Hardware
Hardware
PE microplastics Content Detection Kit
1 The design of our Kit
2 Previous Results
3 Industrialization thoughts
References
1 The design of our Kit
1.1 Overview

The assessment of microplastics content in the environment is an indispensable part of environmental governance. Currently, common methods include laser infrared (LDIR), gas chromatography-mass spectrometry (GC-MS), and scanning electron microscope - energy dispersive x-ray spectroscopy (SEM-EDS). However, these methods either have the risk of misjudgment or have a long detection cycle, and the detection cost is generally expensive, which is not conducive to long-term evaluation of the effectiveness of governance for topics that require it.

Based on the problems of the current Polyethylene(PE) microplastics content detection methods, we designed a portable low-cost PE microplastics Content Detection Kit, which continuously supplies diluted soil test solution to the sample inspection center pool through a peristaltic pump. The working electrode in the sample inspection center pool is covered with functional supported lipid bilayers (SLBs), and the electrode, counter electrode, and reference electrode form a three-electrode system that outputs different electrochemical parameters depending on the different microplastics content of the test solution. By building a standard model in advance, we can decode these electrochemical parameters and obtain the microplastics content in the test solution.

1.2 Structure

Our PE microplastics content detection kit is dedicated to providing a portable and low-cost microplastics detection solution. In the design andassembly of this kit, we have fully, we have fully considered the possibility of different tasks for each component. The modular components can not only be used for microplastics detection, but also for other tasks.Microplastics content detection assembly can be divided into different new equipment, including a peristaltic pump, a sample center pool, and a portable electrochemical workstation.

1.2.1 Peristaltic pump
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Figure 1 3D solid model of a peristaltic pump

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Figure 2 Peristaltic pump engineering drawing

In our PE microplastics content detection kit, we have designed a peristaltic pump that is responsible for delivering the test solution to the sample inspection center pool. Our peristaltic pump consists of four main components: an electric motor, a speed control circuit board, a rotating central axis, and a protective casing. Compared to traditional peristaltic pumps, we have introduced more flexibility in selecting peristaltic pump pulsation and rotation speed to cater to different operational requirements. In terms of pulsation selection, we have installed 12 detachable rolling shafts on the rotating central axis of the peristaltic pump to compress the delivery tube. Depending on research needs, the output pulsation of the peristaltic pump can be adjusted by detaching or attaching these rolling shafts. As for speed selection, we have incorporated a speed control circuit board between the power supply and the motor. Through the knob on the speed control circuit board, we can stepless adjust the pump's speed from 37 revolutions per minute (RPM) to 13 RPM.

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Figure 3 3D solid model of peristaltic pump central shaft

The central shaft of the peristaltic pump will provide the power required for the pump's operation. The central shaft is fastened using screws and is easy to assemble and disassemble. The central shaft motor part is connected to the turntable part through motor fasteners, and users can choose different motors for replacement according to their own needs. The number of sub-axes can be changed in the rotary table part to achieve the selection of different pulses for the pump.

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Figure 4 3D solid model of peristaltic pump cover

The peristaltic pump cover consists of two parts, top and bottom, and is the central location for the peristaltic pump to generate pulse output.

To achieve quantitative selection of peristaltic pump speed and adapt to different use environments, we selected a speed-regulating electrode plate.

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Figure 5 Speed control board PCB diagram

1.2.2 Sample Inspection Center Pool
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Figure 6 3D physical diagram of sample inspection center pool

The sample inspection center pool will provide a central place for the three-electrode system to detect microplastics content.

1.2.3 Portable Electrochemical Workstation
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Figure 7 Portable electrochemical workstation

Record the electrical signals of the three-electrode system and output them.

