Membraneless Gas-Separation Microfluidic Paper-Based Analytical

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Membraneless Gas-Separation Microfluidic Paper-Based Analytical Devices for Direct Quantitation of Volatile and Non-Volatile Compounds Piyawan Phansi, Saichon Sumantakul, Thinnapong Wongpakdee, Nutnaree Fukana, Nuanlaor Ratanawimarnwong, Jirayu Sitanurak, and Duangjai Nacapricha Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02103 • Publication Date (Web): 28 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

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Analytical Chemistry

Membraneless Gas-Separation Microfluidic Paper-Based Analytical Devices for Direct Quantitation of Volatile and Non-Volatile Compounds Piyawan Phansi,†,‡ Saichon Sumantakul,†,‡ Thinnapong Wongpakdee,‡ Nutnaree Fukana,‡ Nuanlaor Ratanawimarnwong,†,§ Jirayu Sitanurak,*,†,‡ and Duangjai Nacapricha*,†,‡

†

Flow Innovation-Research for Science and Technology Laboratories (FIRST labs.)

Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand ‡

§

Department of Chemistry, Faculty of Science, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand

*Corresponding authors’s emails: [email protected] and [email protected] (D. Nacapricha); [email protected] (J. Sitanurak); fax: +6622015127

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ABSTRACT: This work presents new chemical sensing devices called ‘membraneless gasseparation microfluidic paper-based analytical devices (MBL-GS µPADs). MBL-GS µPADs were designed to make fabrication of the devices simple and user friendly. MBL-GS µPADs offer direct quantitative analysis of volatile and non-volatile compounds.

Porous

hydrophobic membrane is not needed for gas-separation, which makes fabrication of the device simple, rapid and low-cost. A MBL-GS µPAD consists of three layers: ‘donor layer’, ‘spacer layer’ and ‘acceptor layer’. The donor and acceptor layers are made of filter paper with printed pattern. The donor and acceptor layers are mounted together with a spacer layer in between. This spacer is a two-sided mounting tape, 0.8 mm thick, with a small disc cut out for the gas from the donor zone to diffuse to the acceptor zone. Photographic image of the color that is formed by the reagent in the acceptor layer is analyzed using ImageJ program for quantitation. Proof of concept of the MBL-GS µPADs was demonstrated by analyzing standard solutions of ethanol, sulfide and ammonium. Optimization of the MBL-GS µPADs was carried out for direct determination of ammonium in wastewaters and fertilizers to demonstrate the applicability of the system to real samples.


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INTRODUCTION SECTION Since the first communication in 2007 by Whitesides and co-workers,1 analytical devices made of patterned paper known as ‘paper-based microfluidic devices’ or ‘µPADs’ have gained growing interest.2 Most of µPADs are fabricated using filter paper as substrate for the flow of liquid sample and reagent. The filter paper is printed with a hydrophobic compound with a pattern that defines the flow path. The hydrophobic barrier constrains the liquid within the flow channel and guides the liquid flow in a controllable manner.3,4 To date, µPADs have been applied in broad fields such as point of care diagnosis, forensic analysis and food safety,3,5 as well as environmental monitoring.6 There have been a rapid growth in the publications of µPAD technology in terms of new fabrication techniques, new hydrophobic materials for the pattern, incorporated functionality, detection and quantitation, as well as development of applications.3,5,6 The attractiveness of µPADs includes low cost, suitable for use in developing world,7 portability1 and biodegradability,8 for environmentally friendly devices. The volume of reagents can be as low as 0.3 µL1 and thus producing low amount of chemical waste.9 Movement of liquid in µPADs is by capillary-flow inside the porous network of cellulose, eliminating the use of pumps required for microchips or lab-ona-chip.10 However there are many areas of µPAD that still needs to be addressed.5 In-situ sample preparation is an important factor in development of µPADs, particularly for point of care purposes.11 In this work, we are interested in development of in-device sample preparation employing gas-liquid separation. In conventional fluidic-based analysis such as flow injection (FI) and its related technique, there are two commonly used methods for online gas-liquid separation. These are the ‘gas-diffusion (GD)’ device12-14 and the ‘pervaporation (PV)’ device15-17. With these membrane-based devices, gaseous analyte 3 ACS Paragon Plus Environment


