Microfluidic Assays for DNA Manipulation Based on a Block Copolymer Immobilization Strategy

Abstract

Methods to manipulate and visualize isolated DNA and oligonucleotide strands are important for investigation of their biophysics as well as their interactions with proteins. Herein, we report such a method by combining a block copolymer surface functionalization strategy with microfluidics. The copolymer poly(L-lysine-graftpolyethylene glycol) (PLL-g-PEG) coated one surface of the microfluidic channels, rendering it passive to adsorption and thus minimizing any noise arising from nontargeted adsorbed molecules. Single λ-phage DNA molecules were immobilized and were extended by molecular combing. Their extension did not exceed their contour length, which we attribute to the low surface tension of the coated surface. To demonstrate further the applicability of our method, the anchored DNA was extended by hydrodynamic flow. We propose this method for exploring DNA-protein interactions due to the copolymer's enhanced capacity for single-molecule detection, stability under wet or dry conditions, hydrophilicity, full compatibility with microfluidics and simplicity being a one-step process.

“Click” chemistry‐based surface modification of poly(dimethylsiloxane) for protein separation in a microfluidic chip

“…Physisorption methods are simple and rapid. However, these methods are relatively limited since the coated modifiers are not durable during the protein separation [5][6][7]. On the contrary, modification of PDMS microchannels by chemical grafting using polymer brushes exhibits high stability and repeatability [8].…”

Enhancing Single Molecule Imaging in Optofluidics and Microfluidics

“…Physisorption with PEG copolymers is one of the simplest ones, and involves only the incubation of aqueous buffers of the copolymer. Poly( l -lysine)- g -poly(ethylene glycol) (PLL- g -PEG) is one such copolymer, which has found numerous applications in both oxide and PDMS based microfluidics for cell cultures, protein studies and single molecule manipulation and imaging [40,153,158,159] (Figure 7). An alternative PEG surface functionalization is based on the covalent bonding of PEG to albumin microgels, which are recognized by the surface [160].…”

“…Right: An optically magnified image of an individual nucleic acid; the scale bar is 1 μm. In these experiments, the copolymer aided both the prevention of non-specific interactions, but also the stretching of the DNA below its contour length due to the hydrophilic nature of the PEG [40]. …”

Recent advances in single‐molecule detection on micro‐ and nano‐fluidic devices

“…In order to work at the physiological pH 7.4 and to avoid the overstretching of DNA molecules, Dukkipati et al introduced a protein-assisted DNA immobilization and stretching approach, in which the proteins on the DNA-protein complex adsorbed on the channel surface to stretch the DNA under a hydrodynamic flow [103]. In another study, the surface of microchannel was rendered passive to adsorption by functionalizing with a block copolymer, poly(L-lysine-graft-polyethylene glycol), thus minimizing the fluorescence background from the nontargeted adsorbed molecules, and more importantly, the channel surface properties were stable under either wet or dry conditions [104]. Manipulation of DNA molecules has also been demonstrated in free solution without immobilization.…”