucleotides. The control of the organization state of this functionalized monolayer upon compression was achieved by recording surface pressure-area (pi-A) isotherm diagrams, presenting a specific shape with a typical liquid expanded-liquid condensed phase transition on a pure water subphase. In the presence of various dsDNA concentrations in the subphase, the isotherms were markedly modified in a time and concentration-dependent manner. The main modifications, corresponding to a large shift towards higher molecular areas and a clear fading of the phase transition, were corroborated by the fine analysis of the monolayer compressibility profile, thus suggesting a characteristic change in the monolayer fluidity as a function of both time and DNA concentration. Moreover, an ATR-Fourier transform infrared (ATR-FTIR) characterization showed evidences for the adsorption of DNA strands onto the lipidic matrix. The direct observation of the mixed monolayer morphology by Brewster angle microscopy (BAM) strongly suggests that DNA adsorption induces a reorganization of lipids at the interface, as evidenced by the change in the condensed lipidic domains morphology in the presence of DNA in the subphase. The immobilization of various polynucleotidic probes of 4000, 400 and 22 base length, confirmed by fluorescence microscopy, had similar effects on DOGS interfacial properties. Preliminary studies are finally presented to explore the possibility of using this system for the study of hybridization between complementary strands. Hence, this study demonstrates this functionalized matrix behaves as a fluid support where polynucleotide immobilization induces interfacial properties modifications, which could be further exploited through the experimental characterization of Faraday instabilities sensitive to visco-elasticity variations.
Thomas
Daphné L
DL
Laboratoire de Génie Enzymatique et Biomoléculaire, UMR 5013/EMB2-CNRS/UCBL, Université Claude Bernard Lyon 1, 43 Bd du 11 novembre 1918, F-69622 Villeurbanne Cedex, France.
Blum
Loïc J
LJ
Girard-Egrot
Agnès P
AP
D000327:Adsorption; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D017403:In Situ Hybridization / Q000295:instrumentation / Q000379:methods*; D008563:Membrane Lipids / Q000032:analysis / Q000737:chemistry; D008567:Membranes, Artificial; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods; D015345:Oligonucleotide Probes / Q000032:analysis* / Q000737:chemistry; D011119:Polynucleotides / Q000032:analysis* / Q000737:chemistry; D013095:Spermidine / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000737:chemistry; D013500:Surface Tension
D008563:Membrane Lipids; D008567:Membranes, Artificial; D015345:Oligonucleotide Probes; D011119:Polynucleotides; C446820:dioctadecylamidoglycylspermidine; D004247:DNA; D013095:Spermidine