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Hardware

Overview

Project Introduction

Manufacture of microfluidic devices

Chip operation

Summary

1.Overview

To assist in biological design, a high-throughput microfluidic chip was manufactured for directed evolution in fatty acids sensing module. In high-throughput arrays, bacteria that have the target gene required directed evolution will be captured, enabling us to use a single cell as the templet to obtain a mutation library of FadR by error-prone PCR.

This will greatly save the time and cost of experiments and facilitate the quantitative analysis of directed evolution for this is the first step to conduct directed evolution at the single-cell level. If time permits, additional experimental methods will be designed.

For detailed fabrication of microfluidic chips and 3D printed models, see Hardware.

2.Project Introduction

Directed evolution (DE) is a method used in protein engineering that harnesses the power of selection to evolve molecules, typically proteins or nucleic acids, from large, stochastically permuted pools or combinatorial libraries, to have desired properties. In fatty acids sensing module

It mainly comprises the following steps:

(1) Gene diversification by random mutagenesis and/or gene recombination to generate a diverse library of variants;

(2) Screening/selection to obtain variants with improved phenotypes.

(3) The improved variant will serve as a new starting point for the next round of gene diversification.

We have obtained a mutation library by error-prone PCR. In order to turn this process to a single cell level, we designed the microfluidic chip. We use gravity, positive and negative pressures to encapsulate bacteria in water-in-oil droplets in the microarray. Then, beads containing unique primers are merged, separately split and labeled. The barcode is spliced onto the genomic DNA of cells through a single overlapping stretch PCR. EP-PCR (error-prone PCR) was realized by changing the proportion of dNTP and reagent content.

As for the model’s advantages, for one thing, coupled with the fact that we are single-cell capture, we can perform a series of quantitative operations on it to better explore the conditions of directed evolution.

For another, after obtaining the mutation library by using our physical model, we can combine it with the microfluidic chip which has been already designed for expression and screening for directed evolution. [1] That is, after amplification, the droplets will be fused one-to-one with droplets containing a cell-free coupled transcription–translation (IVTT) system and then screened by fluorescence intensity. We plan to do it in the future to completely build an assembly, formation, detection, and screening integration microfluidic system for directed evolution.

Experimental process:

2.1.Oil-phase coated EP-PCR

A. Design and develop open microfluidic array chips
B. Drop beads marked with primers into each array by gravity deposition.
C. Drop single bacteria into the array by negative pressure.
D. Positive and negative pressure interlaced to complete the passage and separation of bacterial lysis fluid, and the reagents required by EP-PCR
E. Form oil-in-water droplets by wrapping them up and down, and complete the directed evolution process outside the equipment

2.2.High-throughput droplet separation

F. After directed evolution of promoters, they will express stronger fluorescent proteins. Droplet fluorescence or absorption can be monitored on the chip, and another set of integrated electrodes can classify droplets in different channels based on readings to screen for droplets that have evolved specifically to target genes.

3.Manufacture of microfluidic devices

3.1. Use computer Aided Design (L-Edit) software (provided as. DWG; See supplementary documentation) prepare microfluidic mask designs. And use lithography machine to print the corresponding chrome plate.

Note: For multilayer microfluidic devices, the corresponding mask contains alignment markers.

The first air chamber:

The second array:

3.2.For each device, make the SU-8 main die as follows (Figure 1A).

The height of the gas chamber is 50μm and the height of the double chip is 40+40.

First. Cleaning silicon wafer time: 2h

1. Take 6 new disposable silicon wafers and 5 glass rods, put them indirectly into a 500ml beaker, and add 500ml water to cover the wafers
2. Put the beaker into the ultrasonic cleaning machine and ultrasonic for 20min
3. Pour out 500ml water and replace it with a new 500ml water
4. Repeat steps (2) and (3) three times

Second. Hot plate dehumidification time: 3.5h

1.Dry four silicon wafers using nitrogen
2.Place the silicon wafer with the mirror facing upwards on the hot plate
3.Set hot plate parameters:

Step	Temperature	Rate of temperature increase	Time
1	150	120	1h
2	28	450	24h

Third.Time of pouring glue evenly: 1h

1.Plasma was used to treat the silicon wafer for 2min
2.Pour microchem 2015 SU8 glue 3ml into two silicon slices, and microchem 2050 SU8 glue 3ml into the other two silicon slices
3.Set the speed of the glue homogenizer

For microchem 2015 SU8:

Step	Rotate speed	Time
1	500	30s
2	500	10s
3	1500	30s

For microchem 2050 SU8:

