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Simple and Equipment-Free Paper-Based Device for Determination of Mercury in Contaminated Soil

This work presents a simple and innovative protocol employing a microfluidic paper-based analytical device (µPAD) for equipment-free determination of mercury. In this method, mercury (II) forms an ionic-association complex of tetraiodomercurate (II) ion (HgI42−(aq)) using a known excess amount of iodide. The residual iodide flows by capillary action into a second region of the paper where it is converted to iodine by pre-deposited iodate to liberate I2(g) under acidic condition. Iodine vapor diffuses across the spacer region of the µPAD to form a purple colored of tri-iodide starch complex in a detection zone located in a separate layer of the µPAD. The digital image of the complex is analyzed using ImageJ software. The method has a linear calibration range of 50–350 mg L−1 Hg with the detection limit of 20 mg L−1. The method was successfully applied to the determination of mercury in contaminated soil and water samples which the results agreed well with the ICP-MS method. Three soil samples were highly contaminated with mercury above the acceptable WHO limits (0.05 mg kg−1). To the best of our knowledge, this is the first colorimetric µPAD method that is applicable for soil samples including mercury contaminated soils from gold mining areas.


Method S1
Preliminary test for selection of the color scale 2

Figure S1
Calibration graphs obtained from preliminary tests using the iodine-starch reaction on a single-layer paper device with the ImageJ analysis of the plot between (a) grey intensity, (b) red intensity, (c) green intensity and (d) blue intensity against the concentration of iodide. 2

Part 2
Method S2 Control of light illumination and calibration of camera 3

Figure S2
Effect of pH of standard 150 mg L -1 Hg solutions on the green intensity value.
4 Figure S3 Examples of photographic images of the colored product formed in the acceptor reservoirs during analysis of soil and water samples. Note: Soil samples S1-S3 were highly contaminated with mercury whereas the levels of mercury in other samples of soils and waters are below the detection limit.

Method S1: Preliminary test for selection of the color scale
Preliminary experiments were carried out using a single-layer paper device. This device was produced with the same pattern as the "acceptor layer A" of the µPAD (see Fig. 1a(ii)). Reagent A (2 µL of a solution mixture of 0.2 mol L -1 IO3and 0.2 mol L -1 H + ) was first dispensed onto the circular reservoir and allowed to dry for 5 min before dispensing a series of I --starch solution with varying concentrations of I -(2-9 mmol L -1 ) but fixed starch at 1 %(w/v). After dispensing the I --starch solution, the circular reservoir turned from white to purple color. The intensity of the purple color complex of I3 --starch formed on the reservoir corresponds to the concentration of Iin the mixture. After recording the images, the image was analyzed using ImageJ (version v1.35e) to obtain the intensity values of grey-and RBG-scales and to plot the calibration graphs. The preliminary results in Figure S1 show that the plot obtained using the green intensity values of the RGB-scales gave the highest sensitivity with r 2 close to 1. (The green color corresponds to the color absorbed when the color observed is purple,) We therefore used the green intensity values for the calibration plots of the 3-layer PADs for this work.
. Figure S1. Calibration graphs obtained from preliminary tests using the iodine-starch reaction on a single-layer paper device with the ImageJ analysis of the plot between (a) grey intensity, (b) red intensity, (c) green intensity and (d) blue intensity against the concentration of iodide.

Part 2 Method S2: Control of light illumination and calibration of camera
On the stage for placing the µPAD for photography, a red circular zone (6 mm diameter) was employed to calibrate the camera light sensing system before recording the images of the µPAD. The following is the protocol for recording the image of the acceptor reservoir 'ar'.
Step 1: The camera (Canon IXUS 125 HS, Japan) is first set to ISO 200. The shutter button is pressed halfway until the focusing frame appears covering the red circular reference zone. The values of ISO 200, speed shutter 1/80 and f-number (aperture) 5.0 are then displayed.
Step 2: Move the focusing frame to cover the area of the acceptor reservoir 'ar' and record the image of the purple-colored zone.
Step3: The obtained image is exported to a computer for analysis by ImageJ.  Figure S3. Examples of photographic images of the colored product formed in the acceptor reservoirs during analysis of soil and water samples. Note: Soil samples S1-S3 were highly contaminated with mercury whereas the levels of mercury in other samples of soils and waters are below the detection limit.