Platform Development

Our research interests are frequently application-oriented, focusing on cutting-edge technologies and biologically-important analytes. These efforts apply state-of-the-art optical and miniature tools to study fundamental building blocks in chemistry and biology, in particular, single molecules and single cells. We explore and evaluate a wide range of technologies with an interest in addressing practical challenges in throughput, multiplexing, cost, and robustness. We have special interest in high-throughput, high-resolution tools, such as microfluidics, optical tweezers, TIRF, and flow cytometry, that can study and manipulate microscale objects.

Oil Sealing for Single Molecule Analysis

We developed a liquid-based method for sealing femtoliter microwell arrays used for single-molecule analysis. This development may have wide impact in several important factors for a successful implementation of the SiMoA technology, such as increasing robustness of the sealing procedure, lowering equipment cost, and allowing for scale-up. The liquid-based method also promises synergy by combining microwell arrays with other fluid manipulation techniques, such as microfluidics.

We used a contact printing method to selectively modify the surface of the material between the microwells with a hydrophobic silane. This modification made it possible to fill the wells with an 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 converted the substrate into a fluorescent product that can be readily detected because a high local concentration of the product is achieved. We demonstrated that the percentage of wells displaying 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 microwell 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.

oilsealing

Figure 1. Fiber modification and oil sealing. (i-vii) The core material (brown) of the optical fiber bundle is selectively etched to form an array of femtoliter-sized wells (i), and a contact printing method (ii) is used to selectively modify the surface of the cladding material (yellow) with a hydrophobic silane (blue) (iii). The modified fiber is dipped into an aqueous dye solution to fill wells (iv); a drop of excess solution remains on the fiber (v). When the fiber is dipped into a reservoir of oil, the excess solution can be removed by the shear force generated by stirring the oil (vi); the aqueous dye solution is sealed in the wells by a layer of oil (vii). Photograph of a droplet of water applied to a freshly etched fiber; the contact angle is 50° (a); Photograph of a droplet applied to a fiber modified with OTS; the contact angle is 116° (b). Fluorescence micrographs taken before (c), immediately after (d), and 30 minutes after (e) photobleaching a portion of the dye-loaded array with high-intensity light. The bleached pattern (dark area, d-e) remained sharp after 30 minutes, indicating the integrity of sealing between microwells.

Optical Tweezers

Optical tweezers use the energy of a highly focused laser beam to trap small dielectric particles, such as microspheres or cells. The trapped particles can be precisely manipulated within small sample volumes, such as microfluidic channels. We are investigating the use of time-shared optical tweezers to create dynamic arrays of sensing beads. Using galvano-mirrors, the position of the trapping beam can be switched rapidly between different positions, creating an array of optical traps. Each trap can be modified on-the-fly, and is individually addressable. Microspheres functionalized to detect the presence of a wide range of analytes, such as nucleic acids or proteins, can thus be dynamically arrayed, exposed to a sample, and then read out using fluorescence. The bead array can then be released by turning off the trapping source and a new array can be assembled in situ, and analyzed. Using this technique also allows us to manipulate micron sized cells such as E. coli or S. cerevisiae

Publications:

“Oil-sealed femtoliter fiber-optic arrays for single molecule analysis”
H. Zhang ,  S. Nie ,  C.M. Etson ,  R.M. Wang and D.R. Walt, Lab Chip, 2012,12, 2229-2239