High-Precision Dispensing of Nanoliter Biofluids on Glass Pedestal Arrays for Ultrasensitive Biomolecule Detection

Precise dispensing of nanoliter droplets is necessary for the development of sensitive and accurate assays, especially when the availability of the source solution is limited. Conventional approaches are limited by imprecise positioning, large shear forces, surface tension effects, and high costs. To address the need for precise and economical dispensing of nanoliter volumes, we developed a new approach where the dispensed volume is dependent on the size and shape of defined surface features, thus freeing the dispensing process from pumps and fine-gauge needles requiring accurate positioning. The surface we fabricated, called a nanoliter droplet virtual well microplate (nVWP), achieves high-precision dispensing (better than ±0.5 nL or ±1.6% at 32 nL) of 20-40 nL droplets using a small source drop (3-10 μL) on isolated hydrophilic glass pedestals (500 μm on a side) bonded to arrays of polydimethylsiloxane conical posts. The sharp 90° edge of the glass pedestal pins the solid-liquid-vapor triple contact line (TCL), averting the wetting of the glass sidewalls while the fluid is prevented from receding from the edge. This edge creates a sufficiently large energy barrier such that microliter water droplets can be poised on the glass pedestals, exhibiting contact angles greater >150°. This approach relieves the stringent mechanical alignment tolerances required for conventional dispensing techniques, shifting the control of dispensed volume to the area circumscribed by the glass edge. The effects of glass surface chemistry and dispense velocity on droplet volume were studied using optical microscopy and high-speed video. Functionalization of the glass pedestal surface enabled the selective adsorption of specific peptides and proteins from synthetic and natural biomolecule mixtures, such as venom. We further demonstrate how the nVWP dispensing platform can be used for a variety of assays, including sensitive detection of proteins and peptides by fluorescence microscopy or MALDI-TOF.