A plug-in electrophoresis microchip with PCB electrodes for contactless conductivity detection

A plug-in electrophoresis microchip for large-scale use aimed at improving maintainability with low fabrication and maintenance costs is proposed in this paper. The plug-in microchip improves the maintainability of a device because the damaged microchannel layer can be changed without needing to cut off the circuit wires in the detection component. Obviously, the plug-in structure reduces waste compared with earlier microchips; at present the whole microchip has to be discarded, including the electrode layer and the microchannel layer. The fabrication cost was reduced as far as possible by adopting a steel template and printed circuit board electrodes that avoided the complex photolithography, metal deposition and sputtering processes. The detection performance of our microchip was assessed by electrophoresis experiments. The results showed an acceptable gradient and stable detection performance. The effect of the installation shift between the microchannel layer and the electrode layer brought about by the plug-in structure was also evaluated. The results indicated that, as long as the shift was controlled within a reasonable scope, its effect on the detection performance was acceptable. The plug-in microchip described in this paper represents a new train of thought for the large-scale use and design of portable instruments with electrophoresis microchips in the future.


Introduction
Microchip electrophoresis (ME) is emerging as a highly promising method for rapid analysis with a minimum amount of analytes [1][2][3][4]. Compared with other ion detection methods such as laserinduced breakdown spectrometry [5] and inductively coupled  were purchased from Sinopharm Chemical Reagent Co. Ltd, China. All reagents were of analytical grade, and deionized water was used throughout. Two high-voltage modules (DW-P102-1C32 and DWP502-1C0F; Dongwen Cop., China; http:// www.tjindw.com) were used to supply the injection and separation voltages for the electrophoresis experiments. A CNC milling machine (SXX05; Sharpe CNC Co. Ltd, China; http://www.sharpecnc.com) was employed to fabricate the steel template with a convex microchannel pattern. An ultrasonic cleaner and a Harrick Plasma Cleaner (PDC-002) were used to clean and active the surfaces of the PMMA plates. A self-made hot-embossing device and a heating furnace were introduced to copy microchannels from the steel template and seal the microchannel layer.

Apparatus
A CNC graphing tool (Mastercam 9.1) was employed to draw the microchannel pattern and generate the CNC programs for the manufacture of the steel template. Because the pattern of the steel template was very elaborate, a milling cutter with a diameter of 0.5 mm and a high rotation speed of 15 000 r.p.m. were used. The hot-embossing and the hot-bonding processes are illustrated in figure 1a. A PMMA plate with thickness 5 mm was put on the steel template at a pressure of 0.6 MPa and temperature of 103°C, a little lower than the glass transition temperature of PMMA, for 40 min. Then it was naturally cooled to room temperature and the pattern of the steel template was copied to the PMMA plate. An ultrasonic cleaner was employed to rinse the PMMA plate and another 200 µm thickness PMMA thin film with deionized water for 15 min. The cleaning process was repeated three times and the deionized water was changed each time. Before bonding the two PMMA plates, they were treated with a plasma cleaner (1 min, high power) to improve the surface activity. Finally, the two PMMA plates were subjected to a pressure of 0.6 MPa and temperature 90°C for 30 min to finish the hot-bonding process. A sealed microchannel layer was achieved after naturally cooling to room temperature. The size of the microchannel layer was 30 mm × 70 mm and the effective separation length was approximately 45 mm, as shown in figure 1c. The designed width and depth of the microchannels were 100 µm and 100 µm, respectively. The length of the injection channel was 8 mm. The diameter of the reservoirs was 2 mm.
The PCB plate with detection electrodes was manufactured by J&C Co. Ltd, China. The PCB manufacturing process is a fairly mature technology and has been widely used for decades. The manufacturing cost of common PCB plates is cheap because of their mass production. The detection electrodes were patterned on the PCB plate. We chose a three-electrode structure containing a signalsending electrode, a signal-receiving electrode and a grounding electrode (see figures 1 and 2). The three-electrode structure can provide a better detection performance than the two-electrode structure [24,27,28]. The height of the three electrodes was 35 µm. The widths of the signal-sending electrode and  the signal-receiving electrode were both 1 mm. The width of the grounding electrode was 0.2 mm. The gaps between the three electrodes were both 0.3 mm. A U clamp was designed and manufactured to secure the microchannel layer. As shown in figure 1d, in order to fix the microchannel layer, the inner sides of the clamp were designed as a 1 : 50 incline. Two stop lugs were also employed to limit the movement of the microchannel layer in the vertical direction. The clamp was assembled on the PCB plate by four plastic bolts firstly. And then we just need to insert the microchannel layer or pull it out of the clamp to install or remove the microchannel layer.

