284605 A Peg-DA Microfluidic Device for the Study of Cellular Chemotaxis

Monday, October 29, 2012
Hall B (Convention Center )
Mahama A. Traore and Bahareh Behkam, Mechanical Engineering, Virginia Tech, Blacksburg, VA

A PEG-DA MICROFLUIDIC DEVICE FOR THE STUDY OF

CELLULAR CHEMOTAXIS

 

Mahama A. Traore1, Bahareh Behkam1, 2

  1. Mechanical Engineering Department, Virginia Tech, Blacksburg, VA
  2. School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA

Corresponding Author: Bahareh Behkam, Email: Behkam@vt.edu

  <>INTRODUCTION

Microfluidic devices have been extensively used for the study of both prokaryotic and eukaryotic cells under well-defined chemical and mechanical conditions. In many cases, the establishment of steady-state (temporally invariant) spatially varying chemical gradients within which cells can be studied over extended periods of time and in absence of any fluid flow is of outmost importance.

In this work, we have microfabricated a microfluidic device made of the biocompatible and photopolymerizable polyethylene glycol diacrylate (PEG-DA). We have demonstrated that PEG-DA of different molecular weight can be used in order to achieve customizable chemical gradients. This microfluidic device is composed of three channels fabricated using photolithography methods and operates under the principle of diffusion as shown in Fig 1.

Figure 1. Schematic showing a cross-section of microdevice and SEM image of a 2000 Da molecular weight porous gel

A solution of a chemical agent of choice (e.g. casamino acids) in a buffer (e.g. PBS) is flowed in one of the outer channels while just the buffer solution is flowed in the other outer channel. Cells (i.e. subject of study) reside in the buffer solution in the center channel. The chemical agent diffuses through the porous gel wall to the center channel, generating a well-defined spatially varying concentration field. The gel wall thickness and porosity can be designed to accommodate for the required chemical diffusion rate. COMSOL multiphysics simulations, diffusion coefficient measurements by optical spectrophotometry and diffusion characterization using fluorescence microscopy were employed to develop custom-devices with desired gel wall characteristics to determine the threshold chemical gradient at which Escherichia coli (E. coli) strain RP437 begin to exhibit a chemotactic behavior towards casamino acids. <>  <>METHODOLOGY

 

Fabrication of microfluidic device

The microfluidic device is fabricated using a two-step photolithography process (Fig 2). Briefly, the glass surface was first rendered hydrophilic using oxygen plasma for duration of 5 minutes and then functionalized using a TPM (3-(Trichlorosilyl)propyl methacrylate) solution. PEG-DA gels of a desired molecular weight (700 Da, 2000 Da, 6000 Da or 10000 Da) were exposed to UV light (EFOS Ultracure, UV spot lamp, Mississauga, ON, Canada) at a wavelength of 365nm for 20 s through a photomask with the chosen pattern.

 

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Figure 2. Process flow fabrication of patterned PEG-DA gel in microfluidic device

Figure 3. Image of a 700 Da MW PEG-DA microfluidic device. For demonstration purposes, red and green fluids are flown in the two outer channels.

The fabricated gel pattern is assembled with the other layers of the device; Figure 3 shows an assembled microfluidic chemotaxis assay device.

Diffusion properties characterization

The diffusion coefficients of the desired chemical agent through the fabricated gels were determined using a Franz diffusion cell1 as seen in Fig. 4. Known concentration of a chemical agent in buffer present in the donor compartment diffuses over time across the porous PEG-DA membrane and into the acceptor chamber. Samples from the acceptor compartment are collected over time and their absorbance was measured using a UV spectrophotometer. For chemical agents that cannot be detected by spectrophotometry, the chemical gradient in the center channel of the microfluidic device was quantified using a different method2. A fluorescent dye solution of known concentration with molecular weight close to the molecular weight of the chemical agent of interest was flown in the outer channel and diffused through the PEG-DA porous walls to the center channel. Fluorescence microscopy images were used to find the chemical concentration profiles within the center channel and thus the diffusion coefficient values. A picture of the fluorescence profile within the center channel can be seen in Fig 4.

