Introduction

This work presents a microfluidic device that has the unique capability of automating combinatorial processes such as protein refolding and bioconjugate synthesis. Conventionally, these processes are done by hand-pipetting or using robotic systems. The microfluidic approach offers the advantages of automation, cost-effectiveness, compatibility with optical detection, and a million-fold reduction in sample volumes. Successful devices will greatly reduce the cost of realizing biopharmaceuticals.

In this work, we develop a device designed specifically for protein refolding applications. Protein refolding has been a bottleneck in the production of biopharmaceuticals on a large scale. In order to maximize the production of proteins, bacterial hosts are induced with promoters, resulting in the production of aggregated, over-expressed proteins. The process of refolding, which involves the conversion of these inactive aggregated proteins into their functional native state, is a highly empirical process. Determination of solution conditions to properly refold a protein is a combinatorial process which can be automated using our microfluidic device.

This microfluidic approach also has potential applications in bioconjugate synthesis for gene silencing. Short-interfering RNAs (siRNA) are small nucleotide chains which associate with the corresponding mRNA and suppress the expression of specific disease-causing proteins. Introduction of siRNA into cells needs a delivery vector. The combinatorial process of creating polymeric delivery vectors can be automated using our microfluidic device.

Lysozyme Refolding

Lysozyme refolding has been chosen as the model system for evaluating device performance, as its refolding protocols and activity assays have been widely developed [1]. We implement a combinatorial protocol using 16 refolding chaperones (Table 1). The inactive aggregated lysozyme is solubilized in a strong denaturing medium (8M Guanidinium Chloride, 1mM EDTA, 50mM Tris HCl and 16mM Dithiothreitol). This solubilized lysozyme along with the denaturants is diluted in a renaturing medium, which consists of the renaturing buffer (50mM Tris HCl, 1mM EDTA, 5mM Glutathione, 2 mM Dithiothreitol, 0.85M Guanidinium Chloride) and a mixture of three artificial chaperones, chosen combinatorially. The 16 chaperones are used in two different concentrations and this results in a total of 4480 combinations.

Reagent aliquoting and mixing is achieved using microfluidic control [2]. The device consists of two layers of PDMS (Poly (dimethylsiloxane)) on a glass slide. The schematic of the channel layout is shown in Figure 1. The layout consists of input channels for protein and reagent solutions, an annular mixer and an output channel for the refolded protein. High pressure air is passed through control channels to control fluid flow in flow channels. The device is integrated with a control unit which enables automation of the processes (Figure 2).

Results and Discussion

Peristaltic pumps present on each flow channel allow precise control of the amount of fluid through the channel. The flow rate of the fluid in the flow channel depends on the frequency of actuation of the control channels. The volume injection rate as a function of the actuation frequency was calibrated by monitoring the dye-water interface (Figure 3). Dye-water mixing experiments performed at different frequencies resulted in a maximum flow rate of 1.07 nL/s.  This corresponds to an actuation frequency of 25 Hz and a mixing time of 45s.

Preliminary experiments with protein solutions were performed on a three-input device (Figure 4). A solution of the solubilized lysozyme in denaturing medium was metered through one input. Solutions of urea (46.88mg/mL) and guanidinium chloride (55.96 mg/mL), used as the artificial chaperones, were prepared independently in the renaturing buffer and were metered through the other two inputs. All the flow channels connected to the annular mixer were closed with control valves and the peristaltic pump on the annulus was actuated to mix the solutions. A solution of partially refolded lysozyme was obtained from the output channel.

Future work includes thorough calibration of the flow channels to meter precise fluid volumes into the mixer and scaling up of the device to include more chaperone inputs and combinatorial capabilities.

Conclusion

In conclusion, by integrating the control capabilities with the PDMS device, we can facilitate automation of combinatorial protein refolding protocols. The system is compatible with on-chip detection using terahertz spectroscopy [3], which gives the potential for real-time monitoring of kinetic intermediates.

 

Lithium Chloride	α – Cyclodextrin
Sodium Chloride	L – Arginine
Sodium Sulfate	Urea
Glycerin	Guanidinium Chloride
Sorbitol	Polysorbate 20
Methylcellulose	Polysorbate 80
PEG	CHAPS
Dextran	Triton X-100

Table 1: 16 chaperones at two different concentrations are used in refolding lysozyme.

Figure 1(a): Schematic of the channel layout with the red lines representing flow channels and the green lines representing control channels. (b): Peristaltic pump action in the device. (c): Valve action in the device. The microfluidic device is a two-layered PDMS (Poly (dimethylsiloxane)) on glass device. (1 - 5 mm thick flow layer made of PDMS; 2 - 30 μm thin control layer made of PDMS; 3 – Glass).

Figure 2: Microfluidic device integrated to an eight-valve manifold through tygon tubing. This manifold is supplied with shop air and is controlled through a Fluidigm circuit which is operated using a Lab-View interface.

Figure 3: Volume injection rate in a flow channel plotted against the frequency of actuation of control channels. The injection rate is calculated from the flow velocity which is estimated by monitoring the dye-water interface in the flow channel.

Figure 4: Micrograph of the device with three inputs (1, 2 and 3), the annular mixer and an output (4). The mixer is controlled by a peristaltic pump. A mixture of solubilized lysozyme along with the denaturing medium (8M Guanidinium Chloride, 1mM EDTA, 50mM Tris HCl and 16mM Dithiothreitol) is metered through input 1. Stock solutions of Urea (chaperone 1) and Guanidinium Chloride (chaperone 2) prepared independently in the renaturing buffer (50mM Tris HCl, 1mM EDTA, 5mM Glutathione, 2 mM Dithiothreitol, 0.85M Guanidinium Chloride) are metered through inputs 2 and 3.

References:

1. Hevehan D.L., et al., Oxidative renaturation of lysozyme at high concentrations. Biotechnology and Bioengineering, 1997. 54(3): 221-230

2. Unger, M.A., et al., Monolithic microfabricated valves and pumps by multilayer soft lithography. Science (Washington, D. C.), 2000. 288(5463): 113-116.

3. George PA, Rana F, Erickson D, Kirby BJ, Terahertz microfluidics for on-chip detection and identification of bio-molecular compositions and conformations IEEE-LEOS Summer Topicals: Optofluidics, Quebec City, July 2006.