Oil-sealed femtoliter fiber-optic arrays for single molecule analysis

Abstract

This paper describes a novel method for fabricating and sealing high-density arrays of femtoliter reaction chambers. We chemically etch one end of a 2.3 mm diameter glass optical fiber bundle to create an array of microwells. We then use a contact printing method to selectively modify the surface of the material between microwells with a hydrophobic silane. This modification makes it possible to fill the wells with aqueous solution and then seal them with a droplet of oil, forming an array of isolated reaction chambers. Individual β-galactosidase molecules trapped in these reaction chambers convert a substrate into a fluorescent product that can be readily detected because a high local concentration of product is achieved. This binary readout can be used for ultra-sensitive measurements of enzyme concentration. We observed that the percentage of wells showing enzyme activity was linearly dependent on the concentration of soluble β-galactosidase in the picomolar range. A similar response was also observed for streptavidin-β-galactosidase captured by biotinylated beads. These arrays are also suitable for performing single-molecule kinetics studies on hundreds to thousands of enzyme molecules simultaneously. We observed a broad distribution of catalytic rates for individual β-galactosidase molecules trapped in the microwells, in agreement with previous studies using similar arrays that were mechanically sealed. We have further demonstrated that this femtoliter fiber-optic array can be integrated into a PDMS microfluidic channel system and sealed with oil on-chip, creating an easy to use and high-throughput device for single-molecule analysis.

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

“…A similar microsphere-based protein array can be used on other platforms such as microfluidic devices. The encoding and immobilized methods could also be applied to microspheres made with other materials 9 . Table 4.…”

“…Antibody microarrays capable of simultaneously detecting multiple proteins in diagnostic fluids, such as blood, have important applications in clinical diagnostics and biomarker screening [2][3][4][5][6][7][8][9][10] . Saliva, containing many of the same analytes as blood, has been considered as a preferable alternative to blood because saliva collection is safe, noninvasive, and can be carried out by minimally-trained medical personnel [11][12][13] .…”

Salivary Diagnostics Using a Portable Point-of-Service Platform: A Review

“…SiMoA technology was developed in our laboratory and recently extended to nucleic acids, thereby providing a sensitive detection alternative to existing technologies that utilize amplification, such as the polymerase chain reaction (PCR) (53). To summarize, SiMoA technology is being used to detect virus particles, proteins, and nucleic acid biomarkers (52, 54) and has the potential to improve the sensitivity of our POS device for future applications.…”

Oil-Isolated Hydrogel Microstructures for Sensitive Bioassays On-Chip

“…For example, femtoliter-sized microwells, which are large enough to hold a single 3 – 5 micron diameter bead, have been fabricated using etched optical-fiber bundles or injection molding of polymers. 5–7, 12 In other systems, similarly sized bead-filled droplets have been arrayed on hydrophobic surfaces patterned with hydrophilic wells. 8–9 Individual beads with target-enzyme complexes and the enzymatic substrate solution are then confined into the compartments and sealed using mechanical force or, in more recent work, inert fluorinated oil.…”