Team:BIT/Hardware-Microarray
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Microarray
We have proposed a microarray chip processing process here because the microarray chip is very suitable for throughput detection. In addition, we shared two optimized microfluidic chip liquid drive methods. We found that the current production process and specific parameters of the chip are rarely mentioned in literatures, which are the key points to determine whether the chip can be used, so we also share the production steps.
Coupling
In the biological part, we use aptamers to complete the detection of various disease markers. Because the joint detection of disease markers is very important for the correct rate and reliability of disease diagnosis. For example, h-FABP joint detection cTnI, Mb and CK-Mb has important clinical value in early diagnosis of childrenren with HFMD myocarditis, improvement of the diagnostic rate and lessening of the misdiagnosis rate, et al. [1]br>
Therefore, in the hardware part, we chose the microarray chip as the detection platform to complete the flux detection of the sample after the reaction. We have proposed two different materials - PMMA based and negative photoresist SU-8 based microarray chip processing. They have their own advantages and can adapt to different testing needs. At the same time, in order to verify that they can achieve basic functions, we have made chips of different sizes and tested them with our device.
So what is a microarray chip? The essence of microarrays is the parallel analysis of biological signals, which concentrates a large amount of biological information on a small solid-phase matrix, so that some traditional biological analysis methods can minimize the consumption of reagents and try to minimize the amount of reagents in the smallest possible space. Fast speed is completed. Fluorescence-based microarray chips provide us with a portable, fast, and high-throughput assay that effectively complements the clinical diagnostic process. As shown in the figure (Fig. 1), a chip that realizes flux detection based on the aperture shape. 【2】
Fig.1 Porous microwells for microparticle arrays
PMMA Based Processing Platform
The PMMA flux detection chip can achieve detection requirements for <10 samples. PMMA material, namely polymethyl methacrylate, commonly known as plexiglass, is the highest quality and price-appropriate variety of synthetic transparent materials. Its light transmittance is as high as 93%, which is friendly to fluorescence detection.
At present, we have designed three sizes of chips, each chip has a total size of 30 × 30mm, and the height is determined by the thickness of the acrylic. In use, the reacted biological sample is injected into the chip, and each chip can be used to detect a plurality of biological samples. Taking a 9×9mm chip as an example, the chip has a total of 9 liquid passages, one of which is used as a control when used, and the other can detect one sample.
The processing of PMMA chips is relatively simple. If only less than ten disease markers need to be detected, it is more convenient to use PMMA chips. Figure (Fig. 2) is our production process.
Fig.2 Schematic digram of PMMA chip production.(a)Draw a chip digram on the computer;(b)Export the chip map to the laser cutting machine;(c)Using a laser cutting machine to complete the preliminary production of the chip;(d)Arranging, aligning, and, fixing the layers of the chip in accordance with the design;(e)Completing the bonding of the chip using an oven;(f)Completion of chip production.
- Draw a designed chip map using SOLIDWORKS and export it to DXF file format;
- Use RDWorksV8 to import the drawn print files into the laser cutting machine.
- Place a 2mm thick acrylic plate in the laser cutting machine, set the cutting speed to 5mm/s, start the laser cutting machine, and wait for the chip to be cut.
- After cutting the layers of the chip, wash and dry with deionized water;
- Arrange, align and align the layers of the chip according to the design;
- Clamp the chip with two aluminum plates to make the layers of the chip closely fit together;
- Put the aluminum plate and chip into the oven at 170 °C, control the temperature change within ±5 °C, and heat for 14 min.
- Take out the heated aluminum plate and chip, and apply a small amount of water to the outside of the aluminum plate with a test tube brush to slowly cool it to a free evaporation state.
- Place the cooled aluminum plate and chip in a ventilated place, and then take out the chip after natural air cooling to check whether the bonding is successful. If successful, the chip is completed.
- When cutting the chip, complete it as much as possible, and repeating the cutting multiple times may cause the chip cavity error to become larger;
- Check whether the layers of the chip are aligned before bonding. Use a small amount of tape when fixing to avoid the indentation on the surface of the chip.
- When bonding, pay attention to the stability of the bonding temperature. If the temperature is too low, the bonding may fail. If the temperature is too high, the chip may melt.
Fig.3 The schematic diagram mainy shows the main production process of the PMMA chip.(a)Cutting the PMMA board with a laser cutting machine to complete the preliminary production of the chip;(b)Cleaning the chip with deionized water;(c)Removing the surface water of the chip with filter paper;(d)Fix the aligned chips with tape and aluminum plate;(e)Heat the chip in an oven at 170℃ for 14 min;(f)Get the chip.
