Comparing microfluidic performance of three-dimensional (3D) printing platforms

Three-dimensional (3D) printing has emerged as a potential revolutionary technology for the fabrication of microfluidic devices. A direct experimental comparison of the three 3D printing technologies dominating microfluidics was conducted using a Y-junction microfluidic device, the design of which was optimized for each printer: fused deposition molding (FDM), Polyjet, and digital light processing stereolithography (DLP-SLA). Printer performance was evaluated in terms of feature size, accuracy, and suitability for mass manufacturing; laminar flow was studied to assess their suitability for microfluidics. FDM was suitable for microfabrication with minimum features of 321 ± 5 μm, and rough surfaces of 10.97 μm. Microfluidic devices >500 μm, rapid mixing (71% ± 12% after 5 mm, 100 μL/min) was observed, indicating a strength in fabricating micromixers. Polyjet fabricated channels with a minimum size of 205 ± 13 μm, and a surface roughness of 0.99 μm. Compared with FDM, mixing decreased (27% ± 10%), but Polyjet printing is more suited for microfluidic applications where flow splitting is not required, such as cell culture or droplet generators. DLP-SLA fabricated a minimum channel size of 154 ± 10 μm, and 94 ± 7 μm for positive structures such as soft lithography templates, with a roughness of 0.35 μm. These results, in addition to low mixing (8% ± 1%), showed suitability for microfabrication, and microfluidic applications requiring precise control of flow. Through further discussion of the capabilities (and limitations) of these printers, we intend to provide guidance toward the selection of the 3D printing technology most suitable for specific microfluidic applications

Exploring chip-capillary electrophoresis-laser-induced fluorescence field-deployable platform flexibility: Separations of fluorescent dyes by chip-based non-aqueous capillary electrophoresis

Microfluidic chip electrophoresis (chip-CE) is a separation method that is compatible with portable and on-site analysis, however, only few commercial chip-CE systems with laser-induced fluorescence (LIF) and light emitting diode (LED) fluorescence detection are available. They are established for several application tailored methods limited to specific biopolymers (DNA, RNA and proteins), and correspondingly the range of their applications has been limited. In this work we address the lack of commercially available research-type flexible chip-CE platforms by exploring the limits of using an application-tailored system equipped with chips and methods designed for DNA separations as a generic chip-CE platform - this is a very significant issue that has not been widely studied. In the investigated Agilent Bioanalyzer chip-CE system, the fixed components are the Agilent chips and the detection (LIF at 635nm and LEDIF at 470nm), while the chemistry (electrolyte) and the programming of all the high voltages are flexible. Using standard DNA chips, we show that a generic CE function of the system is easily possible and we demonstrate an extension of the applicability to non-aqueous CE (NACE). We studied the chip compatibility with organic solvents (i.e. MeOH, ACN, DMF and DMSO) and demonstrated the chip compatibility with DMSO as a non-volatile and non-hazardous solvent with satisfactory stability of migration times over 50h. The generic CE capability is illustrated with separations of fluorescent basic blue dyes methylene blue (MB), toluidine blue (TB), nile blue (NB) and brilliant cresyl blue (BC). Further, the effects of the composition of the background electrolyte (BGE) on the separation were studied, including the contents of water (0-30%) and buffer composition. In background electrolytes containing typically 80mmol/L ammonium acetate and 870mmol/L acetic acid in 100% DMSO baseline separation of the dyes were achieved in 40s. Linearity was documented in the range of 5-28μmol/L, 10-100μmol/L, 1.56-50nmol/L and 5-75nmol/L (r2 values in the range 0.974-0.999), and limit of detection (LOD) values were 90nmol/L, 1μmol/L 1.4nmol/L, and 2nmol/L for MB, TB, NB and BC, respectively. © 2013 Elsevier B.V

The necessity of and strategies for improving confidence in the accuracy of western blots

Western blotting is one of the most commonly used laboratory techniques for identifying proteins and semi-quantifying protein amounts, however, several recent findings suggest that western blots may not be as reliable as previously assumed. This is not surprising since many labs are unaware of the limitations of western blotting. In this manuscript we review essential strategies for improving confidence in the accuracy of western blots. These strategies include selecting the best normalization standard, proper sample preparation, determining the linear range for antibodies and protein stains relevant to the sample of interest, confirming the quality of the primary antibody, preventing signal saturation and accurately quantifying the signal intensity of the target protein. Although western blotting is a powerful and indispensable scientific technique that can be used to accurately quantify relative protein levels, it is necessary that proper experimental techniques and strategies are employed

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist