Development of a Gas-Diffusion Microfluidic Paper-Based Analytical

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Technical Note

Development of a gas-diffusion microfluidic paper-based analytical device (µPAD) for the determination of ammonia in wastewater samples Badra Manori Jayawardane, Ian D. McKelvie, and Spas D Kolev Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 09 Apr 2015 Downloaded from http://pubs.acs.org on April 9, 2015

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Analytical Chemistry

Development of a gas-diffusion microfluidic paper-based analytical device (µPAD) for the determination of ammonia in wastewater samples Badra Manori Jayawardanea, Ian D. McKelviea,b and Spas D. Koleva, ∗ a

School of Chemistry, The University of Melbourne, Victoria 3010, Australia

b

School of Geography, Earth and Environmental Sciences, Plymouth University, Plymouth PL4 8AA, United

Kingdom

∗

Corresponding Author. Tel. +61 3 8344 7931. Fax +61 3 9347 5180. Email: [email protected]

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Abstract An inexpensive, disposable and highly selective microfluidic paper-based analytical device (µPAD) is described for the determination of ammonia (molecular ammonia and ammonium cation) in wastewaters which implements for the first time a gas-diffusion separation step on a paper-based platform. Its hydrophilic reagent zones were defined by printing filter paper with a hydrophobic paper sizing agent using a conventional inkjet printer. The sample was introduced into the impregnated with sodium hydroxide sample zone of the µPAD. This allowed the quantitative conversion of the ammonium ion to molecular ammonia which diffused across the hydrophobic microporous Teflon membrane of the device into an adjacent hydrophilic reagent zone containing the acid-base indicator 3-nitrophenol or bromothymol blue. The change in indicator color was measured using a desktop scanner for ammonia quantification. Under optimal conditions, the µPAD is characterised by a limit of detection of 0.8 and 1.8 mg N L-1 and repeatability of 3.1% and 3.7% (n ≥ 10, 20 mg N L-1), expressed as relative standard deviation, in the case of 3-nitrophenol or bromothymol blue, respectively. This µPAD was used successfully for the determination of ammonia in sewage and soil water samples. The small dimensions, minimal reagent consumption, low cost, simplicity of operation, and possibility of using a portable scanner make the proposed µPAD an attractive sensor for on-site ammonia monitoring in contaminated environmental waters and domestic, agricultural and industrial wastewaters. The successful implementation of the gas-diffusion approach on a paper-based platform is expected to result in the development of other µPADs for volatile analytes.


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Introduction Ammonia contamination of both freshwater and marine aquatic systems is of considerable environmental concern because as a source of nitrogen it fuels algal blooms and is toxic to fish and other aquatic organisms at relatively low concentrations1. The development of simple and inexpensive methods for routine measurement of the ammonia concentration in domestic, agricultural and industrial effluents, which are the main sources of ammonia contamination of the hydrosphere, is therefore of great importance for ammonia pollution control of environmental waters. Numerous methods for the determination of ammonia have been developed for samples with complex matrices with those involving sample distillation with subsequent colorimetric, potentiometric or titrimetric measurement being the most frequently used2. Most of these methods are either laborious, expensive or include reagent manipulation steps. Colorimetric analytical detection test strips for on-site monitoring of ammonia are commercially available 3

. However, they are relatively expensive and can be imprecise due to human error and

interference by suspended solids and colored compounds. One revolutionary approach to low cost qualitative analysis based on the use of impregnating paper strips was first reported by Yagoda in 19374. Recent research in this area started in 2007 with the pioneering work of Martinez et al5 on the development of microfluidic paperbased analytical devices (µPADs). In these devices, the hydrophilic cellulose support acts as a network of capillaries along which liquids are transported without the need of an external driving force (e.g. propelling device)6, 7. Paper-based devices are of low cost, disposable, portable, easy to store because of their small size, and provide high optical contrast for colorimetric detection8. Unlike Lab-on-a-chip and Micro Total Analysis Systems (µTAS), the hydrophilic channels, and reagent and sample zones are usually in the millimeter range while at the same time these dimensions still ensure that the consumption of samples and reagents is in the low microliter range. This fact has allowed simple, rapid and inexpensive fabrication of µPADs based on the use of conventional inkjet printing techniques involving paper-sizing agents. µPAD can be configured for simultaneous multiple testing of a single analyte9, 10 or multi-analyte determinations on a single device11-14. Martinez et al. have demonstrated that the processing of µPADs data can be done remotely because the colorimetric testing results could be scanned or photographed and transmitted electronically , which is particularly beneficial in environmental monitoring and analysis in remote areas by unskilled staff.15-17 The most commonly used spectrophotometric color reactions in ammonia monitoring are 3 ACS Paragon Plus Environment