SU-8 Based Processing Platform
The SU-8 flux detection chip can achieve detection requirements for 10 to 1000 samples. We made microchip chips based on solid particles. The principle of flux detection is very simple. The area is divided on the microarray chip, and the shape of the holes of each piece is different. Each shape of the hole can only accommodate the solid particles of the corresponding shape. Before the test, it is only necessary to process different samples to be tested into gel particles of a specific shape in a certain step.
If you need to detect the detection of multiple disease markers, it is more convenient to use the SU-8 chip. Figure (Fig. 4) is our production process.
Fig.4 The schematic diagram mainy shows the main production process of the SU-8 chip.(a)Hydroxylation;(b)Silicon;(c)Lithography;(d)Wash;(e)Put in PDMS;(f)Peel the PDMS layer off;(g)Put in SU-8;(h)Lithography;(i)Microarray chip.
1.silicon pretreatment
(1) Cleaning of single crystal silicon wafer: using deionized water + detergent, cleaned and dried with nitrogen.
(2) Add the solution to the large beaker as concentrated sulfuric acid: hydrogen peroxide = 7:3, put it into the washed and dried silicon wafer, and seal the beaker mouth with a plastic film (when there is no plastic mold, the glove can be covered) The beaker is placed on a heating table and heated at 100 ° C for 15 min. If the silicon wafer is used for a long time, it takes a long time to clean.
(3) After the cleaning, the silicon wafer was clamped with rubber tweezers, washed with deionized water, dried with nitrogen, and placed on a hot plate on which aluminum foil was laid, and the water was evaporated to dryness at 200 ° C for 10 min. (This can be done depending on the situation, such as nitrogen has dried the moisture in the silicon wafer, this step can be simplified)
(4) Put the processed silicon wafer in a clean petri dish and wait for use
2.spin-coating
(1) In the darkroom, open the homogenizer, check if the vacuum pump is connected, and adjust the temperature to 65 °C.
(2) Turn off the light, turn on the red light, and use the centrifuge tube to pour the SU8 photoresist according to your needs.
(3) Turn on the pump, check if it can be sucked, put the silicon wafer, tilt the glue slowly onto the silicon wafer, suck the film, start
(4) After the silicone is finished, the silicon wafer should be taken out after 10 minutes (to remove the silicon wafer, first push it with a tweezers)
(5) Remove the silicon wafer on the heating table on the left and raise the temperature to 95 ° C (average 1 min / 1 ° C, put it at 75 ° C for 10 min) and rise to 95 ° C, then cool to below 75 ° C.
3.lithography
(1) It can be used after turning on the lithography machine for 15 minutes.
(2) Turn on the power of the lithography machine and set the time to 1 min (the specific time should be determined by the required process)
(3) Remove the silicon wafer, cover the mask, and add it with a clip. Suction, alignment (manual), do not look directly at the UV light. Protect from light after removal. Heating on a hot plate (75 ° C for 1 min and 105 ° C for 10 min)
(4) Remove and place in the culture dish
4.development
(1) Pour the developer into the Petri dish, submerge the silicon wafer, and gently oscillate
(2) Rinse the silicon wafer with isopropyl alcohol to remove residual photoresist. If it cannot be removed, put it into the developer and shake it. Then continue to rinse with isopropyl alcohol.
(3) After rinsing, rinse with deionized water and blow dry with nitrogen.
5.silanization
(1) Make a good mold, 20μl pipette, centrifuge tube, tip
(2) Take 2 drops of C2H6Cl2Si (very toxic), place in a centrifuge tube, insert the tube into the foam board, and place the silicon wafer flat.
(3) Align the grooved side with the tube, open the vacuum pump, and evacuate for 10 minutes.
(4) Turn off the vacuum pump, remove the cover and let it stand for 1 hour, it can put overnight.
6.molding
(1) Dispense PDMS and curing agent in the required amount at 10:1 and 5:1, place in a centrifuge tube, vacuum with a vacuum pump, observe at any time to prevent foam overflow, when no bubbles are drawn. You can stop.
(2) Wrap the aluminum plate with tape around, do not have gaps around, cut off the unsuitable parts with scissors, and place the silicon on the aluminum plate. A channeled silicon wafer was cast with a 10:1 PDMS mixture and a 5:1 PDMS mixture was used to cast a silicon wafer without channels. Be careful not to show bubbles during the casting process. After casting, it is best to place it on the water platform for 10 minutes, and then put it into the oven at 100 °C for 90 minutes.
7.Preparation
(1)Use a channel layer mold made by standard photolithography of SU8-2025 with negative photoresist on a glass wafer.
(2)Silanize two to three chips.
Fig.5 SU-8 microarray chip production process.(a)Stirring the PDMS and the curing agent evenly;(b)Extracting the air with a vacuum pump;(c)Uniformly pouring the PDMS mixture into the prepared silicon wafer mold;(d)Placing the chip in an oven for heat curing;(e)Removing the PDMS mold from the silicon wafer;(f)Placing the mold into the porous film;(g)Adding a negative photoresist to the chip injection port;(h)Curing the negative photoresist;(i)Get the chip.