Step	Rotate speed	Time
1	500	30s
2	500	10s
3	2500	30s

4th.Pre-baking time: 3h

1. Place the silicon wafer on the hot plate with the mirror facing upwards
2. Set hot plate parameters

For microchem 2015 SU8:

Step	Temperature	Rate of temperature increase	Time
1	28	120	1h
2	95	120	5min
3	28	120	24h

For microchem 2050 SU8:

Step	Temperature	Rate of temperature increase	Time
1	28	120	1h
2	65	120	5min
3	95	120	5min
4	28	120	24h

5th.Exposure time: 1.5h

1. Turn on the mercury lamp to preheat for 30 minutes
2. Place the silicon wafer in the center of the lithography machine, turn on the air pump, and press the suction plate to fix the silicon wafer
3. Take the Indrop O+S mask, tear off the protective film, and clean the mask with nitrogen
4. Put the mask into the mask card slot, press the mask switch to fix the mask, and place the selective transparent glass sheet, iron ring, elastic PDMS, and fixed clip successively
5. Push the mask to the top of the wafer and lock it tightly to lift the wafer platform.
6. Set the exposure time (600MJ /cm2~90s, 6.67MJ /cm2~1s) :
(1) For Microchem 2015 SU8(30μm) : 155MJ /cm2~24s
(2) For Microchem 2050 SU8(120μm) : 240MJ /cm2~36s
7. Turn off the power of the mercury lamp in advance after exposure, take out the silicon wafer and recycle the mask

6th. Post baking time: 2h

1. Place the silicon wafer on the hot plate with the mirror facing upwards
Set hot plate parameters:

For microchem 2015 SU8:

Step	Temperature	Rate of temperature increase	Time
1	95	120	5min
2	28	120	24h

For microchem 2020 SU8:

Step	Temperature	Rate of temperature increase	Time
1	65	120	5min
2	95	120	10min
3	28	120	24h

7th Development time: 1h

1. Put the silicon chip into a 10cm glass dish and pour it into PGMEA to immerse the chip
(1) For Microchem 2015 SU8: PGMEA 10min, diacetone alcohol 5min, isopropyl alcohol terminated
(2) For MicroChem 2050 SU8: PGMEA 10min, diacetone alcohol 5min, isopropyl alcohol terminated
2. Dry the silicon wafer with nitrogen

8th. Hard film time: 2.5h

1. Place the silicon wafer with the mirror facing upwards on the hot plate
2. Set hot plate parameters:

Step	Temperature	Rate of temperature increase	Time
1	150	120	5min
3	28	450	24h

Gas cell:

Array:

Fig. 3 Diagram of finished silicon wafer

3.3.PDMS reverse mold production

After the molds are prepared in Step 3.2， proceed with the equipment fabrication using the PDMS castings.[1]

1. PDMS were prepared by combining organosilicon base with curing agent at a mass ratio of 8:1.Manually mix the silicone base and hardener using a stir bar.

2. Degassing PDMS by placing them in the degassing chamber and applying a vacuum. Allow the PDMS to be degassed until the bubbles are no longer visible (usually 30 minutes).

3. Carefully pour the degassed PDMS onto the master plate, resulting in a PDMS layer thickness of about 5 mm. The PDMS is degassed again to ensure that any bubbles are removed.

4. After degassing, bake PDMS and main at 80°C for 80 minutes.

5. Use a razor blade to carefully remove the cured PDMS plate from the grill.Make sure all the cuts are at the top of the wafer.
Note: Any cut in the silicon wafer may cause the lip rim to prevent uniform bonding.

6. Punch inlet and outlet holes using a 0.75mm biopsy punch. Remove any dust and stray PDMS using packing tape on the function side of the device.

7. Prior to plasma treatment of equipment, rinse and dry with isopropyl alcohol to clean 50 mm x 75 mm slides.

8. For plasma treatment, place PDMS plate and glass slide in plasma bonding machine, feature up. Plasma treatment was performed with 1 mbar O 2 plasma for 1 min. Glue the device to the slide by putting the exposed or face-up sides together.

9. After plasma treatment, bake the device at 80°C for 40 minutes.

10. Finally, a glass surface treatment solution is injected into one of the inlets to make the microfluidic channel hydrophobic. Make sure all channels are completely filled with solution and repeat for each dropmaker. Bake the treated equipment at 80° C for 10 minutes to evaporate the excess solvent.