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those proposed by Nessler and Berthelot2. However, these reactions cannot produce reliable results if directly applied to samples containing suspended particulate matter or colored compounds. Therefore, in such cases sample preteatment to separate ammonia from the sample matrix is required. A frequently used separation method is distillation2. However, as mentioned earlier, this method is generally time-consuming and laborious which leads to low sample throughput and relatively high cost of analysis. It is also not suitable for on-site determination of ammonia. With the introduction of flow analysis techniques over 40 years ago it has become possible to simplify and speed up ammonia separation from the samples with complex matrices by using a separation technique known as gas-diffusion18. The ammonia sample is introduced into an alkaline donor stream where the ammonium ion is quantitatively converted to molecular ammonia which diffuses across a hydrophobic microporous membrane (e.g. Teflon membrane) into an acceptor stream where detection of ammonia takes place19. Gas-diffusion separation increases substantially the selectivity of ammonia determination by eliminating interferences from suspended particulates and nonvolatile solutes. This allows the use of simple and non-selective analytical reactions such as those involving the protonation of molecular ammonia by acid-base indicators or mixtures of such indicators which result in a color change. This paper describes the development of a low cost µPAD for accurate and reproducible quantification of ammonia in wastewaters based on gas-diffusion separation, applied for the first time on a paper-based platform. The determination of ammonia in the proposed gasdiffusion µPAD involves mixing of an ammonia sample or standard with excess solid NaOH deposited in a hydrophilic reagent zone to produce molecular ammonia which then diffuses across a polytetrafluoroethylene (PTFE) hydrophobic microporous membrane into a detection zone containing an acid- base indicator solution. Change in colour intensity of the detection zone can be related to the ammonia concentration in the original sample or standard. This process is highly selective to basic volatile species such as molecular ammonia, since nonvolatile species and particulates in the sample matrix cannot cross the membrane into the detection zone.


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Experimental Fabrication of the gas-diffusion µPAD Ink-jet printing was used to reproducibly define the circular hydrophilic sample and reagent zones of the proposed µPAD according to a previously reported method20. The zone pattern was designed by using the drawing facility in Microsoft OfficeTM, and printed on Whatman Grade 4 filter paper (0.205 mm thick) using a Canon™ iP4700 inkjet printer with a customfilled printer cartridge containing 4 % (v/v) alkenyl ketene dimer (AKD, Precis 900, Hercules Chemicals Australia) in n-heptane (Scharlau, 99 %). The printed paper was heated at 105 oC for 30 min to make the printed hydrophilic pattern permanent. The proposed µPAD was credit card size (78 mm x 58 mm) and contained 15 pairs of circular hydrophilic sample and detection zones impregnated with sodium hydroxide and indicator solution, respectively (Fig. 1). It was fabricated by assembling the following three layers, as shown schematically in Fig. 1: (1) a layer of patterned filter paper containing a set of fifteen 7 mm in diameter hydrophilic sample zones (Zone 1); (2) a PTFE hydrophobic microporous membrane layer applied as 3 individual strips (12 mm x 78 mm, 0.1 mm thickness); and (3) a layer of patterned filter paper containing a set of fifteen 3 mm in diameter hydrophilic detection zones (Zone 2). Prior to assembling the µPAD 10 µL of 2 mol L-1 NaOH solution were spotted on Zone 1 and oven dried for 5 min at 40 oC (More details about the reagents used are provided in the Supporting Information). The sample zones were covered with 3 strips of the PTFE membrane mentioned above and a layer of patterned filter paper with the detection zones was placed on top of it after carefully aligning the sample (Zone 1) and detection (Zone 2) zones. This was followed by the deposition of 1 µL of indicator solution into each one of the detection zones (Zone 2) and laminating (GBC HeatSealTM H65) the µPAD to maintain the alignment of the sandwiched patterned paper and prevent the evaporation of the indicator solution from the detection zones. The deposited volumes of sodium hydroxide and indicator solutions ensured uniform distribution of these reagents within the corresponding hydrophilic zones. A tissue biopsy punch was used to punch a sample insertion hole of 2 mm in diameter in the plastic cover over the center of each sample zone (Zone 1).



