Coaxial Flow System for Chemical Cytometry

Marc, Paul J.; Sims, Christopher E.; Allbritton, Nancy L.

doi:10.1021/ac7017519

Cited by 17 publications

(20 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Maximum sampling efficiency was found using a 3-ns, 2-μJ laser pulse positioned directly under the cell. Another approach for cell disruption utilized a coaxial buffer exchange system at the capillary inlet that delivered a sheath flow of electrophoretic buffer (cell lysing agent) at the moment of turning on the separation electric field (159). To perform a cell injection and lysis, the capillary tip was lowered until it was ∼10 μm above the cell, and the sheath-flow and electric voltage were turned on simultaneously, exposing the cell to both the electrophoretic buffer and the electric field, thereby lysing the cell; other cells were in the same preparation were not affected by the lysis of the selected cell.…”

Section: Single-cell Analysismentioning

confidence: 99%

Capillary Electrophoresis in Bioanalysis

2008

View full text Add to dashboard Cite

Science reports ∼4800 hits on capillary electrophoresis (CE) including 410 reviews. Because of the prominence of CE techniques in bioanalysis, we decided make them the focus of this review and selected 200 hundred papers representing advances in this field. The selected papers cover advances in CE theory, instrumentation, and methodologies that are specific to various analytes of biological origin or relevance. The group of analytes includes nucleic acids, proteins and peptides, carbohydrates, lipids, single cells, and bioparticles. In addition, we have included advances in the use of CE to define functional assays or to investigate biomolecular interactions. The use of microfabricated devices for CE analysis was not included because this is already covered in other review. Technique Developments Separation SchemesDetermining the velocity of the electroosmotic flow (EOF) and how it changes during an electrophoretic separation is still an important research topic. A simple method for EOF measurements using so-called thermal marks was reported (1). Here, a tungsten filament caused punctual heating at the capillary wall and caused a perturbation in the electrolyte concentration. A sequence of these "thermal marks" then migrated with the EOF until each mark reached and was detected by a conductivity detector. The feasibility of using thermal marks as internal EOF standards in different separation systems was thereby demonstrated.Isoelectric focusing separates amphoteric analytes such as proteins or peptides by the differences in their isoelectric points. Most of the reports on capillary isoelectric focusing (cIEF) describe an initial focusing phase after which the focused zones are mobilized and detected. A dynamic cIEF method for protein analysis was reported (2). This technique made it possible to control each protein's position and focused width by moving the pH gradient within the capillary through manipulation of the electric fields. An important advantage of this approach is the capability of collecting focused analytes from the central section, suggesting that there may be great potential for introducing selectively focused proteins to a second separation dimension such as LC or CE.Micellar electrokinetic capillary chromatography (MEKC) is typically incompatible with electrospray mass spectrometry (ESI-MS) because the nonvolatile surfactants in the micellar phase result in complicated adduct formation and loss of sensitivity during the electrospray process and because the presence of the organic solvent needed for electrospray may cause instability in the micellar phase. These drawbacks were overcome by using synthetic polymeric surfactants that can work as a pseudostationary phase and provide stable electrospray (3). The polymeric surfactant was made by polymerizing three amino acidderived (L-leucinol, L-isoleucinol, L-valinol) sulfated chiral surfactants. These polymeric

show abstract

Section: Single-cell Analysismentioning

confidence: 99%

Capillary Electrophoresis in Bioanalysis

2008

View full text Add to dashboard Cite

show abstract

“…This cell buffer velocity was less than one-tenth of that required for the aforementioned coaxial system (1.7 mm/s). [29] Additionally when fluorescein (1 nM) was added to the cell buffer and the capillary was placed in the electrophoretic channel 1.5 cm from the channel intersection, no increase in fluorescence was observed in the capillary.…”

