ABSTRACT
Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.
Subject(s)
Electroporation/methods , Gene Expression Regulation , Oligonucleotide Array Sequence Analysis/methods , Animals , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Gene Silencing , Green Fluorescent Proteins/metabolism , Intracellular Space/metabolism , Microelectrodes , Nucleic Acids/metabolism , Oligonucleotides/metabolism , RNA, Small Interfering/metabolism , Time Factors , TransfectionABSTRACT
Ion sensitive field effect transistors (ISFET) are candidates for a new generation of fully electrical DNA sensors. To this purpose, we have modified ISFET sensors by adsorbing on their Si(3)N(4) surface poly-L-lysine and single (as well as double) stranded DNA. Once coupled to an accurate model of the oppositely charged layers adsorbed on the surface, the proposed sensor allows quantitatively evaluating the adsorbed molecules densities, as well as estimating DNA hybridization kinetics.
Subject(s)
Biosensing Techniques/instrumentation , DNA, Complementary/analysis , Nucleic Acid Hybridization , DNA, Complementary/metabolism , KineticsABSTRACT
Floating Gate (FG) nonvolatile memories are based on a tiny polysilicon layer (the FG) which can be permanently charged with electrons or holes, thus changing the threshold voltage of a MOSFET. Every time a FG is hit by a high energy ion, it experiences a charge loss, depending on the ion linear energy transfer (LET) and on the transistor geometrical and electrical characteristics. This paper discusses the opportunities to use this devices as single an ion dosemeter with sub-micrometer spatial resolution and capable of distinguish the impinging ion LET.
Subject(s)
Microchemistry/instrumentation , Nanotechnology/instrumentation , Radiometry/instrumentation , Semiconductors , Signal Processing, Computer-Assisted/instrumentation , Computer Storage Devices , Dose-Response Relationship, Radiation , Equipment Design , Equipment Failure Analysis , Ions , Microchemistry/methods , Miniaturization , Nanotechnology/methods , Radiation Dosage , Radiometry/methods , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
UVPROM memory devices employing FGMOS transistors as memory cells make excellent dosemeters for applications involving ionising radiation. With proper preparation and programming, these devices can be used in remote-sensing applications in high-radiation environments with no power required during exposure.
