High-throughput Protein Expression Generator Using a Microfluidic Platform

Summary

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Abstract

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays¹. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration^2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein ⁴, protein-RNA⁵ or protein-DNA⁶ interactions.

The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins⁷, DNA⁸, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

Protocol

1. Device Fabrication

1. Purchased DTPA-D SU-8 control mold and SPR220-7 flow mold from the Stanford Microfluidics Foundry (www.stanford.edu/group/foundry).
2. Expose the silicone molds to chlorotrimethylsilane (TMCS) vapor for 10 min to promote elastomer release after the baking steps⁹.
3. Prepare a mixture of silicone based elastomer and curing agent (mix well) in two different ratios 5:1 and 20:1 for the control and flow molds, respectively. The different ratios are necessary for proper bonding of multiple layers.
4. Pour the 5:1 PDMS on the control....

Discussion

In this paper we present a method for generation protein arrays in high-throughput using a microfluidic platform. The array generation is based on microarray printing of DNA templates and in vitro protein expression from the DNA within the microfluidic device.

Our novel microfluidic platform has several important advantages over currently used methods, which make it a promising and general tool for proteomics. One advantage is with membrane-bound proteins. In vitro protein s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PDMS- SYLGARD 184	Dow Corning USA	ESSEX-DC
Chlorotrimethylsilane (TMCS	Sigma-Aldrich	C72854
Epoxy coated glass substrates	CEL Associates USA	VEPO-25C
Poly ethylene glycole (PEG)	Sigma-Aldrich	81260
D-trehalose dihydrate	Sigma-Aldrich	T9531
Biotinylated-BSA	Pierce, Thermo Scientific	PIR-29130
Neutravidin	Pierce, Thermo Scientific	31050
penta-His-biotin	Qiagen	34440
Hepes	Biological Industries	03-025-1B
TNT-T7	Promega Corp.	L5540
C-myc Cy3 antibody	Sigma-Aldrich
Control box	Stanford Microfluidics Foundry
Mold	Stanford Microfluidics Foundry
Pin	New England Small Tubes Corporation
Tygon microbore tubing	Tygon	S-54-HL
Microarrayer	Bio Robotics	MicroGrid 610
Silicone pins	Parallel Synthesis	SMT-S75