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diffuses from the donor stream (sample) through the porous hydrophobic membrane into the acceptor stream (carrier).12-17,18 The hydrophobic membrane prevents mixing between the donor and acceptor liquids and allows only gas to diffuse across the membrane for selectivity prior to detection. Recently there was a report of the incorporation of a hydrophobic membrane into the fluidic unit for gas-liquid separation on a centrifugal microfluidic platform.19 Due to various drawbacks of these membrane-based devices, e.g. aging of membrane, membrane clogging and contamination, we presented in 2006 an on-line membraneless device for the separation of gas from liquid samples.18 The two reservoirs are connected by a common air headspace, across which the volatile analyte diffuses from the donor reservoir (sample reservoir) to the acceptor reservoir (carrier and/or reagent). Dissolution of the gas into the acceptor liquid leads to detectable changes in its physical property. The concept of membraneless gas-separation (MBL-GS) has been efficiently utilized in fluidic-based techniques to replace the conventional membrane-based devices (GD and PV).18,20,22,23 Various designs of the membraneless device, suitable for use in fluidicbased techniques, have been presented for direct analysis of liquid18,22,24,25 and solid samples.21,26 In this paper, we have incorporated the concept of membraneless gas diffusion into the µPAD framework. We have called the devices ‘membraneless gas-separation microfluidic paper-based analytical devices’ (MBL-GS µPADs). To our knowledge, this is the first time that such a membraneless gas-separation concept is reported for µPAD technology. Since our MBL-GS µPAD comprises of only three layers, the fabrication is therefore less complex than the fabrication of the five-layered gas-diffusion µPAD, with porous Teflon tapes stretched inside the device.27 In our device, a circular hole, cut out of the middle layer (spacer), provides an air space through which the gaseous analyte diffuses from the bottom layer to the top layer, in which donor and acceptor zones are respectively located. Demonstration of the 4 ACS Paragon Plus Environment

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concept was made for three analytes, viz. ethanol, sulfide ion and ammonium ion. Finally, a MBL-GS µPAD unit was developed with two detection chemistries for the quantitative analysis of ammonium ion in wastewaters and fertilizers.

EXPERIMENTAL SECTION Reagents. All chemicals were of analytical reagent grade and solutions were prepared in deionized Milli-Q® water. For ethanol analysis, the detection reagent (0.4 mol L-1 dichromate reagent) was prepared by dissolving 2.9 g of potassium dichromate crystal (Ajax Finechem, Australia) in 25 mL of 4 mol L-1 sulfuric acid (Merck, Germany).23Working standard solutions of ethanol were freshly prepared in deionized water by appropriate dilution of 99.5% (v/v) ethanol (Lab Scan, Ireland). The solutions were kept in tightly sealed containers to prevent loss of ethanol. The detection reagent used in analysis of sulfide was prepared by dissolving 0.07 g of N, N-dimethyl-p-phenylenediamine or DMPD (Sigma-Aldrich, USA) and 0.14 g of iron(III) chloride hexahydrate (Sigma-Aldrich, USA) in 10 mL of 1 mol L-1 HCl (Lab Scan, Ireland). Standard stock solution of sulfide ion (50 mmol L-1) was freshly prepared by dissolving approximately 0.6 g sodium sulfide (Sigma-Aldrich, Germany) in 50.00 ml of 25 mmol L-1 NaOH solution. This stock solution was standardized by recommended standard method.28 Appropriate dilution was made from this stock solution to prepare the sulfide working solutions. The solution of 2 mol L-1 HCl, used as the gas generation reagent, was prepared by appropriate dilution of concentrated hydrochloric acid (37 %, Lab Scan, Ireland). In the analysis of ammonium ion, two types of detection chemistries were used: ‘Nessler’ and ‘3-nitrophenol’ or NTP. The Nessler reagent was prepared by dissolving 0.9 g of mercury (II) iodide (Acros organics, USA), 0.7 g of potassium iodide (Merck, Germany), and 0.3 g of 5 ACS Paragon Plus Environment