Electrophoresis procedure
Before electrophoresis, the microchannels were washed with deionized water and running buffer (20 mM MES/His-0.7 mM 18-crown-6) for 20 min, respectively. All ion samples, including potassium chloride, sodium chloride and lithium chloride, were dissolved in running buffer. Sample injection was performed electrokinetically using the normal cross-injection method [29]. A voltage of 500 V was applied between reservoir S and reservoir SW for 10 s, as shown in figure 1c. Sample separation was performed by applying a voltage of 1000 V between reservoirs B and BW. The voltage and frequency of the excitation signal were 5 V pp and 800 kHz, respectively.

Electrophoresis experiment for the installation shift
The plug-in structure brings excellent maintainability; however, it causes another problem with the installation shift between the separation channel and the electrodes because the plug-in structure cannot guarantee precision assembly of the microchannel layer and the electrode layer. To evaluate the effect of installation shift on the detection performance, a set of comparison experiments were conducted.
As shown in figure 2, a series of installation shifts were defined from −1.5 mm to 1.5 mm, and electrophoresis detection was implemented for each installation shift, respectively. A total of 0.5 mM mixed solution of K + , Na + , Li + was chosen to assess the effect of the installation shift on the test performance. The experimental method and parameters were as described in §3.3.

Quality of the microchannels
To evaluate the quality of the microchannels made from the metal template, the upper layer of the PMMA microchannel layer was observed by microscope (Leica DMI3000 M). Several bumps and hollows with a size of several micrometres were observed, as shown in figure 3a. They were copied from the bumps and hollows of the metal template and were inevitable because machining cannot guarantee the excellent surface smoothness that is possible with photolithography. The layer was cut off and smoothed with a piece of fine sandpaper, then the cross section of the microchannel was obtained. It can be seen that the cross section is an approximate trapezoid, as shown in figure 3b. The topline and the baseline are 123 µm and 163 µm, respectively. The angle of the lower corner is approximately 74.6°. The height of the trapezoid is approximately 70 µm. The designed size of the microchannel section was 100 µm × 100 µm and the discrepancy was caused by the hot-embossing and demoulding processes.      the order of K + , Na + and Li + . The peak height and the migration time when the three ions arrived at the detection position were extracted from the experimental data. From figure 4b,c, several features of the ion migration can be seen. At first, the migration speeds of the three ions are diverse and in the order of K + , Na + and Li + , which is consistent with results in the literature [24]. For the same ion at different concentrations, the migration speed slightly decreases with increasing concentration. The peak height of the three ions at the same concentration is in the order of K + , Na + and Li + . The peak height of the same ion at different concentrations increases with the increase in the concentration and shows an acceptable linear relationship between concentration and peak height at concentrations ranging from 0.25 to 1 mM. To assess the test stability of the plug-in microchip, several detection tests of the three ions K + , Na + , Li + at a concentration of 0.5 mM were carried out. The results are shown in figure 5a. The peak height and migration time of each ion were extracted, which indicated a good consistency, as shown in figure 5b-d. The average and the repeatability of the migration time are listed in table 1. The average and repeatability of the peak height are also listed. Compared with [30,31], the repeatability of the migration time and the peak height are at the same level. In addition, the limits of detection (LODs) for K + , Na + and Li + were obtained through experimental detection. In order to obtain more persuasive  shift (mm) K + Na + Li + Figure 6. Electrophoresis test results for K + ,Na + ,Li + with different installation shifts between the microchannel layer and the electrode layer. Experimental conditions were as described in the text. The concentrations of the ions were all 0.5 mM.
results of the LODs for the three ions, several experiments were conducted. The LOD for K + is taken as an example for the explanation of the experiments. The detection experiments of K + at concentrations of 0.25 mM, 0.20 mM and 0.15 mM were implemented, respectively. K + at concentrations higher than 0.20 mM could be detected; however, K + at a concentration of 0.15 mM could not be recognized clearly in the electrophoretogram. We also considered 0.20 mM as the LOD for K + . However, the real LOD for K + was between 0.15 and 0.20 mM, and we chose the higher one for a conservative evaluation. Similar detection experiments were conducted for Na + and Li + . In summary, the LODs for K + , Na + and Li + were 20 µM, 25 µM and 40 µM, respectively. Compared with other microchips [30,31], the LODs of the plug-in microchip were several times higher because of the thick insulating film used to seal the microchannel layer, the rough inner face of the microchannels and the assembled structure. Also some experimental parameters including buffer concentration and structure parameters were not systematically optimized.