1.2.4 PE microplastics-Responsive Three-Electrode System

1.2.4.1 Spatial distribution of the three electrodes

To meet the requirements of our system for gravitational potential, we chose to change the traditional vertical equidistant arrangement of the three-electrode system. We place the working electrode (glassy carbon electrode) horizontally, and place the counter electrode ( platinum wire electrode ) horizontally directly above the working electrode, reducing the distance between the working electrode and the counter electrode to reduce the impact of electrolyte resistance on potential control. On the contrary, the reference electrode (silver/silver chloride electrode) is placed vertically to avoid mutual interference with the reaction substances on the working electrode.

1.2.4.2 Counter electrode and reference electrode

The counter electrode, also known as the auxiliary electrode, is used to form a series circuit with the working electrode, and only plays a conductive role. In our system, we have chosen a platinum wire electrode.

The reference electrode is used as a reference to measure the potential difference relative to the research electrode. We selected a silver/silver chloride electrode as the reference electrode based on the pH value of the test solution.

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Figure 8 Ag/AgCl electrode & platinum wire counter electrode

1.2.4.3 Modified Working Electrode

We have chosen a glassy carbon electrode as our functional working electrode substrate. This electrode is a good inert electrode with good electrical conductivity, high hardness, high smoothness, high hydrogen overpotential, wide polarization range, and stable chemical properties, making it suitable for chemical modification electrodes.

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Figure 9 Modified glassy carbon working electrode

To correlate the microplastics content with the electrical signals on the glassy carbon electrode, we chose to construct a SLBs on the surface of the glassy carbon sheet, and coupled the membrane with PE binding peptides and MscS transmembrane proteins.

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Figure 10 Schematic diagram of the working principle of functional SLBs membranes

This membrane can convert microplastics content into electrical signal through the following pathway: when microplastics are present in the environment, the PE binding peptide on the functional SLBs specifically captures PE microplastics and conducts membrane pressure signals to the MscS protein. Then the MscS protein opens a positive ion channel and causes changes in the electrical potential on the glassy carbon sheet.

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Figure 11 MscS & PEBP-PEase

Click to Read More

MscS:
The Mechanosensitive Channel of Small Conductance (MscS) is an ion channel in bacteria that rapidly responds to changes in membrane tension. When a cell is swelling, MscS opens to release ions, preventing the cell from bursting.

PEBP:
We initially use a Support Vector Machine (SVM) model to learn from existing PE-binding peptides, creating a predictive filter. We then expand the dataset, converting it into graph structures for learning with a Graph Neural Network (GNN). Additionally, we've developed a more accurate predictor that incorporates an attention mechanism and the BLOSUM62 matrix. By integrating the predictions from the SVM filter, GNN, and the attention mechanism, and rigorously validating the model, we've optimized its performance for practical application

1.2.5 Workflow
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Figure 12 Workflow chart

2 Previous Results
2.1 Peristaltic pump
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Figure 13 Speed control board

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Figure 14 Peristaltic pump

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Figure 15 Working Peristaltic pump

The peristaltic pump can still operate smoothly after running at the highest speed and highest pulse for 24 hours.

2.2 PE microplastics-Responsive Three-Electrode System

2.2.1 Hydroxylation of Pyrolytic Graphite

To ensure that the SLBs can be tightly adsorbed on the glassy carbon sheet, we treated the glassy carbon sheet with Piranha Solution for hydroxylation, and preliminarily characterized the results of the hydroxylation.

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Figure 16 The effect diagram of glassy carbon piece wetting

On the left is a piece of glassy carbon that has not undergone hydroxylation treatment. It can be seen that after a drop of water is added with a dropper, the water droplet does not wet the surface. On the right is a piece of glassy carbon that has been treated with hydroxylation, and the water droplet has wetted the surface of this glassy carbon.

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Figure 17 Glassy carbon floatation test

2.2.2 Construction of Supported Lipid Bilayers(SLBs)

After completing the hydroxylation of the glassy carbon sheet, we began to construct SLBs.

We used DOPC as the basic material for SLBs, DGS-NTA(Ni) as the PE-bound peptide coupling substrate, and Texas Red ®DHPE dye as an indicator of SLB construction.

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Figure 18 Electrochemical characterization images of SLBs

From the above pictures, it can be seen that the SLBs prepared by this method are densely and evenly distributed.

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Figure 19 Electrochemical workstation characterization results
(a) Cyclic voltammentric responses of 0.5 mM K3Fe(CN)6 on a s-BLM modified glassy carbon electrode in 0.5 M NaCl solution, (b) AC impedance spectroscopy of glassy carbon electrode in 0.5 mM K3Fe(CN)6 solution

By comparing the test results of the blank background CV and EIS, it can be seen that after covering the working electrode with SLBs, the insulation is good, which proves that the prepared SLBs are functional and the chemical modification of the glassy carbon electrode is complete.

2.2.3 Loading of PE-conjugated Peptides and MscS Protein

After obtaining SLBs, we loaded PE binding peptide and MscS protein.

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Figure 20 Fluorescence microscope image of SLBs loaded with proteins
(a) Fluorescence microscope image of SLBs with proteins, (b) Fluorescence microscope image of proteins

Based on the experimental results, it can be concluded that the PE binding peptide and MscS protein have been successfully loaded onto SLBs.

3 Industrialization thoughts

In the later stage, we will use the PE microplastics content detection kit to calibrate the instrument for various concentrations of microplastic solutions. Through regression analysis of the data, obtain a model that can analyze the unknown concentration of microplastics content.

We will cooperate with the electrochemical workstation development company to delete unnecessary functions of the electrochemical workstation to further reduce the cost of the entire equipment.

References
  • 1. 邹卉,吴建华 & 方颖.(2019).支撑脂双层膜的制备及其功能化研究.生物医学工程学杂志(01),85-93.
  • 2. 张迎.(2016).磷脂双层膜阵列的构建及其内部带电磷脂在电场下的行为(博士学位论文,哈尔滨工业大学).博士
  • 3. Kefauver, J. M., Ward, A. B., & Patapoutian, A. (2020). Discoveries in structure and physiology of mechanically activated ion channels. Nature, 587(7835), 567-576.
Hardware
Electron Transport Detection System
1 Structure
2 Results
References
1 Structure

To validate the eEET in our project, we need to build a two-chamber u-tube biofuel cell(MFCs). However, during our procurement process, we found that it was more expensive to have the company produce according to our experimental plan. In order to effectively reduce the cost of experiments, we have developed an MFC detection device using 3D printing technology.

1.1 Dual-chamber sample pool

The electron transport detection system consists of a double-chamber sample cell, graphite sheet, anode film, resistor, copper wire, multimeter, and other components.

Figure 1. Double Chamber Pool 3D Solid Model

Figure 1 Double Chamber Pool 3D Solid Model

This part is made up of several resin boards fastened together with butterfly screws, which has the advantage of being easily disassembled and portable. In the center of the sample pool, we designed two grooves to accommodate four porous plates. The porous plates can be placed with ion membranes in the middle, and users can insert 1 to 3 ion membranes according to their needs.

1.2 Conductive Component

This part consists of graphite sheets, resistors, and copper wire parts. The graphite sheets are punched by a punching machine, and 12 copper wires are twisted into a strand and soldered with tin to a 1kΩ resistor. The two parts are then connected by conductive epoxy resin.

1.3 Multimeter
Figure 2. Multimeter

Figure 2 Multimeter

In order to obtain the electrical signal output from a biofuel cell, we have chosen a multimeter to utilize its output voltage signal.

2 Results

After negotiating with material manufacturers, we obtained the entire set of equipment, with a cost (excluding the multimeter) of 120 CNY, which is only 15% of the custom-made price.

Figure 3. Electron transport detection system

Figure 3 Electron transport detection system

The testing phase also achieved the expected results.

Figure 4. Test results

Figure 4 Test results

References
  • 1. Liu, X., Wang, S., Xu, A., Zhang, L., Liu, H., & Z, L., MA. (2018). Biological synthesis of high-conductive pili in aerobic bacterium Pseudomonas aeruginosa. Applied Microbiology and Biotechnology, 103(3), 1535–1544.
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