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potassium hydroxide (Merck, Germany) in 10 mL deionized water.29 The NTP reagent was prepared by dissolving 0.07 g of 3-nitrophenol powder (BDH, England) in 10 mL of deionized water.27 Stock solution of ammonium ion (1,000 mg N L-1) was prepared by dissolving accurately weighed ammonium chloride (Ajax Finechem, Australia) of approximately 0.4 g in deionized water and the solution made up to 100.00 mL. Appropriate dilution was made from this stock solution to obtain the standard working solutions. The gas generation reagent (1.0 mol L-1 NaOH) was prepared by dissolving 1 g of sodium hydroxide pellet (Merck, Germany) in water and made up to 25 mL. Sample Preparation. Wastewater samples were collected from an ammonium fertilizer factory. These water samples were directly analyzed by our MBL-GS µPAD method without filtration. However for the analysis of the samples using the reference method, gas-diffusion flow injection (GD-FI),30 wastewater samples were filtered through 0.45 µm cellulose acetate membrane (Sartorious Stadium Biotech, Germany) prior to injection into the GD-FI system. Liquid and solid fertilizers were purchased from local agricultural suppliers. Liquid fertilizers were diluted with deionized water to be within the linear working range of analysis for our method and the reference method. Approximately 1 g of a solid fertilizer was accurately weighed and 50.00 mL of deionized water added. The mixture was stirred for 5 min and then filtered (Whatman No. 1 filter paper, UK). Appropriate dilution of the filtrate was made prior to analysis by our method and the GD-FI method. Design and Fabrication of the Devices. There are two designs of our µPAD, designated as ‘Device I’ and ‘Device II’ in Figure 1. Both devices are rectangular with detailed dimensions given in Figure S1 (supporting information). The photographs in Figure 1a show the actual size relative to human fingers. The photographs also show the two opposite sides of a single MBL-GS µPAD, with the donor side labeled ‘D’ and acceptor side labeled ‘A’. Figure 1b shows the schematic 3D drawings of ‘Device I’ and ‘Device II’, respectively. Each 6 ACS Paragon Plus Environment

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device is approximately 1.2 mm thick. The white areas are bare filter paper (Whatman No. 4, UK). The black lines and letterings (D, A) are hydrophobic fabric ink screen-printed on to the filter paper. As shown in Figure 1, the hydrophobic barriers, for containment of liquids, are either circular or dumbbell shape located in the center of the filter paper. The black lines along the edges of the pad are used only to assist in the final cutting step to give separate devices. Figure 1c shows an enlarged view of the three layers that comprise the GS µPAD. The top and bottom layers are the ‘acceptor layer and ‘donor layer’, respectively. The middle or ‘spacer layer’ is made of mounting tape with a circular hole to form a small air space between the donor reservoir DR and the acceptor reservoir AR (Figure 1c).Volatile compound diffuses across this gap from the donor reservoir DR to the acceptor reservoir AR. Operating Procedures. The MBL-GS µPADs in Figure 1 were employed to analyze volatile and non-volatile substances. An ethanol solution was chosen to demonstrate the analysis of volatile compounds in conjunction with ‘Device I’. The operating procedure for ethanol is shown in Figure 2a. In step 1, the device is held with the acceptor side A facing up. A 2-µL volume of the dichromate reagent is placed on the acceptor reservoir with an autopipette (Rainin Instrument, Mettler Toledo, USA). The acceptor zone is then covered with a transparent tape to prevent vaporization of the reagent (step 2). The device is then turned over (step 3) and an aliquot of ethanol standard solution added to the donor reservoir, which is then covered with a transparent tape to prevent loss of ethanol (step 4). The pad is turned over and immediately placed inside the illuminated photographic box (step 5). Vaporization of ethanol then takes place inside the air gap. Ethanol vapor from the donor reservoir reduces the dichromate in the acceptor reservoir leading to a change in its color. The image of the colored acceptor zone is recorded with a digital camera (IXUS 125 HS, Canon, Japan) exactly 3 min after addition of the standard ethanol solution (step 4, Figure 2a). 7 ACS Paragon Plus Environment


