On-chip MIC by Combining Concentration Gradient Generator and Flanged Chamber Arrays

Minimum inhibition concentration (MIC) of antibiotic is an effective value to ascertain the agent and minimum dosage of inhibiting bacterial growth. However, current techniques to determine MIC are labor intensive and time-consuming, and require skilled operator and high initial concentration of bacteria. To simplify the operation and reduce the time of inhibition test, we developed a microfluidic system, containing a concentration generator and sub-micro-liter chambers, for rapid bacterial growth and inhibition test. To improve the mixing effect, a micropillar array in honeycomb-structure channels is designed, so the steady concentration gradient of amoxicillin can be generated. The flanged chambers are used to culture bacteria under the condition of continuous flow and the medium of chambers is refreshed constantly, which could supply the sufficient nutrient for bacteria growth and take away the metabolite. Based on the microfluidic platform, the bacterial growth with antibiotic inhibition on chip can be quantitatively measured and MIC can be obtained within six hours using low initial concentration of bacteria. Overall, this microfluidic platform has the potential to provide rapidness and effectiveness to screen bacteria and determine MIC of corresponding antibiotics in clinical therapies.


Microfluidic Chip Preparation
The bottom and top layer were prepared by standard soft lithographic process. The middle layer was borosilicate glass with 36 holes punched by laser, and the culture chamber was 1.2 mm deep, 1 mm diameter. SU-8 2015 and 2050 (Microchem Corp., Newton, MA, USA) were respectively molded to fabricate the bottom layer and top layer. A 10:1 mixer of PDMS (Sylgard 184, Dow Corning, Midland, MI, USA) prepolymer and curing agent were poured onto SU-8 mold after degassing. After baking at 70 °C in oven, the PDMS mold was peeled off and used to bond with glass.
The borosilicate glass with 36 holes was cleaned in organic and metallic contamination in sequence for 5 min in following solutions, H2SO4:H2O2 (4:1), H2O: H2O2: NH4OH (50:10:1), H2O:H2O2: HCl (5:1:1). After washing the substrate, a H2O/H2O2/NH4OH solution (7:2:1) was used to increase the hydrophilicity of the glass plates. The top layer PDMS and the middle layer glass were bounded to each other by using UV o-zone cleaner (Jelight) for 5 min and baking for 70 °C for 15 min. The bottom layer was reversibly adhered to the upper two layers.
In the chamber layer, the diameter of each culture chamber is 1 mm. On the gas channel layer, the length of each side of the gas channel array is 2.8 mm, which is much larger than the culture chamber. The alignment of the two layers is quite easy. Although the diameter of the flanged channel on top of the chamber is 2 mm, which is also larger than the size of the chamber, comparing to the above alignment, it is a little difficult to handle the top layer and the middle layer. The fabricated chips will be checked by the microscope. Some shift of the alignment could be ignored (as shown in Figure 4). The chip with bad alignment cannot be used. Anyway, the fabrication is only suitable for the laboratory.

The Gas Permeation
PDMS is used as the main part of this chip, because it is transparent and gas permeable material, with the performance of gas sorption, diffusion, and permeation, which follow Henry's Law [1,2]. It has a high oxygen diffusivity, which is necessary for bacterial growth [3], and the amount of maximum oxygen Fmax diffusing through the PDMSs can be estimated by the following diffusion equation [4]: Fmax ≈ DPDMS (△C/△z) DPDMS, the diffusivity of oxygen in PDMS is 4.1 × 10 −9 m 2 /s. △C, the oxygen concentration gradient across the PDMS layer is 0.2 mol O 2 /m 3 . △z denotes the thickness of PDMS, which is 5 mm in this design. Fmax is approximated 1.29 × 10 −13 mol/s. On the other hand, it is reported that the oxygen consumption rate of E.coli is 1.78 × 10 −18 mol/cell/s. According to this value, the amount of oxygen in one culture chamber can supply 10 5 cells E.coli to grow. In general, the number of bacteria in one culture chamber with the volume of 1 μL is less than 10 4 cells. Therefore, sufficient oxygen dissolved in PDMS can keep the bacteria in good condition during the culture.

Modeling of bacterial growth curve
We applied Logistic model to bacterial growth curve. The equation: The modified equation [5] is: