Diffusional Microtitration: Reagent Delivery by a Diffusional Microburet into Microscopic Samples

Abstract

Diffusional microtitration can be used to precisely titrate extremely small samples from tens of microliters to a few hundred femtoliters. A cylindrical diffusion membrane of macroscopic dimensions is used for reagent delivery into macroscopic samples (>100 nL). Microscopic samples (<100 nL) are titrated with a microscopic device called a diffusional microburet, DMB. This is a pulled glass capillary filled with the reagent solution and a miniature agar gel plug in its conical tip. Apart from a negligibly short initial transient period, the reagent diffuses through this plug into the sample at a constant rate. This results in a linear DMB calibration with a negligible offset term. This experimental observation is quantitatively predicted by a mathematical model derived in this work for diffusive mass transport through a conical membrane. The delivery rate is limited by the taper angle and inner diameter

Continuous and Quantitative Delivery of Molecules into Individual Cells with a Diffusional Microburet

“…These circumstances taken together will ensure that diffusional transport through the hydrogel plug is at quasi-steady state during the entire delivery process. This has been established in earlier work on DMB delivery into microscopic droplets2 and lends support for the steady state treatment of diffusional delivery in Equation 1.…”

“…In the tip of a pulled micropipette the shape of the gel plug will resemble more closely to a truncated cone (see Figure 1). When such a conical plug is much longer than its radius at the truncated end, the transport coefficient can be simplified2:…”

“…Finally, the mass transport limiting region in the DMB is restricted to within a few micrometers of the conical diffusion plug from the pipette tip for a DMB made with the usual fabrication protocol1. Diffusional relaxation of an eventual transient in the concentration profile over such a small distance happens on the subsecond time scale2. Moreover, intracellular delivery has time constants in the order of minutes.…”

“…Thus concentrations on both sides of the junctions are available and can be correlated with the corresponding states of the gap junctions observed in the recordings. The absolute flux of dye delivery into the primary cell can also be calculated from the intracellular concentration in the impaled cell using Equation 1 if the transport coefficient, B, of the DMB is known from precalibration performed in a droplet of known volume, as described in reference 2. From the integral of this flux the total amount of dye that has passed through the junctions in any given time period into the secondary cell can be derived, provided that the ratio of the volumes of the two cells is made available.…”

Study of magnetohydrodynamic driven flow through LTCC channel with self-contained electrodes

“…More recently, researchers [9][10][11][12][13] have reported the use and construction of MHD micropumps with silicon and ceramic substrates. They have demonstrated movements of liquids around small conduits using MHD micro-pumps.…”