4.Chip operation

4.1.Chip matching

The air chamber chip and the array chip are aligned under a microscope. Due to the different PDMS ratio between the gas chamber chip and the array chip, the two layers are adhered to each other, vacuumed for 30min and kept at 70 ℃ for 4 hours. After PLASMA is applied, the gas chamber outlet is sealed with glass sheet. Placed under a microscope

4.2.Chip path detection

The suction port is filled with water, and liquid seeps from the surface, proving that the two chips are connected.

4.3.Pre-experiment with ball capture

Experimental conditions: 1um polystyrene ball, 0.08mpa pressure
Results: Beads have little flow and do not flow into the airway through the suction port
Improvement: Negative pressure too low? With subsequent use of 0.5Mpa negative pressure, liquid can be seen to be drawn through the suction port and polystyrene pellets can be detected in the suction liquid.

4.4.Beads loading

Experimental conditions: 30-50umbeads.No negative pressure, gravity deposition. Diameter of beads is 50 micro and marked with green fluorescence

Operation steps: See Appendix 1

Experimental results: The capturing probability of beads is high, and single beads is captured in a single chamber

Fig. 6 The chip captures microbeads and in fluorescence

4.5.Bacteria loading

Experimental conditions: After beads loading is completed, drop 20ul bacterial suspensions (40000) on the chip surface, shake in the X-Y direction for 1min, add negative pressure, and observe under a microscope
Results: The number of bacteria captured in a single chamber was more than 1
plan: dilute bacteria, mean =1

Fig. 7 One array one beads one bacterium

4.6.The 3D-printed scaffold was viewed under an inverted fluorescence microscope

Experimental conditions: The droplet flow in the chip was observed in real time under inverted fluorescence microscope
Experimental results:
1. The chip can not be fixed and can not be observed by negative pressure at the same time when viewed from the front up beyond the observation range of inverted fluorescence microscope (about 7000μm)
2.The back of the chip can be observed normally and clearly by looking up.However, there are gaps between the chip and the bottom support glass, which may cause the beads and cells to pour out and not in the same glue plane.
Solution: The scaffold was designed by 3D printing to match the inverted fluorescence microscope and meet the demand of frontal and upward observation.

We found a specialized 3D printing company to print our models using photocuring technology.

Fig. 9 3-D design

We found a specialized 3D printing company to print our models using photocuring technology. The 3D-printed stand has grooves that fits the pipe, allowing the chip and channel to fit perfectly, making it easy to observe the capture performance of the chip.

Conclusion: Through the use of 3D printing model, we successfully solve the problem that it is difficult to observe the chip under the microscope. The chips and tubes will be embedded in the grooves of the 3D-printed model, and the glass slides used to seal it will fit perfectly. Our 3D printed model helps us to realize real-time fluid observation under the microscope, which will better detect the chip performance and carry out subsequent experiments.

4.7.Formation of water-in-oil droplets

Experimental conditions: A fluorescent dye is inserted into the friendly gas cell of the chip to fill the array, and the surface dye is absorbed with absorbent paper. Fluorinated oil containing surfactants is dropped on the surface of the chip, and oil is passed through the airway, and the two sides are wrapped to form droplets

Experimental principle: See Appendix 2

Result: water is pushed out to form droplets

Fig. 11 Fluorescent droplets of water in oil

5.Summary

In conclusion, we have successfully designed and manufactured a microfluidic chip for EP-PCR in the directed evolution pipeline for fatty acid sensing module. We have verified the usability of the chip through some experiments. We hope that the chip will become a high-throughput platform for directed evolution in the future, combined with the microfluidic chip which has been already designed for expression and screening in previous studies. In addition, 3D printing plays an indispensable role in making the results visible.

Reference

[1] Fallah-Araghi A, Baret JC, Ryckelynck M, Griffiths AD. A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution. Lab Chip. 2012 Mar 7;12(5):882-91.
[2] Demaree, B., Weisgerber, D., Lan, F., Abate, A. R. An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing. J. Vis. Exp. (135), e57598, doi:10.3791/57598 (2018).
[3] Gierahn TM, Wadsworth MH 2nd, Hughes TK, Bryson BD, Butler A, Satija R, Fortune S, Love JC, Shalek AK. Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput. Nat Methods. 2017 Apr;14(4):395-398.
[4] Shemesh J, Ben Arye T, Avesar J, Kang JH, Fine A, Super M, Meller A, Ingber DE, Levenberg S. Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis. Proc Natl Acad Sci U S A. 2014 Aug 5;111(31):11293-8.

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