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Sulfide ion was chosen as one of the two compounds representing non-volatile substances. In this case ‘Device II’ with the dumbbell shape donor reservoir is employed. The operating steps for analysis of sulfide is shown in Figure 2b. The donor side D is first held facing up for application of the gas generation reagent (2 µL of 2 mol L-1 HCl) on to the dumbbell region opposite the acceptor reservoir (step 1, Figure 2b). The device is then turned over for adding the DMPD reagent (2µL; step 2 Figure 2b) to the acceptor reservoir and taping of the zone with transparent tape (step 3, Figure 2b). The device is turned over a second time with the dumbbell shape donor side D facing up. After inspecting that the gas generation reagent in the donor reservoir is dry, a transparent tape is then placed on top of this lobe (step 4, Figure 2b) Then 10 µL of standard sulfide solution is applied to the adjacent reservoir zone (step 5, Figure 2b). ‘Device II’ is inverted and immediately placed inside the illuminated photographic box (step 6, Figure 2b). The generated sulfur dioxide gas diffuses across the air gap to react with the DMPD reagent at the acceptor zone. The color image of the acceptor side is recorded using the digital camera exactly 2 min after addition of the standard sulfide (step 5, Figure 2b). Ammonium ion is the second non-volatile compound and ‘Device II’ (Figure 1) is employed. The procedure for ammonium analysis is shown in Figure 2c. Standard ammonium solution is aliquoted (3.5 µL) onto the section of the donor reservoir D opposite the acceptor region (step 1, Figure 2c). This aliquot is blow-dried for 2 min at room temperature (step 2, Figure 2c). A second aliquot of ammonium solution is added and blowdried (steps 3 and 4, Figure 2c). This lobe of the donor reservoir is then covered with transparent tape but leaving the adjacent lobe uncovered (step 5, Figure 2c). The device is turned over with the acceptor side A facing up and 2.5 µL of the detection reagent (Nessler reagent or NTP reagent) applied (step 6, Figure 2c). The acceptor reservoir is taped (step 7, Figure 2c). The µPAD is turned over and 12 µL of 1 mol L-1 NaOH added (step 8, Figure 2c) 8 ACS Paragon Plus Environment

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on to the un-covered lobe of the donor reservoir. Sodium hydroxide solution flows along the donor reservoir to react with the ammonium ion, generating volatile ammonia gas which diffuses across the air gap to the acceptor reservoir. Reaction of ammonia gas with the adsorbed reagent leads to a change in the color of the detection reagent. The color image of acceptor reservoir is recorded (step 9, Figure 2c) with the digital camera at exactly 6 min (Nessler) and 5 min (NTP) after the application of the NaOH solution (step 8, Figure 2c).

RESULTS AND DISCUSSION Proof of concept. ‘Device I’ and ‘Device II’ (Figure 1) were used to show that the concept and design of our device can be used to analyze both volatile and non-volatile compounds. Ethanol was chosen as a representative of volatile analytes, whereas sulfide and ammonium were used as representatives of non-volatile analytes. The design of ‘Device I’ is suitable for a compound that vaporizes at room temperature. In ‘Device I’, there is only one circular reservoir on the donor layer D. After step 3 in Figure 2a in which the sample was applied to the donor reservoir, ethanol vaporized from the donor, crossing the air gap of the spacer to react with the pre-deposited dichromate reagent in the acceptor reservoir. As a result, the dichromate ion was reduced to chromium (III) leading to a change in the color of the acceptor reservoir AR, from yellow to greenish brown. The insets of Figure 3a shows the color of the acceptor reservoirs corresponding to concentrations of ethanol from 0 to 7% (v/v), respectively. The color intensity was measured using the Blue intensity scale of the ImageJ program, since it gave the best sensitivity when compared with both the Red and Green scales. Figure 3a shows an example of a calibration line for five concentrations of ethanol. These results clearly demonstrate that the design of our membraneless paper device is successful for separation of ethanol gas from the solution contained within the pores of the