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B

A Figure 1: (A) Schematic fabrication diagram of the proposed gas-diffusion µPAD (one pair of a sample and detection zones is only shown). The diameters of Zone1 and Zone 2 are 7 and 3 mm, respectively. (B) Photograph of the detection zone side of a bromothymol blue µPAD (right) and a 3-nitrophenol µPAD (left). Analytical procedure The proposed paper based method was evaluated by measuring ammonia in influent streams, supernatant and effluent streams from a sewage treatment plant near Melbourne (Victoria, Australia) and soil water samples, obtained from an experimental site in the State of Victoria (Australia). The freshly collected soil water samples (400 g) were centrifuged at 1500 rpm for 15 min using a GT20 Spintron centrifuge

21

. The µPAD results for samples that did not

contain suspended particulate matter and colored compounds were compared with those obtained by the standard spectrophotometric salicylate batch method22. However, it should be pointed out that the use of the µPAD does not require removal of the solid particles in the soil sample by filtering or centrifugation because they are retained in sample zone (Zone 1) and do not reach the detection zone (Zone 2) (Fig. 1). The µPAD results for ammonia in the remaining samples were compared with those produced by a gas-diffusion flow injection analysis (GD-FIA) method based on a previously developed pervaporation flow injection method for ammonia23. Both the salicylate and the GD-FIA experimental procedures are described briefly in the Supporting Information.


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The analytical procedure was conducted at room temperature and involved the deposition of 10 µL of sample or standard solutions into the individual sample insertion holes of the laminated µPAD, which were subsequently covered with a masking tape to prevent sample or molecular ammonia evaporation. Molecular ammonia, produced in the sample zones, diffused across the Teflon membrane and changed the indicator color in the detection zones which were scanned after a predetermined period of time (detection time, Table 1) using a conventional flatbed (Canoscan

TM

Lide 700f) or portable scanner (Pico). These scanners

were used for capturing the color image of the µPAD due to the high precision of their light sources. Another advantage of this approach is that it is not necessary to use a mounting box to get constant illumination, usually required for using a smartphone or digital camera.24-27 Scanned images of the µPAD were stored in JPEG format at 600 dpi. The intensity of the color formation in the indicator zones was measured using Image J software (National Institute of Health USA, http://imagej.nih.gov./ij). The RBG (red, green, blue) color intensity profile plot was obtained for a chord passing through the centre of each detection zone. The highest sensitivity was obtained for blue and red colors for 3-nitrophenol and bromothymol blue, respectively. The measured color intensity was converted to absorbance by the method of Birch and Stickle 28 as Absorbance = - log (I/I0), where I is the mean color intensity of the sample or standard and I0 is the mean color intensity of the blank. Optimisation of the µPAD Table 1 presents a list of the main design and operational µPAD parameters which were optimized in the present study together with their working ranges and optimal values in the order in which the optimization was conducted. Each optimal parameter value corresponds to the maximal absorbance obtained by varying this parameter. More details about the optimisation procedure are provided in the Supporting Information.

