What is microfluidic chip?
Microfluidic chip technology is a technology that uses microfabrication technology to create micro-channel network structures and other functional units on a chip of several square centimeters, which integrate the basic operating units involved in the biological and chemical fields as much as possible in a small operating platform to complete different biological or chemical reaction processes and analyze their products.
Why use it?
Our project focuses on the use of E. coli for the detection of oxidative damage, and in practice, users often need to work outdoors for a long time, so we are required to provide portable, low-cost stable culture container for E. coli. At the same time, since we have chosen fluorescence detection as the final detection method, there is a high requirement for the light transmittance of the culture container. Taking into account various factors, microfluidic chips have become our first choice.
Which material should we use?
Comparison of various aspects ofdifferent materials is shown in Figure 1:
Both PMMA (polymethyl methacrylate) and PDMS (polydimethylsiloxane) have good optical properties, toughness and low cost, so they can be used as our options. PMMA chips are relatively less expensive, and the advantage of PDMS chips is that it is easier to copy submicron-sized microstructures.Based on the characteristics of two materials, we designed the chip for static culture and dynamic culture.
How to make it (in laboratory)?
Our production solutions are based on the existing conditions of our laboratory and not on the factory. Learn more about our production process: Click here.
What is our design:
PDMS chips: This topic uses the Adobe Illustrator software to design a microfluidic chip mask, as shown in the following figure. The chip contains 6 experimental channels and 6 control channels, each of which has 5 branches with a diameter of about 150 μm. It serves as a chamber for microbial perfusion culture as well as a detection site for fluorescent proteins expressed by microorganisms. The experimental channel is connected to the upper two inlets through a mixed grid. When two inlets input different concentrations of the target, a concentration gradient is formed in the lower experimental channel, which is used to explore the expression pattern of the fluorescent product as well as subsequent high-throughput testing.
We designed an arrow-shaped ratchet structure between the main channel and the microbial culture chamber, with an intermediate channel width of about 15 μm. Due to the inherent tendency of E. coli to swim to the right, most of the cells that travel outside the chamber along the linear channel are redirected and confined within a circular culture chamber. The ratchet structure not only guides and concentrates the moving microorganisms in the main channel, but also prevents trapped microorganisms from escaping into the main channel. Further, the liquid flow on the chip is driven by an external force to infuse the nutrients and the target molecules into the culture chamber, while flushing the metabolites from the chamber to the main channel without being restricted by the ratchet structure. Thereby, a simple and convenient microorganism-cultivating scheme of a chemostat-like type can be realized, as shown in Figure 2:
PMMA chips: In the PMMA chip we apply static culture. The main mode of static mode culture is to inoculate the culture into the finished chip, provide a culture solution for growth, and place it in a carbon dioxide incubator or a self-made device for cultivation.
The frequency of replacement of the culture medium is one day or longer, which is useful for observing the natural morphology of the cells. We have designed single-chamber and multi-chamber chips, in which multi-inlet multi-chamber chips can simultaneously culture several bacteria, reducing the burden on the operator. The chip design based on this is as shown in Figure 3,4,5,6. The radius of the culture chamber is 4mm and the width of the channel is 0.7mm
How to prove them?
We conducted a variety of detailed experiments on different design options.
PDMS chip verification
Using the above design and production method to obtain PDMS chip. Chip picture and ratchet structure picture (ten times bright field) as shown in Figure 7 ,8:
We used a syringe to extract 800μl of bacteria liquid in the logarithmic phase and connected the flow channel of injection irrigation. Under a microscope, we observed the process of fluid perfusion into the ratchet and the condition of the culture chamber. And the records were recorded as shown in the figure. The magnification of microscope is 10 times and the perfusion speed of peristatic pump is 333.3μl/h，as shown in Figure 9.
We also obtained images of E.coli moving within the ratchet, demonstrating that the ratchet not only directs and aggregates the active microorganisms in the main channel, but also prevents trapped microorganisms from escaping to the main channel, as shown in Figure 10.
It has been verified that when the narrowest channel is 17μm wide and the spines are 30μm apart, the self-movement characteristics of E. coli can be fully utilized to prevent the escape of e. coli from the chamber on the condition of good material exchange between the chamber and the main channel.
Bacterial culture and gene expression
We took 30μl of antibiotics and 100μl of cryopreservation bacteria solution and added them to 100ml of sterile culture solution. The culture solution was then incubated in a 37℃ constant temperature shaking table (120rpm). After that, we measured its absorbance (OD) every hour and drew the growth curve as shown in Figure 11. This curve was used as a standard reference curve to evaluate E. coli growth in subsequent studies.