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filter paper (donor reservoir). The linear calibration line in Figure 3a shows that the sensitivity of ‘Device I’ is sufficient for the determination ethanol in beer and wine. Prevention of losses of ethanol vapor was ensured by sealing the exposed side of the paper with transparent tape. Photographic image of the acceptor zone was taken through the transparent tape covering the reservoir. ‘Device II’ was designed for analysis of non-volatile compounds. ‘Device II’ differs to ‘Device I’ only in the pattern of donor layer, which has two circular areas of the filter paper connected to each other in a dumbbell shape (Figure 1c).With this design, a suitable nonvolatile analyte can be converted into a gaseous form in the section of donor reservoir DR located opposite the acceptor reservoir (the circle next to letter D). In the example of sulfide analysis, the gas forming reagent (2 mol L-1 HCl in step 1, Figure 2b) is applied to this region. Standard sulfide solution (or sample containing sulfide) is added on to the adjacent area (step 5, Figure 2b). The sample flows by capillary force from this area to the reservoir DR. Once the sample enters the DR area hydrogen sulfide gas is rapidly generated. The volatile gas diffuses across the air gap of the spacer layer to react with the colorimetric reagent (DMPD) that had been previously loaded in the acceptor reservoir AR (step 2, Figure 2b. The insets in Figure 3b show the color changing from purple to blue with increasing concentration of sulfide ion (0 to 100 mg L-1). The linear calibration in Figure 3b was constructed using Red intensity scale of the ImageJ image processing program. These results demonstrate the viability of ‘Device II’ for converting non-volatile analyte such as sulfide to its volatile form and subsequent detection at the upper acceptor layer. The procedure for analysis of ammonium ion employing ‘Device II’ was developed to demonstrate the situation where greater volume of a non-volatile analyte is required to be loaded in order to achieve the required sensitivity of detection. The sample is first applied and dried twice (steps 1 to 4, Figure 2c) to the donor reservoir DR. The gas generation reagent (1 10 ACS Paragon Plus Environment

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mol L-1 NaOH) is loaded onto the adjacent area and flows to react with the sample (step 8, Figure 2c) to form volatile ammonia gas. Reaction of ammonia gas with the Nessler reagent that was pre-deposited leads to formation of the yellow color in the acceptor reservoir AR. The increasing intensity of the yellow color shown in the insets of Figure 3c and the calibration line in Figure 3c show that the design of MBL-GS µPAD can be successfully employed for quantitation of ammonium ion. Spacer thickness. The effect of spacer thickness on sensitivity was investigated. In our design the spacer thickness is determined by the thickness of the mounting tape. When the thickness was increased from 0.8 to 3.2 mm sensitivity of analysis dramatically decreased, as shown in Figure 4, for all three analytes. Increase in the spacer thickness increases the volume of the air gap (headspace) and hence decrease in the partial vapor pressure of the gas, resulting in lower sensitivity. For this work, a spacer thickness of 0.8 mm was selected. Development of MBL-GS µPADs for determination of ammonium ion in real samples. In order to demonstrate the performance of our membraneless gas separation device for real samples, the design of ‘Device II’ was chosen for further development to measure ammonium ion in wastewaters and fertilizers. Detection chemistry based on the standard Nessler method29 was employed. As discussed above two inter-connected donor zones are required for a non-volatile analyte that can be converted to a volatile compound. The size of the donor reservoir DR (the area directly opposite the acceptor zone) was varied to optimize the sensitivity of the ammonium measurement. Increasing this area will allow larger volume of sample to be applied. The diameter of the cylindrical air gap was also increased to the same width of the donor zone. This leads to a corresponding increase of the volume of the air gap. 11 ACS Paragon Plus Environment


