Performance of multistep immunochemical reactions by counterflow isotachophoresis on nitrocellulose membranes—I. Immunoblotting

1000-fold sample focusing on paper-based microfluidic devices

“…16 In the 70's and 80's, renewed interest in ITP led to its first implementation on paper substrates including cellulose acetate membrane (CAM) and filter paper. [17][18][19] ITP has been used to directly focus or separate proteins of interest from urine, 20 as well as to establish electroosmotic flow (EOF) patterns for delivery of target proteins to immunosensing sites. To mitigate excessive Joule heating and evaporation, experimental setups either housed the membrane in a closed chamber, 21,22 used external cooling components as part of their apparatus, 17,21 or used cellogel film which holds high water content in its gel matrix.…”

“…22 In addition, experiments were run at relatively low electric fields, resulting in several hours of analysis time. 20 Other electrokinetic techniques have been demonstrated on paper substrates. Mandal et al 23 demonstrated electrokinetic control of liquid transport in a paper-based device, and Ge et al 24 were the first to demonstrate electrophoretic separation on μPAD.…”

Dynamic control of capillary flow in porous media by electroosmotic pumping

“…45 Integration of EO-pumps for liquid manipulation can potentially enhance the functionality of paper-based devices, reducing assay time, and enabling simple automatic sequential delivery of biological samples, conjugates, reagents, and wash solutions. Furthermore, once the paper is entirely wet, EO-pumping provides a mean to continue flowing liquid as previously shown by Abelev and Karamova 25,26 .…”

“…Abelev and Karamova implemented EO-pumping on cellulose acetate and nitrocellulose porous membranes, as a mechanism for driving immunoreactions. 25,26 In recent years, EO-pumps receive increasing attention in microfluidics since they have several unique features compared to other micropumps: they generate constant pulse-free flows, their flow magnitude and direction are convenient to control, they are readily integrated with lab-on-chip devices, and they have no moving parts. [27][28][29][30][31][32] In contrast to previous work that utilized EO-flow (EOF) to drive flow in already wet membranes, we here investigate the use of EO pumping for dynamic control of capillary flows during the filling process in paperbased devices.…”

New Approaches for the Detection and Characterization of Alpha-Fetoprotein Epitope Variants

“…The separated proteins were transferred onto Hybond C nitrocellulose membrane (NCM; Amersham, UK) by electrotransfer [17] using a Mini Transblot Electrophoretic Transfer Cell (Bio-Rad, USA). After blocking with 0.5% casein, the blots were treated with HRP poly-aAFP by counterflow immunoblotting [18] to detect the total AFP fractions. The antigen-bound HRP poly-aAFP was detected by enhanced chemiluminescence (ECL; Amersham, UK).…”