Section: Resultsmentioning

confidence: 99%

“…To develop serial analysis of adherent cells, Marc et al designed a coaxial system to rapidly modify the buffer composition surrounding the inlet of a capillary from a physiological buffer to a separation buffer. [29] The separation capillary mated with a coaxial capillary were placed adjacent to cells cultured in a physiologic buffer. After injection of the cell into the capillary, separation buffer flowed through the coaxial capillary providing a 100% exchange of separation buffer to the inner capillary during electrophoresis.…”

Section: Introductionmentioning

confidence: 99%

Microelectrophoresis platform for fast serial analysis of single cells

2010

Self Cite

View full text Add to dashboard Cite

A capillary-based microelectrophoresis platform for fast serial analysis of single cells is described. In this system, the capillary remains fixed and a two-channel flow system is used to rapidly switch the buffer surrounding the capillary inlet from a physiological buffer to an electrophoretic buffer. Single cells are retained in the physiologic-buffer channel utilizing an array of cell microwells patterned into the channel floor. The defined addresses of the cells on the array enable the sequential delivery of individual cells to the inlet of the capillary, where a focused laser pulse lyses the cell. The cell chamber is moved along a preordained route so that the inlet of the capillary is located in the electrophoresis buffer for separation and the physiological buffer during cell sampling. The throughput of the current system is limited by peak overlap between successive samples. Key characterizations of this system including the fluid flow rates, the cell array dimensions, and laser energies were performed. To demonstrate this system, 28 cells loaded with Oregon green and fluorescein were serially analyzed in under 16 min, a rate of 1.8 cells/min.

show abstract

“…However, conventional western blots require large numbers of cells and also entail disruption of the cell-matrix interactions by proteolytic and/or harsh mechanical conditions. Efforts to append an electrophoretic (capillary) separation to adherent cell lysis have shown promise; 32 however, serial cell analysis limits throughput. Moreover, in all the aforementioned assays, protein isoforms that have small molecular mass differences are not separable.…”

Section: Introductionmentioning

confidence: 99%

Electrophoretic cytometry of adherent cells

Herr

2017

Lab Chip

View full text Add to dashboard Cite

Cell-matrix and cell-cell interactions influence intracellular signalling and play an important role in physiologic and pathologic processes. Detachment of cells from the surrounding microenvironment alters intracellular signalling. Here, we demonstrate and characterise an integrated microfluidic device to culture single and clustered cells in tuneable microenvironments and then directly analyse the lysate of each cell in situ, thereby eliminating the need to detach cells prior to analysis. First, we utilise microcontact printing to pattern cells in confined geometries. We then utilise a microscale isoelectric focusing (IEF) module to separate, detect, and analyse lamin A/C from substrate-adhered cells seeded and cultured at varying (500, 2000, and 9000 cells per cm2) densities. We report separation performance (minimum resolvable pI difference of 0.11) that is on par with capillary IEF and independent of cell density. Moreover, we map lamin A/C and β-tubulin protein expression to morphometric information (cell area, circumference, eccentricity, form factor, and cell area factor) of single cells and observe poor correlation with each of these parameters. By eliminating the need for cell detachment from substrates, we enhance detection of cell receptor proteins (CD44 and β-integrin) and dynamic phosphorylation events (pMLCS19) that are rendered undetectable or disrupted by enzymatic treatments. Finally, we optimise protein solubilisation and separation performance by tuning lysis and electrofocusing (EF) durations. We observe enhanced separation performance (decreased peak width) with longer EF durations by 25.1% and improved protein solubilisation with longer lysis durations. Overall, the combination of morphometric analyses of substrate-adhered cells, with minimised handling, will yield important insights into our understanding of adhesion-mediated signalling processes.

show abstract

Coaxial Flow System for Chemical Cytometry

Cited by 17 publications

References 48 publications

Capillary Electrophoresis in Bioanalysis

Capillary Electrophoresis in Bioanalysis

Microelectrophoresis platform for fast serial analysis of single cells

Electrophoretic cytometry of adherent cells

Contact Info

Product

Resources

About