In order to achieve our goal of sperm detection for household use, we need to design a convenient equipment with simple operation and cheap cost. In the hope of realizing the whole process of sperm detection on the same equipment, including screening for motile sperms, integrating two different protein signals representing sperm motility and fertility respectively through an engineered bacterial system, and developing visualizable color representation to show results, we applied microfludic technology as an ideal tool for integrating multiple experimental processes into a chip to build a microbiological laboratory. In this project, we designed a microfluidic chip driven by a finger-actuated operation for screening and detection of sperm quality to meet the needs of household use.

Schematic Design

The schematic diagram illustrates the structure of our microfluidic chip, which could achieve the purpose of sperm separation and screening. We hope that by forming a chemokine concentration gradient in the main channel of the chip, the motile sperms in the sample well can swim towards the detection well where the engineered bacteria is located, and the fertility of sperm can be detected successfully by those bacteria.

Our chip consists of four parts:

1. Sample well:
   Under the suggestion of Shangjian Liu, a student from Tian-Ling Ren's lab, we planned to pre-mix the sperm samples with the test solutioin containing antibodies and other detection molecules before loading into the sample well. In this process, the semen sample will bind to two groups of detection molecules, Sp10 antibody and NisA-affibody, respectively.
   
2. Gradient generator:
   The serpentine channel gradient generator is designed to produce diffusive mixing of two inlet media under laminar flow in sequentially branching and converging microfluidic channels.^[1] Here we add two media containing 0 and 100% chemokine in sperm culture media into two wells. The continuous branching and converging of liquids with different concentrations can finally form a chemokine concentration gradient at the main channel.
   
3. Main channel:
   Motile sperms will be examined and screened within the main channel. Only motile sperms can be induced by the chemokine gradient, swimming upstream towards the detection area via the main channel. In contrast, immotile sperms will be discharged from the exit in the other direction.
   
4. Detection well:
   The engineered bacteria was placed in the detection well, outputting corresponding sginals after recognizing the detection molecules bound to the sperms.
   

Finger-pressing Pump

Microfluidic chips often need external devices to provide power for the flow of liquid, but it would bother our customers if they need to connect external fluid control devices to the microfluidic chip at home. In order to solve this problem, liquids with different concentrations of chemokine will be driven by a finger-pressing pump to form a concentration gradient. The finger-actuated operation is two elastic pouches bulge above the chip. After being pressed, the deformed membrane extrudes the corresponding solutions. In this way, chemokine concentration gradient can be established by simply pressing two buttons on the chip.

Prototype

We fabricated the prototype of our detection chip using polydimethylsiloxane (PDMS). PDMS is an elastic material that could be deformed with gentle press, suitable for the fabrication of our finger-pressing pump. Other characteristics of PDMS such as low cost, good biocompatibility, low toxicity and chemical inertness also make it an ideal material for our household sperm quality detection chip.

We have tried two methods to built the channel of our detection chip. At first, we used photolithography and etching to fabricate the pattern of channel to the wafer before pouring with PDMS.

Later on, we changed our design as described above. The external pump used by traditional microfluidic technology was replaced with the finger-pressing pump, and the chemokine loading wells in the gradient generator became much larger to fit in the size of human fingers. Due to time limitation, we used PolyJet 3D printing technology to make the channel bulge from a matrix, and then casting the shape of the channel onto the PDMS while pouring with PDMS. (See our Engineering Page for the change of design)

The size of our chip is 3 cm in diameter, making it convenient for household use. The length from the center of sample well to that of detection well is 2 cm. Given that the average moving rate of sperm is 25 μm/s^[2], it will take less than 15 min for sperms to swim from the sample well to the detection well.

Concentration Gradient Formation

The core of the chip design of this project is to induce the movement of motile sperms against the concentration gradient to complete the sperm screening. Therefore, the experimental verification of constructing the concentration gradient part is an important part of our experiment. We printed a PDMS chip to form the concentration gradient according to the existing design. Water and 1mg/mL brilliant blue dye solution were introduced into the two flow inlets respectively, and liquid was collected at the outlet respectively. Gradient of solution color can be observed, verifying the feasibility of forming a concentration gradient scheme within the channels in the microfluidic device.

We performed a modeling analysis to further characterize the range of concentration gradients that the device can form and the time it required to reach stabilized concentration. (See our Modeling Page for details)

Instructions of our chip-based microfluidic device are very simple.

• Collect semen sample.
• Mix the sample with the test solution containing detection molecules.
• Load the mixture into the sample well
• Press the two elastic buttons to let the pre-stored solution flow into the gradient generator to form a chemokine concentration gradient.
• Results will be presented in the detection well.

Several improvements can be made to further modify our microfluidic detection chip.

In order to examine the feasibility of using the chemokine concentration gradient to induce motile sperms, we established a gradient marker model to discuss the formation mechanism of concentration gradient in the microfluidic chip. Details on this model can be found on Modeling Page. Further improvements can be made to simplify the concentration gradient forming device to occupy less space, reducing the production cost and chip volume.

Another problem need to be solved is how to pre-store the solution needed for the formation of the concentration gradient to avoid leakage. We have found an article published by Beihang University, which is very helpful to solving this problem.^[3] We have contacted the first author of the article.

How to prevent the engineered bacteria from leaking out the chip is also an aspect to be optimized. We will further explore ways to make the engineered bacteria work safely on the chip with better detection performance. We hope that this chip will be able to developed into a real commercial product.

[1] Liu, C. S., Liu, J. J., Gao, D., Ding, M. Y., & Lin, J. M. (2010). Fabrication of Microwell Arrays Based on Two-Dimensional Ordered Polystyrene Microspheres for High-Throughput Single-Cell Analysis. ANALYTICAL CHEMISTRY, 82(22), 9418-9424. https://doi.org/10.1021/ac102094r
[2] World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th ed., Geneva, World Health Organization, Cop, 2010.
[3] Wang, Z., Wang, Y., Lin, L., Wu, T., Zhao, Z., Ying, B., & Chang, L. (2022). A finger-driven disposable micro-platform based on isothermal amplification for the application of multiplexed and point-of-care diagnosis of tuberculosis. Biosens Bioelectron, 195, 113663. https://doi.org/10.1016/j.bios.2021.113663