DIFFUSIONAL MICROTITRATION - REAGENT DELIVERY BY A DIFFUSIONAL MICROBURET INTO MICROSCOPIC SAMPLES

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 of the tip of the pipet. Consequences are as follows: (1) there is no need for the membrane material to exhibit low, rate limiting diffusivity to ensure reproducible reagent delivery, and (2) the delivery rate is not affected by membrane thickness, These predictions were experimentally verified using acid/base titrations of nano-, pico-, and femtoliter samples. DMB's are used for analysis, or fine chemical manipulation, of tiny droplets, and for reagent/drug delivery into single biological cells.