Single-cell quantification of ribosome occupancy in early mouse development.

Translation regulation is critical for early mammalian embryonic development. However, previous studies had been restricted to bulk measurements, precluding precise determination of translation regulation including allele-specific analyses. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development.

Isolation of enriched small RNA from cell-lysate using on-chip isotachophoresis.

In spite of the growing interest in the roles and applications of small RNAs (sRNAs), sRNA isolation methods are inconsistent, tedious, and dependent on the starting number of cells. In this work, we employ ITP to isolate sRNAs from the cell-lysate of K562 (chronic myelogenous leukemia) cells in a polydimethylsiloxane (PDMS) mesofluidic device. Our method specifically purifies sRNA of <60 nucleotides from lysate of a wide range of cell number spanning from 100 to 1 000 000 cells.

Simultaneous RNA purification and size selection using on-chip isotachophoresis with an ionic spacer.

We present an on-chip method for the extraction of RNA within a specific size range from low-abundance samples. We use isotachophoresis (ITP) with an ionic spacer and a sieving matrix to enable size-selection with a high yield of RNA in the target size range. The spacer zone separates two concentrated ITP peaks, the first containing unwanted single nucleotides and the second focusing RNA of the target size range (2-35 nt).

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring.

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR.

Increasing hybridization rate and sensitivity of DNA microarrays using isotachophoresis.

We present an on-chip electrokinetic method to increase the reaction kinetics and sensitivity of DNA microarray hybridization. We use isotachophoresis (ITP) to preconcentrate target molecules in solution and transport them over the immobilized probe sites of a microarray, greatly increasing the binding reaction rate. We show theoretically and experimentally that ITP-enhanced microarrays can be hybridized much faster and with higher sensitivity than conventional methods.

Integration of rapid DNA hybridization and capillary zone electrophoresis using bidirectional isotachophoresis.

We present a method for rapid, sequence-specific detection of multiple DNA fragments by integrating isotachophoresis (ITP) based DNA hybridization and capillary zone electrophoresis (CZE) using bidirectional ITP. Our method leverages the high preconcentration ability of ITP to accelerate slow, second-order DNA hybridization kinetics, and the high resolving power of CZE to separate and identify reaction products. We demonstrate the speed and sensitivity of our assay by detecting 5 pM, 39 nt ssDNA target within 3 min, using a molecular beacon probe.

Rapid hybridization of nucleic acids using isotachophoresis.

We use isotachophoresis (ITP) to control and increase the rate of nucleic acid hybridization reactions in free solution. We present a new physical model, validation experiments, and demonstrations of this assay. We studied the coupled physicochemical processes of preconcentration, mixing, and chemical reaction kinetics under ITP.

Extraction of DNA from malaria-infected erythrocytes using isotachophoresis.

We demonstrate a technique for purification of nucleic acids from malaria parasites infecting human erythrocytes using isotachophoresis (ITP). We release nucleic acids from malaria-infected erythrocytes by lysing with heat and proteinase K for 10 min and immediately, thereafter, load sample onto a capillary device. We study the effect of temperature on lysis efficiency.