Single-cell analysis has attracted increasing attention because of cell heterogeneities. Various strategies have been developed for analyzing single cells, but most of these analytical processes kill the cells. Tools that can qualitatively and quantitatively measure the cellular contents without killing the cell are highly demanding because they enable us to conduct single-cell time-course studies (e.g., to examine how a cell responds to a therapy before, during, and after a treatment). Here we develop a femto-liter (fL) pipet to serve this purpose. To ensure that we can accurately and precisely pipet fL solutions, we fill all conduits with liquid and use an electroosmotic pump (EOP) as the driving force to facilitate withdrawal of cellular contents from single cells. We tentatively term this device an EOP-driven pipette or EDP. We characterize the EDP for accurately and precisely withdrawing solution from ∼250 fL to 80 nL; a volume range that covers the applications for most types of cells. To demonstrate the feasibility of utilizing the EDP for a single-cell time-course study, we utilize the EDP to take the cellular contents out at different times during the course of a zebrafish embryo development for cholesterol measurements. More than 50% of the embryos survive after each pipetting and analysis step, and this number will increase considerably as we improve our cell manipulation skills and reduce the pipet-tip diameter. We expect this EDP to become an effective tool for single-cell time-course studies.
Here,
we construct an open-channel on-chip electroosmotic pump capable of
generating pressures up to ∼170 bar and flow rates up to ∼500
nL/min, adequate for high performance liquid chromatographic (HPLC)
separations. A great feature of this pump is that a number of its
basic pump units can be connected in series to enhance its pumping
power; the output pressure is directly proportional to the number
of pump units connected. This additive nature is excellent and useful,
and no other pumps can work in this fashion. We demonstrate the feasibility
of using this pump to perform nanoflow HPLC separations; tryptic digests
of bovine serum albumin (BSA), transferrin factor (TF), and human
immunoglobulins (IgG) are utilized as exemplary samples. We also compare
the performance of our electroosmotic (EO)-driven HPLC with Agilent
1200 HPLC; comparable efficiencies, resolutions, and peak capacities
are obtained. Since the pump is based on electroosmosis, it has no
moving parts. The common material and process also allow this pump
to be integrated with other microfabricated functional components.
Development of this high-pressure on-chip pump will have a profound
impact on the advancement of lab-on-a-chip devices.
Laser-induced fluorescence (LIF) detectors for low-micrometer and sub-micrometer capillary on-column detection are not commercially available. In this paper, we describe in details how to construct a confocal LIF detector to address this issue. We characterize the detector by determining its limit of detection (LOD), linear dynamic range (LDR) and background signal drift; a very low LOD (~70 fluorescein molecules or 12 yoctomole fluorescein), a wide LDR (greater than 3 orders of magnitude) and a small background signal drift (~1.2-fold of the root mean square noise) are obtained. For detecting analytes inside a low-micrometer and sub-micrometer capillary, proper alignment is essential. We present a simple protocol to align the capillary with the optical system and use the position-lock capability of a translation stage to fix the capillary in position during the experiment. To demonstrate the feasibility of using this detector for narrow capillary systems, we build a 2-μm-i.d. capillary flow injection analysis (FIA) system using the newly developed LIF prototype as a detector and obtain an FIA LOD of 14 zeptomole fluorescein. We also separate a DNA ladder sample by bare narrow capillary - hydrodynamic chromatography and use the LIF prototype to monitor the resolved DNA fragments. We obtain not only well-resolved peaks but also the quantitative information of all DNA fragments.
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