Results
We used these two processes to make four sizes of chips. The physical picture of the chip is shown in the figure.Besides, we will take the chip to the conference center to show you!
Fig.6 Chip physical map.(a)The size of both two PMMA chips is 30*30mm;(b)The size of two SU-8 chips’ pore diameter are 150um and 90um.
Additional Work
Microfluidic technology, also known as Lab on a chip , requires only a small amount of sample and reagents and automates the reaction compared to traditional laboratories. In recent years, more and more iGEM teams have chosen to use microfluidic chips to make portable low-cost hardware[3].
However, the size, weight and cost of these instruments are often limited by liquid drive modules. The common liquid driving methods for microfluidic chips are peristaltic pump, air pump and vacuum pump. These pumps need to be connected to large external devices, making the instrument too bulky and unsuitable for promotion. In order to optimize the liquid driving method of the microfluidic chip, at the beginning of the project setting, we proposed two solutions through brainstorming, namely electromagnetic driving based on electromagnet and driving method of integrated peristaltic pump. We have completed the preliminary design and parameter exploration of the chip under the two schemes, hoping to bring some help to the team that might use the microfluidic chip.
Fig.7 Two o optimized liquid drive methods.(a)Electromagnetic drive;(b)Integrated peristaltic pump.
3.1 Driving principle:
The electromagnet is placed on the top of each chamber on the chip. When the electromagnet is energized, the magnetic film layer under the chamber is electromagnetically attracted upward. The liquid in the chip can be driven by controlling the three-phase peristalsis of the magnetic film layer. [4]
3.2 Structural design:
The chip is mainly divided into three materials:
PMMA layer, PDMS-CI mixed layer and pure PDMS layer: The PDMS-CI layer is a magnetic film layer, which is mainly made of CI and PDMS. It is one of the electromagnetically driven control elements and will be control liquid drive under the action of electromagnet. The pure PDMS layer is the main constituent material of the chip, which is used to implement various preset functions in the chip, and the PMMA layer is used for supporting the PDMS chip body.
Fig.8 Principle explanation(a)A schematic illustration of the three- phase peristaltic motion. The fluid could be transported by this motion from the loading chamber to the reaction chamber. [5];(b) From top to bottom is fixed layer, top layer, support layer, magnetic film layer, fixed layer; (c)The mask diagram of the chip design.
3.3 Advantages and disadvantages:
Advantages: Compared to conventional peristaltic pump drives, electromagnetic drives are less expensive and smaller. The basic liquid drive can be achieved by simple electromagnetic action. In addition, the combination of the electromagnet and the magnetic film can also be used for the design of the solenoid valve to controlle the channel switch.
Disadvantages: The chamber size parameter of the chip has a great influence on the liquid driving effect, and the parameter exploration is more complicated. The PDMS-CI hybrid membrane production process is complicated and takes a long time. In addition to this, electromagnetic interference will also affect the experimental results.
3.4 Detailed steps to make the chip
3.4.1. Making CI-PDMS film
1) Add a 2:1 mass ratio CI powder and PDMS prepolymer to the centrifuge tube for thorough mixing and then add and mix the PDMS: curing agent mass ratio of 15:1 (curing agent density close to 1).
2) Vacuum pumping using a vacuum pump
3) Turn off the vacuum pump, deflate, and evacuate
4) Open the vacuum pump Check the operation of the silicone machine Note the change file
5) Use a clean piece of silicon (be careful not to touch the surface with your hand) into the spin coater . Pour the vacuum-filled CI-PDMS mixed glue on it (note the amount of control).
6) After the silicone is finished, place the silicon wafer on the foil paper, put it into a 60 ° C heating box, and heat it for 1 hour.
3.4.2 Make CI-PDMS&PDMS mixed layer and support layer
1) Remove the CI-PDMS film from the wafer and flatten it on the foil
2) Use a 8mm round punch to punch 6 holes on the foil paper
3) Align the holes according to the desired position and place them on top of the mask that has been cut.
4) The mask is reversed and the CI-PDMS layer magnetic film on the mask is attached to the silicon wafer in the original position (note the position of the entire chip)
5) Configure PDMS: curing agent = 10:1 mixture and vacuum (can be operated in parallel with step 4)
6) Place the silicon wafer with the CI-PDMS layer attached to the corresponding position of the silicone machine and pour it into the PDMS to wait for its bubbles to disappear and is placed in a 60 ° C heating box for 1 h.
7) Add some PDMS to another silicon wafer, wait for the bubbles to disappear, and put them in a 90 °C heating box for 1 hour.
3.4.3 First bond
1) Remove the pure PDMS layer from the silicon wafer with a blade and then buckle it on the foil that has been masked.
