A multichannel native fluorescence detection system for capillary electrophoretic analysis of neurotransmitters in single neurons

Lapainis, Theodore; Scanlan, Cory; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

doi:10.1007/s00216-006-0775-9

Cited by 70 publications

(44 citation statements)

References 35 publications

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“…Naphthalene-2,3-dicarboxyaldehyde was used to label amino acids in the cell extract to enable analysis by CE with laser-induced-fluorescence (LIF) detection. Several studies using CE-LIF analysis, focusing among others on analysis of neurotransmitters in single cells, were conducted by the group of Sweedler [29,37]. Chang and Yeung [27] analyzed catecholamines, such as epinephrine and norepinephrine, in single adrenal medullary cells by CE-LIF, taking advantage of the native fluorescence of these compounds.…”

Section: Fluorescence Detectionmentioning

confidence: 99%

Analytical techniques for single-cell metabolomics: state of the art and trends

2010

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Single-cell metabolomics is an emerging field that addresses fundamental biological questions and allows one to observe metabolic phenomena in heterogeneous populations of single cells. In this review, we assess the suitability of different detection techniques and present considerations on sample preparation for single-cell metabolomics. Although targeted analysis of single cells can readily be conducted using fluorescent probes and optical instruments (microscopes, fluorescence detectors), a comprehensive metabolomic approach requires a powerful label-free method, such as mass spectrometry (MS). Mass-spectrometric techniques applied to study small molecules in single cells include electrospray MS, matrix-assisted laser desorption/ionization MS, and secondary ion MS. Sample preparation is an important aspect to be taken into account during further development of methods for single-cell metabolomics.

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Section: Fluorescence Detectionmentioning

confidence: 99%

Analytical techniques for single-cell metabolomics: state of the art and trends

2010

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“…A three-channel LIF detection system was reported (48). The system utilized a single excitation laser emitting at 224 nm; using dichroic mirrors, the fluorescence emission was then spectrally separated into three beams.…”

Section: Detectionmentioning

confidence: 99%

Capillary Electrophoresis in Bioanalysis

2008

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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

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“…13 Because of the ambitious challenge to analyze a complex protein mixture in the minute volume of a single cell lysate, 14 to date mostly single or very few cellular components were investigated and analyzed. Exemplarily, Lapainis et al demonstrated the detection of neurotransmitters, 15 or Wu et al reported the electrophoretic separation of amino acids after cell lysis. 16 Furthermore, quantitative copy numbers of phycobiliproteins (as low as 400 molecules per cell), were obtained via single molecule fluorescence detection.…”

Section: B)mentioning

confidence: 99%

Microfluidic carbon-blackened polydimethylsiloxane device with reduced ultra violet background fluorescence for simultaneous two-color ultra violet/visible-laser induced fluorescence detection in single cell analysis

et al. 2012

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In single cell analysis (SCA), individual cell-specific properties and inhomogeneous cellular responses are being investigated that is not subjected to ensembleaveraging or heterogeneous cell population effects. For proteomic single cell analysis, ultra-sensitive and reproducible separation and detection techniques are essential. Microfluidic devices combined with UV laser induced fluorescence (UV-LIF) detection have been proposed to fulfill these requirements. Here, we report on a novel microfluidic chip fabrication procedure that combines straightforward production of polydimethylsiloxane (PDMS) chips with a reduced UV fluorescence background (83%-reduction) by using PDMS droplets with carbon black pigments (CBP) as additives. The CBP-droplet is placed at the point of detection, whereas the rest of the chip remains transparent, ensuring full optical control of the chip. We systematically studied the relation of the UV background fluorescence at CBP to PDMS ratios (varying from 1:10 to 1:1000) for different UV laser powers. Using a CBP/PDMS ratio of 1:20, detection of a 100 nM tryptophan solution (S/N ¼ 3.5) was possible, providing a theoretical limit of detection of 86 nM (with S/N ¼ 3). Via simultaneous two color UV/VIS-LIF detection, we were able to demonstrate the electrophoretic separation of an analyte mixture of 500 nM tryptophan (UV) and 5 nM fluorescein (VIS) within 30 s. As an application, two color LIF detection was also used for the electrophoretic separation of the protein content from a GFPlabeled single Spodoptera frugiperda (Sf9) insect cell. Thereby just one single peak could be measured in the visible spectral range that could be correlated with one single peak among others in the ultraviolet spectra. This indicates an identification of the labeled protein c-PKC and envisions a further feasible identification of more than one single protein in the future.

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A multichannel native fluorescence detection system for capillary electrophoretic analysis of neurotransmitters in single neurons

Cited by 70 publications

References 35 publications

Analytical techniques for single-cell metabolomics: state of the art and trends

Analytical techniques for single-cell metabolomics: state of the art and trends

Capillary Electrophoresis in Bioanalysis

Microfluidic carbon-blackened polydimethylsiloxane device with reduced ultra violet background fluorescence for simultaneous two-color ultra violet/visible-laser induced fluorescence detection in single cell analysis

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