The growth curve analysis showed that the strain reached the logarithmic growth stage about 2 hours after it was released from the frozen storage state, and the corresponding absorbance was about 0.2. After 4h of culture, the strain entered the stable period. E.coli with an absorbance of 0.2 was introduced into the observation culture system, because E.coli had entered the logarithmic phase of division and had vigorous proliferation and metabolism, which could better meet the conditions required for culture and observation in the experiment.
E.coli liquid in logarithmic phase was introduced into microchip culture system for continuous perfusion culture. During the bacterial culture, we observed the growth of E. coli in the chamber in real time and took photos every hour. The results under the bright field are shown in Figure 12, and the magnification is 40 times.
It can be found from the recorded results that the volume and number of E.coli increased with the culture time. After more than three hours of culture, the bacterial apoptosis had produced filamentous bands. After five hours, the filamentous bands had basically filled the whole culture chamber. By referring to the growth of E.coli under normal conditions in the above studies, it can be found that the growth in the chip is more consistent with the standard growth curve, indicating that the chamber can provide a good growth environment for E.coli and realize the functions designed as expected.
In this experiment, E.coli that can express red fluorescent protein was selected (whose number is Part:BBa_J04450, click here to study more http://parts.igem.org/Part:BBa_J04450) and the growth of the bacteria was judged by observing the fluorescence expression effect. Fluorescence expression results were shown in Figure 13, with a magnification factor of 40 and exposure time of 15s.
It was found that the fluorescence intensity in the culture chamber increased with the passage of time, indicating that the expression of red fluorescent protein was good. This proves that E. coli is growing and multiplying.
We used Image J software to conduct RGB fluorescence intensity analysis on the collected fluorescence photos, and drew the trend chart of fluorescence intensity below, as shown in Figure 14.
Concentration gradient:PMMA chip verification:
Based on the above design and chip fabrication steps we obtained the PMMA chip as shown in Figure 15:
Bacterial culture and expression: E. coli used in the experiment can express red fluorescent protein in the case of natural growth, so adjust the inverted fluorescence microscope to a filter capable of transmitting red fluorescence to observe E-coli. The growth of E. coli at 0 h, 2 h, 4 h, and 6 h was recorded. The experimental results are shown in Figure 16.
It can be seen from the results in the figure that the cultivation of E. coli on the microfluidic chip fabricated by PMMA material has a good effect, and the number of E. coli has increased significantly with the increase of culture time. Under natural light, the growth of E. coli can be clearly seen. Under red fluorescence, the number of red dots in the field of view increases significantly, indicating that the growing E. coli is indeed E. coli capable of expressing red fluorescent protein; Under green fluorescence, E. coli could not be seen in the visual field and did not grow with the increase of culture time. It further showed that E. coli capable of expressing red fluorescent protein grew well in PMMA microfluidic chip. The microfluidic chip can The E. coli culture function was achieved. This result indicates that it is feasible to transfer the microbial sensor to the PMMA microfluidic chip for detection.
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The project team has designed its own fluorescent detection system, which can be operated on smartphones with mini apps. The system is simple in structure, easy to operate, low-cost, and it is available anytime and anywhere. Users can take pictures of the concerned area with their smartphones, collect and process the graphics with a specific mini app, and obtain the result in form of fluorescent. In this simple way, DNA damage is visualized and quantified.
Design & Method
The detector is used to detect and analyze fluorescence expressed by E. coli in the PMMA chip.
- (1) Optical hardware
- ①Optical structure
- ②3D printed housing
- (2) Supporting mobile phone software
The optical structure is shown in Figure 17. The LED light of the smartphone emits white light, which passes through a filter with a center wavelength of 470 nm and turns into blue light, which is conducted by the optical fiber to the detection area of the chip. Fluorescent proteins are excited by blue excitation light, emitting green emission light with 525 nm as the center wavelength. The green light is focused by a plano-convex macro lens, then filtered by a filter with a center wavelength of 530nm, and finally enters the camera of smartphone. The CMOS image sensor converts the optical signal into an electrical signal, which is processed and transmitted to the screen to present the image.
We choose fast, low-cost, and easy-to-use 3D printing to create a shell for optical hardware. Based on the optical structure design and the shape and size of the smartphone, the hardware housing is designed with SOLIDWORKS software, and the required size is measured by the cursor caliper, as shown in Figure 18 and 19.
We assemble lenses, filters, fiber optics, smartphones, and 3D-printed housings and use chips to test the effect.
We choose to write WeChat mini programs to implement software analysis of fluorescent images, due to its ease of use, ease of development, cross-platform operation, and fast distribution and iteration.
The team uses different concentrations of fluorescent microsphere solution to verify the detection system. The result proves linear relation between the G value of the fluorescent image and the concentrations of the fluorescent microspheres. It is a firm proof that our device is able to determine the concentrations of the inducers by testing fluorescence, as shown in Figure 20,21 and 22.
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