2) Remove the extra parts
3) Cover the PDMS layer that needs plasma with tin foil and put it into the plasma cleaner for plasma cleaning.
4) After completing 3), return to the ultra-clean workbench, reverse the layer covered with silicon wafer, and then align the holes, and then bond them. After bonding, the silicon wafers are placed in a 90 °C heating box for 30 minutes, then removed spare.
3.4.4 Channel layer fabrication, second bonding
1) Wrap the channel layer mold with tin foil paper, pour the glue (pay attention to the dosage), put it into the 90 °C heating box and heat it for 1h30min after the bubble disappears, then take it out ( step 4 should start within 30min after step 2 )
2) The 3-step spare bonded part is opposite to the 4-step channel layer chip, plasma-cleaned after plasma plasma cleaning, and then bonded after the chamber is aligned.
2) After heating, put it in a 60 ° C heating box for 1 h, remove, and the experiment is complete
4.1Working principle of traditional peristaltic pump:
A peristaltic pump consists of a motor-driven wheel with peripherally placed rollers and an adjustable compression cam (or band) which is squeezed against the rollers. One or several pump tubes are affixed so that they rest on a minimum of two rollers at all times . The successive peristaltic movements of the tubes force various fluids through the tubes[6]
4.2 Chip design:
With reference to the traditional peristaltic pump construction, we integrate the rotor and hose into the inside of the chip. The driving part is composed of a rotor, a hose and a nut, wherein the nut is a bonding point of the chip and the screwdriver. Rotating the nut with a stepper motor control screwdriver can drive the rotation of the rotor. When the rotor rotates, it squeezes the hose and drives the liquid to flow.
4.3 Advantages and disadvantages:
Advantages: Compared with the electromagnetic interference that may exist in electromagnetic driving, the magnetic strength is difficult to quantify, etc., the integrated peristaltic pump has fewer potential influencing factors, better working stability, short manufacturing cycle and mature process.
Disadvantages: The volume of the integrated peristaltic pump chip is relatively affected by the driving area, and the function is not as flexible as the electromagnetic driving function. The micro-valve is required to complete the control of the liquid flow.
4.4 Detailed steps to make the chip
- Draw a designed chip map using SOLIDWORKS and export it to DXF file format;
- Use RDWorksV8 to import the drawn print files into the laser cutting machine.
- Place a 2mm thick acrylic plate in the laser cutting machine, set the cutting speed to 5mm/s, start the laser cutting machine, and wait for the chip to be cut.
- After cutting the layers of the chip, wash and dry with deionized water;
- Arrange, align and align the layers of the chip according to the design;
- Clamp the chip with two aluminum plates to make the layers of the chip closely fit together;
- Put the aluminum plate and chip into the oven at 170 °C, control the temperature change within ±5 °C, and heat for 14 min.
- Take out the heated aluminum plate and chip, and apply a small amount of water to the outside of the aluminum plate with a test tube brush to slowly cool it to a free evaporation state.
- Place the cooled aluminum plate and chip in a ventilated place, and then take out the chip after natural air cooling to check whether the bonding is successful. If successful, the chip is completed.
- When cutting the chip, complete it as much as possible, and repeating the cutting multiple times may cause the chip cavity error to become larger;
- Check whether the layers of the chip are aligned before bonding. Use a small amount of tape when fixing to avoid the indentation on the surface of the chip.
- When bonding, pay attention to the stability of the bonding temperature. If the temperature is too low, the bonding may fail. If the temperature is too high, the chip may melt.
Reference
[1]Liu A L, Lei Y G, Liu A S. Clinical Value of h-FABP,cTnI,Mb and CK-Mb Joint Detection in Early Diagnosis of HFMD in Children with Myocardial Damage[J]. Journal of Clinical Transfusion & Laboratory Medicine, 2017.
[2]Kim J J, Bong K W, Reátegui E, et al. Porous microwells for geometry-selective, large-scale microparticle arrays[J]. Nature Materials, 2017, 16(1):139-146.
[3]web:https://2017.igem.org/Team:Munich
web:https://2017.igem.org/Team:BIT
[4]Wu J H, Ma Y D, Chung Y D, et al. An integrated microfluidic system for dual aptamer assay utilizing magnetic-composite-membranes[C]// IEEE, International Conference on Nano/micro Engineered and Molecular Systems. IEEE, 2017:438-441.
[5]Wu J H, Wang C H, Ma Y D, et al. A nitrocellulose membrane-based integrated microfluidic system for bacterial detection utilizing magnetic-composite membrane microdevices and bacteria-specific aptamers[J]. Lab on A Chip, 2018, 18(11).
[6]Prados-Rosales R C, Luque-Garcı́A J L, Castro L D. Propelling devices: the heart of flow injection approaches[J]. Analytica Chimica Acta, 2002, 461(2):169-180.