Results and Discussion Optimisation results The amount of NaOH deposited in the sample zone in the range from 2.5 to 80 µmole was found to affect significantly the absorbance value. As expected, by increasing this amount the efficiency of conversion of the ammonium cation to molecular ammonia increased which resulted in higher absorbance values. This relationship reached a plateau value above 20 µmole and therefore this value was selected as the optimum amount of NaOH. 7 ACS Paragon Plus Environment


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Table 1: Summary of the optimised µPAD’s parameters. 3-Nitrophenol Parameter Zone 1:NaOH amount

Working range

Bromothymol blue

Optimal value Working range Optimal value

2.5 – 80

20

2.5 – 80

20

1.0 - 10.5

8.0

0.02 – 0.24

0.24

Sample volume (µL)

5 - 20

10

5 - 20

10

Detection time (min)

2 - 15

2-4

2 - 15

2-6

(µmole ) Zone 2: Indicator concentration (mmol L-1)

Bromothymol blue and 3-nitrophenol were selected as potentially suitable indicators for the detection of ammonia because their transition ranges are around pH 7. 3-Nitrophenol is soluble in water and provides a clear color change from colorless to yellow in its transition range (pH 6.6-8.6)29, 30. The effect of the concentration of this indicator on the absorbance value was studied in the range from 1.0 to 10.5 mmol L-1. The absorbance value increased rapidly with the concentration of the indicator up to 8.0 mmol L-1 and levelled off thereafter. Bromothymol blue changes color from yellow to blue in its transition range (pH 6.0-7.6)31. The dependence of absorbance value on the concentration of this indicator was studied in the range from 0.02 to 0.24 mmol L-1 and the highest absorbance value was reached at 0.24 mmol L-1. It was not possible to use higher concentrations of bromothymol blue due to its limited solubility. The effect of sample volume on absorbance value was also studied in the range from 5 to 20 µL. As expected, the absorbance value increased with the initial increase of the sample volume up to 10 µL and thereafter remained practically unchanged. Hence, 10 µL of sample volume was used in all subsequent experiments with the proposed µPAD. The analytical procedure did not allow color intensity measurements to be carried out with satisfactory repeatability within the first 2 min of sample deposition. The absorbance value was found to remain constant between 2 and 4 min in the case of 3-nitrophenol and between 2 and 6 min in the case of bromothymol blue after which it started to decrease (Supporting Information, Fig. S2). The inter- and intra-device repeatability values in the above mentioned time intervals were similar for both indicators (Table 2). Sample and ammonia evaporation into the ambient atmosphere from the sample zone (Zone 1, Fig. 1) through the sample insertion hole after sample deposition can affect sensitivity and repeatability. For this reason 8 ACS Paragon Plus Environment

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4 different tapes (i.e. common adhesive tape, invisible tape, Parafilm, and masking tape) were tested for their ability to prevent sample and ammonia loss through evaporation. The masking tape was found to be best suited for this purpose by ensuring the highest repeatability and absorbance values. Table 2: Analytical performance of the 3-nitrophenol and bromothymol blue µPADs. Parameter

3-Nitrophenol

Limit of detection (mg N L-1)

0.8

Bromothymol blue 1.8

Inter-device repeatability expressed as RSD (%) for 3 µPADs 3.6, 3.1, 3.0

3.9, 3.4, 3.6

Intra-device repeatability expressed as RSD (%) for 3 µPADs 3.1

3.7

RSD of the slope of 3 calibration curves (%)

0.9

1.4

RSD of the intercept of 3 calibration curves (%)

0.6

2.3

4 days

≥ 3 months

20 days

≥ 5 months

≤ 2 months

≥ 8 months

Room temperature Stability (no interleaving sheet)

(light and dark) Refrigerator (≤ 40C) 0

Freezer (≤ -20 C)

Analytical perfomance

Analytical figures of merit The analytical performance of the 3-nitrophenol and bromothymol blue µPADs was evaluated under optimal conditions (Table 1) and the corresponding data are summarized in Table 2. Deionsed water was used as the blank. All color intensity measurements were replicated 15 times and the calculated absorbance values greater than the 90th or less than the 10th percentiles were excluded in calculating the absorbance mean and standrad deviation. The calibration curves of the 3-nitrophenol and bromothymol blue µPADs for their corresponding linear ranges (10 – 100 and 10 - 50 mg N L-1, respectively) are described by Eqs. (1) and (2), which include the standard errors of the regression coefficients. A (3-nitrophenol) = (9.07x10-4±0.07 x10-4)CAmmonia+(6.58 x10-4±3.64 x10-4)

