A microfluidic dual capillary probe to collect messenger RNA from adherent cells and spheroids.
ABSTRACT Collection of bioanalytes from single cells is still a challenging technology despite the recent progress in many integrated microfluidic devices. A microfluidic dual capillary probe was prepared from a theta (theta)-shaped glass capillary to analyze messenger RNA (mRNA) from adherent cells and spheroids. The cell lysis buffer solution was introduced from the injection aperture, and the cell-lysed solution from the aspiration aperture was collected for further mRNA analysis based on reverse transcription real-time PCR. The cell lysis buffer can be introduced at any targeted cells and never spilled out of the targeted area by using the microfluidic dual capillary probe because laminar flow was locally formed near the probe under the optimized injection/aspiration flow rates. This method realizes the sensitivity of mRNA at the single cell level and the identification of the cell types on the basis of the relative gene expression profiles.
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ABSTRACT: The microfluidic probe (MFP) is a non-contact, scanning microfluidic technology for local (bio)chemical processing of surfaces based on hydrodynamically confining nanoliter volumes of liquids over tens of micrometers. We present here a compact MFP (cMFP) that can be used on a standard inverted microscope and assist in the local processing of tissue sections and biological specimens. The cMFP has a footprint of 175 × 100 × 140 mm3 and can scan an area of 45 × 45 mm2 on a surface with an accuracy of ±15 μm. The cMFP is compatible with standard surfaces used in life science laboratories such as microscope slides and Petri dishes. For ease of use, we developed self-aligned mounted MFP heads with standardized “chip-to-world” and “chip-to-platform” interfaces. Switching the processing liquid in the flow confinement is performed within 90 s using a selector valve with a dead-volume of approximately 5 μl. We further implemented height-compensation that allows a cMFP head to follow non-planar surfaces common in tissue and cellular ensembles. This was shown by patterning different macroscopic copper-coated topographies with height differences up to 750 μm. To illustrate the applicability to tissue processing, 5 μm thick M000921 BRAF V600E+ melanoma cell blocks were stained with hematoxylin to create contours, lines, spots, gradients of the chemicals, and multiple spots over larger areas. The local staining was performed in an interactive manner using a joystick and a scripting module. The compactness, user-friendliness, and functionality of the cMFP will enable it to be adapted as a standard tool in research, development and diagnostic laboratories, particularly for the interaction with tissues and cells.Review of Scientific Instruments 01/2014; 85(3):034301-034301-9. · 1.60 Impact Factor
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ABSTRACT: Neuropeptides are cell to cell signaling molecules that modulate a wide range of physiological processes. Neuropeptide release has been studied in sample sizes ranging from single cells and neuronal clusters, to defined brain nuclei and large brain regions. We have developed and optimized cell stimulation and collection approaches for the efficient measurement of neuropeptide release from neuronal samples using a dual capillary system. The defining feature is a capillary that contains octadecyl-modified silica nanoparticles on its inner wall to capture and extract releasates. This collection capillary is inserted into another capillary used to deliver solutions that chemically stimulate the cells, with solution flowing up the inner capillary to facilitate peptide collection. The efficiency of peptide collection was evaluated using six peptide standards mixed in physiological saline. The extracted peptides eluted from these capillaries were characterized via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with low femtomole detection limits. Using the capillary collection system in small custom-fabricated culturing chambers, individual cultured neurons and neuronal clusters from the model animal Aplysia californica were stimulated with distinct neuronal secretagogues and the releasates were collected and characterized using MALDI-TOF MS.The Analyst 09/2013; · 4.23 Impact Factor
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ABSTRACT: We devised, implemented, and tested a new concept for efficient local surface chemistry that we call hierarchical hydrodynamic flow confinement (hierarchical HFC). This concept leverages the hydrodynamic shaping of multiple layers of liquid to address challenges inherent to microscale surface chemistry, such as minimal dilution, economical consumption of reagent, and fast liquid switching. We illustrate two modes of hierarchical HFC - nested and pinched - by locally denaturing and recovering a 26 bp DNA with as little as 2% dilution and by efficiently patterning an antibody on a surface, with 5 μm resolution and a 100-fold decrease of reagent consumption compared to microcontact printing. In addition, valve-less switching between nL volumes of liquids was achieved within 20 ms. We believe hierarchical HFC will have broad utility for chemistry on surfaces at the microscale.Langmuir 03/2014; · 4.38 Impact Factor