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The top insets in Figure 5a show the three µPADs with DR diameter of 6, 8 and 10 mm, respectively, whilst keeping the diameter of the adjacent area and the acceptor reservoir fixed at 6 mm. The insets in the second row of Figure 5a show 3D-models of the three devices. To show a clearer picture of how the volume of the air gap increases with increase in the donor diameter, the height of the air space is drawn 10 times its actual size (0.8 mm) (Figure S2 in the supplementary information). The air gap volume, for 6, 8 and 10 mm donor diameter, is calculated to be 22.6, 40.2 and 62.8 µL, respectively. A study was first carried out to determine the maximum loading volume of ammonium solution and the gas forming reagent (1 mol L-1 NaOH) for the three sizes of the donor area. For the ammonium analysis, the standard solution is applied directly to donor reservoir DR whilst the NaOH reagent is applied to the adjacent area. Increasing volume of ammonium solution was applied until it was observed that the solution can no longer be contained within the lobe of the application area. Visual observations show that the maximum loading volume of ammonium solution was 2.5, 3.5 and 4.5 µL for donor diameter of 6, 8 and 10 mm, respectively. Since the NaOH reagent must flow into the sample application area, the maximum volume is the volume that completely fill both lobes. The maximum volume of NaOH solution was observed to be 8.0, 10.0 and 12.0 µL for the donor diameter of 6, 8 and 10 mm, respectively. These volumes were used in the following experiments to select the optimal DR acceptor area. A 200 mg N L -1standard ammonium solution was used and loaded only one time (only steps 2 and 3, Figure 2) for each of the three sizes, using the appropriate maximum volume (see above). The corresponding maximum volume of NaOH reagent was then loaded and the color of the Nessler reagent recorded, as described previously. The experiment was repeated 5 times and the mean of the data plotted as bar graphs in Figure 5a. As expected when the diameter of DR zone increased from 6 mm to 8 mm, the yellow color of the Nessler reagent 12 ACS Paragon Plus Environment

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increased (recorded as decrease in the Blue intensity) since more ammonium ion had been loaded. However when the diameter was increased to 10 mm, there was a small decrease in the color (increase in Blue intensity). This can be explained by the decrease in the partial pressure of ammonia in the air space due to the larger volume (62.8 µL), offsetting the increase in the mass of the loaded ammonium ion. For analysis of real samples, the 8 mm diameter of the reservoir DR was selected for ‘Device II’. Concentration of base The effect of concentration of the sodium hydroxide was investigated using the operating procedure for ammonium. The concentration of base was varied from 0.1 to 3 mol L-1 and using standard ammonium of 200 mg N L-1. Results in Figure 5b show that the Blue intensity signal from ImageJ decreased with increasing concentration of sodium hydroxide from 0.1 to 1 mol L-1 and remaining constant for 2 and 3 mol L-1. The yellow color became constant from 1 mol L-1 NaOH and this concentration was therefore chosen for step 8 of the operation procedure (Figure 2c). At this concentration, the amount of base is far in excess even for the maximum concentration of 200 mg N L-1 ammonium ion employed in the calibration (∼100 mole excess). Reaction time The time required for the intensity of the color of the Nessler reagent to become constant was also examined for two concentrations of standard ammonium, 50 and 100 mg N L-1, respectively (Figure 5c). For convenience, we have defined the time interval from step 8 (loading of the sodium hydroxide reagent) to step 9 (recording the image) as the ‘reaction time’. The results in Figure 5c show that color formation reached a constant intensity value after 6 min. Increasing the ‘reaction time’ longer than 6 min did not significantly decrease this value. Thus 6 min was chosen as the optimal reaction time for ammonium analysis. 13 ACS Paragon Plus Environment


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Analytical features The performance of our MBL-GS-µPADs (Device II) is summarized in Table 1. This performance was obtained using the operating procedure shown in Figure 2c and the selected parameters discussed in previous sections (Table S1 of supporting information) for the Nessler reagent. It was necessary to apply the sample solution twice (steps 1- 4, Figure 2c) in order to obtain the low concentration range required for analysis of water samples. The observed linear range for Nessler reagent is 10 - 100 mg N L-1 (Table 1). This performance is better than the commercial test kit that employed the same chemistry (Merck catalog no. 1.16977.0001

for

quantitation

of

NH4+

in

wastewater,

soil

and

fertilizer.