Effects of the installation shift
The effect of the installation shift brought about by the plug-in structure of our electrophoresis microchip, described in §3.4, was evaluated. The experimental results shown in figure 6 indicate that the installation shift between the microchannel layer and the electrode layer lowers the detection precision. To express the relationship between the installation shift and the detection performance, we define a parameter of shift distance that is the absolute distance from the zero shift position. With increasing shift distance, the detected signal intensity decreases obviously. However, the decrease in signal intensity caused by different shift distances was quite different. When the shift distance is greater than 1 mm, the signal intensity falls sharply and is below 50% of that at the zero shift position. However, when the shift distance is less than 0.5 mm, the signal intensity can be maintained within 95% of that at the zero shift position. So it can be concluded that, as long as the shift distance is controlled within 0.5 mm, the test results are acceptable. This is a meaningful result for the plug-in microchip, because it is the basis on which our chip can be effectively used. As long as the U-clamp and the microchannel layer can be fabricated with an accurate size, the assembling accuracy can be controlled within 0.5 mm easily. With the mechanical technology available at present, a machining tolerance of 0.25 mm for the small chip and clamp is easy to attain.

Service life
Six microchips were obtained using the hot-embossing and hot-bonding processes and numbered, respectively.

Conclusion
The proposed method of our plug-in microchip improves maintainability greatly by using a plug-in structure to change the damaged microchannel layer without cutting the circuit wires on the detection component. This method lowers the maintenance cost because only the microchannel layer needs to be changed instead of the whole microchip, including the microchannel layer and the electrode layer. It also lowers the fabrication cost by adopting a steel template and PCB electrodes, avoiding the complex photolithography, metal deposition and sputtering processes. The detection performance of our microchip was also assessed by electrophoresis experiments. The results presented an acceptable gradient and a stable detection performance. The effect of the installation shift between the microchannel layer and the electrode layer brought about by the plug-in structure was also evaluated. The results indicated that, as long as the shift was controlled within a reasonable scope, its effect on the detection performance was acceptable. The LODs for K + , Na + , Li + are relatively higher than previous results for other microchips; however, this does not reduce the application prospect and potential. This microchip can be used in many fields that do not need rigorous detection precision, such as detecting nutrient ions in soil [32] or monitoring the lithium ion concentration in the blood of patients with bipolar disorder who are treated with oral lithium [33]. The plug-in microchip described in this paper indicates a new train of thought for instrument design with electrophoresis microchips and large-scale use with relatively low cost in the future. Developing a detection instrument with a plug-in microchip will be the next step for our research team.
Ethics. Our research focus on the detection of ions and the microchip may be used in environmental monitoring, nutrient detection and other fields. In our experiments, we did not use any animals or any parts from humans.
Data accessibility. The datasets supporting this article have been uploaded as part of the electronic supplementary material.