Figure 4. Franz diffusion cell used for diffusion coefficient measurements.

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Figure 5. Fluorescent gradient profile across microfluidic channel using a 700 Da molecular weight gel

  RESULTS The diffusion coefficient of casamino acid through PEG-DA gels of four different molecular weights (700 Da, 2000 Da, 6000 Da, and 10000 Da) were characterized using a Franz diffusion cell as described above. The diffusion coefficient of the gels was calculated using the mean first passage time measurements1 method; the results are summarized in Table 1.

Table 1. Diffusion coefficient of casamino acid through PEG-DA gel as a function of the gel molecular weight.

PEG-DA gel (Da)

700

2000

6000

10000

Diffusion coefficient

(10-6cm2/s)

1.5±0.5

2.66±0.53

3.61±0.9

5.19±0.98

COMSOL simulations were run to determine the chemical gradient profile in the center channel where the cells will be inserted. The chemical gradient profile in the center channel depends on the dimensions of the device, the molecular weight of the gel used, and the initial chemical concentration in the source channel. In these simulations, all other parameters were kept the same except the molecular weight of the PEG-DA gel used to fabricate the device.

The initial chemical concentration of the chemical diffusing from the source channel was 3.8410-4 M, and the chemical concentration in the buffer channel was maintained at zero. The chemical concentration gradient in the center channel after 1 hour is shown in Fig. 5. As expected the more porous gels have a higher chemical gradient slope. This can be attributed to the fact that the chemical in the source channel diffuses at a faster rate to the center channel, thus creating a steeper linear gradient profile.

Figure 6. Chemical concentration across center channel of microfluidic device

The chemical gradient values obtained above were verified experimentally using a fluorescein solution of identical initial concentration in the source channel. Fluorescent images of the center channel of the device were taken and analyzed using Image J (free software provided by the NIH). Fluorescein gradients within the center channel were then determined and compared within 20% of the chemical concentration gradients obtained through simulations. Table 2 demonstrates the results from the simulations and the experiments. The differences between the experimental and theoretical values can be attributed to the fact that the molecular weight of fluorescein (332 Da) used in the fluorescent quantification of the diffusion coefficient is different from the molecular weight of the casamino acids (250 Da).

Table 2. Comparison between experimental and simulation chemical gradients in the center channel.

A chemotaxis sensitivity assay was conducted using E. coli bacteria strain RP437 (fluorescent bacteria) towards casamino acids. The molecular weight of the gel used in this experiment was 700Da. The lowest molecular weight gel was chosen for this experiment because of the low chemical gradient it generates in the center channel. E. coli bacteria positively responded to the chemical attractant present in the source channel. Fig 6. shows the E. coli bacteria response towards casamino acids in the center channel.

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Figure 7. Response of E. coli bacteria strain RP437 in center channel to a gradient of casamino acids.

DISCUSSION

The fabrication of a microfluidic device with different gradient profiles can be achieved using different molecular weight gels. Bacterial chemotaxis studies usually require smaller chemical gradients to be established in the center channel. The use of the 700 Da molecular weight PEGDA gel can accommodate for the chemotaxis study of bacteria. The ability to control the chemical gradient in the center channel can help determine the threshold chemical gradient needed to provoke the chemotactic behavior of a strain of bacteria as well as the saturation chemical concentration.

CONCLUSIONS

In this work, multiple microfluidic devices were fabricated using a photopolymerizable hydrogel PEG-DA at different molecular weights. Different gel structures lead to significantly different diffusion coefficients and therefore distinctly different chemical gradient field within the main channel. The capability of realizing well-defined and customizable steady-state chemical gradient fields in absence of fluid flow opens many opportunities for the study of various behaviors of bacteria and mammalian cells within a controlled environment.     REFERENCES

1. Wenquan Liang et al. Journal Chem Phys, 2006, 125, 0447071-0447077.

2. Mingming Wu et al. Lab on a Chip, 2007, 7, 763-769.

 


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