http://www.merckmillipore.com/TH/en/product/Ammonium-Test,MDA_CHEM-116977# anchor_TI), which has a lower limit of 15.5 mg N L-1. Apart from Nessler reagent, we also tested the MBL-GS µPAD with the second reagent for ammonia gas, ‘3-nitrophonol’ or NTP.27 The procedure shown in Figure 2c was employed for the NTP reagent. The same parameters for Nessler reagent in Table S1 were applied except the ‘reaction time’. It was found that the ‘reaction time’ for NTP detection was shorter with the optimal ‘reaction time’ of 5 min (Table S1). As shown in Table 1, the working range using NTP detection was the same as that obtained from the Nessler detection. Comparison of the colors and calibration lines for the two reagents is given in Figure S3 (supplement material). Our MBL-GS-µPAD has the same working range as that observed for the membrane-based µPAD presented earlier by the group of Kolev et al.27 Inside their devices, PTFE strips, inserted between two sheets of filter paper, act as membrane allowing only volatile gas to pass through. The analytical features of our device using NTP detection is also summarized in Table 1. The limit of detection using NTP (8.99 mg N L-1) is greater than that for Nessler (3.14 mg N L-1). The precisions are equivalent (approximately 1% RSDs). The


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results in Table 1 show that we can use our MBL-GS-µPADs for quantitation of ammonium using any chemistry for colorimetric detection. However we propose use of the Nessler chemistry since it has been accepted as the standard method for ammonium analysis.29 Applications and validation. ‘Device II’ was applied to determine the ammonium content in three types of samples: wastewater, liquid fertilizer and solid fertilizer. The operating procedure in Figure 2c was employed using the Nessler reagent for colorimetric detection. As shown in Table 2, a gas-diffusion flow injection (GD-FI) method with bromothymol blue detection was employed as the reference method.30 Paired t-test was used to validate our MBL-GS µPAD method with the reference method. There was no significant difference found between the results obtained from the proposed method and the reference method at 95% confidence level by paired t-test (t-stat = 0.99, t-crit = 2.57). Recoveries of our method were found to be 96 -108 % (sample solutions spiked at 10 mg N L-1 ammonium), which indicated that there was no interferences from sample matrices.

CONCLUSIONS This work, present a new concept of gas-liquid separation in µPAD format that can be employed for direct analysis without pretreatment of sample. We call our membraneless paper-based devices incorporating in-device gas separation as ‘membraneless gas separation microfluidic paper-based analytical devices’ or MBL-GS µPADs. The µPAD consists of three layers: the bottom ‘donor layer’ (with donor reservoir), a ‘spacer layer’ (with a cylindrical air gap) and the top ‘acceptor layer’ (with acceptor reservoir). The cylindrical air space allows volatile compound to diffuse from the donor reservoir to the upper acceptor layer where it reacts with a reagent in the acceptor reservoir leading to a change in color.



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Digital images of the acceptor reservoir are recorded and the color intensity quantitated using the ImageJ software. We present two models of MBL-GS µPADs. The first type, named ‘Device I’, is for analysis of volatile compound, whilst the second type, ‘Device II’, is for non-volatile compound that can be converted to a volatile form. These µPADs differ only in the pattern of the donor zone. The pattern of donor reservoir for ‘Device I’ is a simple circle and is employed for volatile samples such as alcohols. In ‘Device II’ there are two connecting circular reservoirs on the donor layer. For ammonium analysis, the sample is loaded and dried twice on to the donor reservoir (designated DR) located directly beneath the acceptor reservoir. After covering this region with transparent tape, the gas forming reagent is then loaded onto the neighboring reservoir. The reason for having a separate area for loading the gas forming reagent on the donor layer is to prevent loss of the gas. With two connecting circular reservoirs, the gas is not formed when reagent is loaded. The gas is produced only when the reagent has moved into the sample reservoir under capillary action. The transparent tape seals the lower layer of the donor reservoir allowing the gas to diffuse only into the air gap and be adsorbed at the upper acceptor layer. Our developed µPADs have several advantages. Fabrication is simple with most of the materials employed in the fabrication available in laboratories and stationery stores, i.e., filter paper, mounting tape and transparent tape. The patterns on the MBL-GS µPADs can be printed using various techniques. With our screening and fabrication method, the production cost per device is approximately 7 US cent (1 US$ for 15 devices). Our MBL-GS µPADs do not require hydrophobic membrane for gas diffusion to separate the gas from sample, resulting in easier fabrication as well as more economic.27


