An All-in-One Solid State Thin-Layer Potentiometric Sensor and Biosensor Based on Three-Dimensional Origami Paper Microfluidics

An origami three-dimensional design of a paper-based potentiometric sensor is described. In its simplest form, this electrochemical paper-based analytical device (ePAD) is made from three small parts of the paper. Paper layers are folded on each other for the integration of a solid contact ion selective electrode (here a carbon-paste composite electrode) and a solid-state pseudo-reference electrode (here writing pencil 6B on the paper), which are in contact with a hydrophilic channel fabricated on the middle part (third part) of the paper. In this case, the pseudo-reference and working electrodes are connected to the two sides of the hydrophilic channel and hence the distance between them is as low as the width of paper. The unmodified carbon paste electrode (UCPE) and modification with the crown ether benzo15-crown-5 (B15C5) represented a very high sensitivity to Cu (II) and Cd2+ ions, respectively. The sensor responded to H2O2 using MnO2-doped carbon paste electrode (CPE). Furthermore, a biosensor was achieved by the addition of glucose oxidase to the MnO2-doped CPE and hence made it selective to glucose with ultra-sensitivity. In addition to very high sensitivity, our device benefits from consuming a very low volume of sample (10.0 µL) and automatic sampling without need for sampling devices.


Indicator electrode
The sample channel which is wider than electrode width; C) Channel width equal to the electrode's and D) The sample channel thinner than the electrode width. Table S1. Effect of the size of sample channel width on the precision of potentiometric measurements (1.0×10 -6 mol.L -1 of Cu 2+ and three times repeat for each design). Experimental conditions: KNO3 0.1 mol.L -1 in HAc-NaAc buffer 0.1 mol.L -1 pH 5.0 and room temperature.

A B
C D

Sample volume
The sample volume which was loaded to the sample channel was set to be as much as the channel becomes saturated from the sample. The needed volume for this purpose was measured by a colorimetric analysis.
Therefore, chromatic substance like murexide was selected to evolution of the volume of the sample channel. Different volumes of murexide solution was loaded into sample channel and then color intensity (R in RGB space) at three parts of channel (start, end and middle sections of the channel) was measured.
The RSD% values of these three R values were calculated. As it is obvious from Figure S3, by increasing the sample volume, the concentration gradient of murexide and therefore RSD% in the channel would be   (Table S2) showed that the best results were obtained when design "B" was employed. In order to choose the optimal design, it should be noted that if the number of layers is even and the paper is folded in the form of an origami, the working electrode (or reference) is facing outside and it is attached to clothespins. From among designs A, B and C, design B was selected.
Design C has a less correlation coefficient than design B; also, when using volumes greater than 50.0 μL (to ensure uniformity of concentration across the channel), hydrophobic parts around the electrode lose their hydrophobic properties. Design A not only has a longer response time than design but also more volume of consumable solution.  Table S2. Analytical characterization of the sensor with different junction layer design. Experimental conditions: KNO3 0.1 mol.L -1 in HAc-NaAc buffer 0.1 mol.L -1 pH 5.0, room temperature and difference concentration of copper (1.0 × 10 -5 -1.0 × 10 -9 mol.L -1 ).     Table S3. Potentiometric selectivity coefficient of the Cd 2+ ion using SSM. Experimental conditions: KNO3 0.1 mol.L -1 in HAc-NaAc buffer 0.1 mol.L -1 pH 5.0, room temperature. 6B pencil as a reference electrode and modified CPE (mixing 71 wt % of graphite powder, 25 wt % of Nujol oil and 4% B15C5) as an indicator electrode.

Determination of the pH value of papers
The paper was cut into pieces of roughly 1.0 cm 2 . The weighted, air-dry paper was transfer to a 100.0 mL beaker and then added 20.0 mL of distilled water and macerated with a flattened stirring rod until the specimen is uniformly wet. After that more 50.0 mL of the distilled water was added, stirred well, then covered with a watch glass, and allow to stand approximately 20.0 hour. The entire procedure is carried out at room temperature. After stirring the mixture once more, the pH of the unfiltered mixture was measured with the pH-meter equipped with a glass electrode [8].
After selecting the design of Figure S4B as the optimal design up to this step, the hydrophobicity of the back of the working electrode was evaluated. As shown in Table S5, back of the working electrode was printed to be hydrophobe. In addition, less volume of solution was consumed and RSD percentage decreased.  To be able to compare the ePAD results with the bulk analysis, the effective parameters (type and concentration of buffer, pH value, electrolyte and carbon paste mixture) were all adjusted the same as the previous reported method for determination of Cu 2+ ion by carbon paste ISE [9].