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ASSOCIATED CONTENT Supporting information Figures S1-S2: Dimension and enlargement view of the MBL-GS µPADs, Figure S3: Calibration curves of Nessler and NTP, Table S1: Selected condition

AUTHOR INFORMATION Corresponding Authors *Tel: +66 2015127. Fax: +66 2015127. Email: [email protected] and [email protected] (Duangjai Nacapricha) [email protected] (Jirayu Sitanurak) Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The scholarships from Royal Golden Jubilee TRF-Ph.D., the Thailand Research Fund for P. Phansi and for J. Sitanurak are gratefully acknowledged. We would like to thank the scholarship from the Development and Promotion of Science and Technology Talents Project (DPST) given to S. Sumantakul. The authors are grateful to the arrangement of the Science Achievement Scholarship of Thailand to support T. Wongpakdee and N. Fukana to work on the proof of concept. The authors acknowledge the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education for the



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support of equipment. Permission for the sabbatical leave from Mahidol University for D. Nacapricha is appreciated. Finally, special thanks go to Prof. Dr. Prapin Wilairat for editing. We would like to thank Ms. Jutamanee Rattana for her photography in production of the graphical abstract.

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Figure 1. Photographs and schematic diagrams showing both sides of ‘Device I’ and ‘Device II’, of the ‘MBLGS µPADs’ for analysis of volatile and non-volatile compounds, respectively.



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Figure 2. The operating procedure employed in the proof of concept of the MBL-GS µPADs for (a) ethanol analysis, (b) sulfide analysis and (c) ammonium analysis. Note: Two reagents are employed for ammonium analysis, ‘Nessler reagent’ and ‘NTP reagent’.


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Figure 3. Calibration curves with the insets of color images of acceptor reservoir of the MBL-GS µPADs for the analysis of (a) ethanol, (b) sulfide and (c) ammonium.



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Figure 4. Effect of spacer thickness of the MBL-GS µPADs on the sensitivity of analysis for (a) ethanol, (b) sulfide and (c) ammonium.


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Figure 5. Investigation of the effect of (a) donor diameter (in millimeter), (b) concentration of NaOH and (c) ‘reaction time’, for the analysis of ammonium ion using the Nessler reagent.



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Table 1. Analytical features of the MBL-GS-µPADs for determination of ammonium

Feature Working range (mg N L-1) Equation (r2) LOD (mg N L-1) RSD, % (25 mg N L-1, n= 10)

Detection chemistry Nessler 3-nitrophenol (NTP) 10 - 100 10 - 100 y = (-0.60 ± 0.05)x + y = (-0.43± 0.02)x (179 ± 2) + (179 ± 1) (r² = 0.999) (r2 = 0.992) 3.14 8.99 1.08 0.97

Note: LOD calculated form (3x(standard deviation of blank)/(slope of calibration line).


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Table 2. Validation data for ammonium quantification in wastewater and fertilizer samples

Sample Wastewater 1 Wastewater 2 Wastewater 3 Liquid fertilizer 1 Liquid fertilizer 2 Solid fertilizer 1 Solid fertilizer 2

Ammonium content GD-FIA30 MBL-GS µPAD (Nessler) -1 11.9 ± 0.8 mg N L 12.1 ± 0.2 mg N L-1 18.4 ± 0.9 mg N L-1 18.2 ± 0.2 mg N L-1 -1 35.0 ± 0.9 mg N L 35.1 ± 0.3 mg N L-1 -1 84.0 ± 29 g N L 83.3 ± 29 g N L-1 -1 0.441 ± 0.1 g N L 0.445 ± 0.1 g N L-1 92.0 ± 2.0 g N kg-1 91.0 ± 1.0 g N kg-1 -1 63.0 ± 2.0 g N kg 64.0 ± 1.0 g N kg-1



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For TOC only


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Membraneless Gas-Separation Microfluidic Paper-Based Analytical

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