(R2= 1.000) (1)

A (bromothymol blue) = (8.10 x10-4±0.05 x10-4)CAmmonia+(8.14 x10-4±1.36 x10-4) (R2=0.999) (2) The limits of detection (n≥10) of the method was determined by the linear regression method of Miller and Miller32. The inter-device repeatability, expressed as RSD, of 3 µPADs with 15 9 ACS Paragon Plus Environment


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detection zones each and 20 mg N L-1 NH4Cl is shown in Table 2. The 3-nitrophenol and bromothymol blue µPADs were calibrated in 3 consecutive days and the RSD values for the slope and the intercept, presented in Table 2, show good repeatability. The applicability of the bromothymol blue µPAD for different temperatures ranging between 16 and 30 oC and for samples with different pH values in the range 2.2 – 9.0 was investigated. The results presented in Figs. S3 and S4 (Supporting Information) illustrate the negligible effect of temperature in the range studied and pH in the range 2.0 - 8.0 on absorbance. A calibration curve for the bromothymol blue µPAD was successfully obtained by a portable scanner thus showing that color intensity measurements of µPADs could be conducted in the field. This calibration curve is compared in Fig. S5 (Supporting Information) with the calibration curve obtained when using the desktop scanner (Eq. (2)). These results show that the error of using a portable scanner is only slightly higher than that in the case of a desktop scanner.

Selectivity The effect of potential interfering ions and water soluble molecules on the determination of ammonia has been eliminated to a greta extent in flow analysis systems by the introduction of a gas-diffusion separation step33. According to previous studies the most likely interfering compound in this case is methylamine34. It was established in the present study that methylamine generated an absorbance value similar to that of ammonia, e.g. the absorbance for a standard containing 50 mg N L-1 of both ammonium chloride and methylamine was 1.93 times higher than that for a standard containing only 50 mg N L-1 of ammonium chloride (Tables S1 - Supporting Information). However, methylamine is usually present in wastewater samples at the µg L-1 level

35

, and therefore it can be expected that with

wastewater samples its interference will be negligible when using the proposed µPAD.

Stability of the µPAD The storage stability is one of the key factors for developing a disposable analytical device for use in the field. Possible reasons for deterioration in the performance of the 3-nitrophenol and bromothymol blue µPADs, described above, are the reaction of atmospheric carbon dioxide or other acidic gases with the sodium hydroxide deposited in Zone 1 and the indicator solution in Zone 2, as well as degradation of the indicators. Therefore, the µPADs were placed in FoodSaver Vacuum zipper bags and vacuum sealed. Their stability was studied 10 ACS Paragon Plus Environment

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under different storage temperatures, i.e. room temperature (light and dark), ≤ 4oC (refrigerator) and ≤ -20oC (freezer). The stability was evaluated on a daily basis by measuring the intensity before and after the addition of a 50 mg N L-1 NH4Cl standard and the stability monitoring continued until the mean concentration value decreased by more than 2 ϭn-1 from the true value. The results of the stability tests are presented in Fig. S6 (Supporting Information) and are summarized in Table 2. In the case of 3-nitrophenol, loss of the stability at room temperature (light and dark) occurred after 4 days, when it was observed that Zone 1 became yellow, presumably as a result of 3-nitrophenol penetrating across the PTFE membrane, a process that has previously been reported23. In order to prevent this process from occurring, a thin sheet of cellulose acetate was interleaved between the PTFE membrane and Zone 2 prior to lamination. The cellulose acetate sheet was removed by cutting off the edge of the laminated card at its shorter side and drawing it out prior to sample deposition. Inclusion of the interleaving cellulose acetate sheet increased stability from 4 to 10 days at room temperature. When the µPADs were stored in a refrigerator or in a freezer without the introduction of an interleaving cellulose acetate sheet, their stability was found to be 20 days and 2 months, respectively. Bromothymol blue did not appear to diffuse across the PTFE membrane into Zone 1 at room temperature (light and dark) due to its larger molecular size and higher polarity, and no deterioration in the performance of the µPAD was observed over a period of 3 months. For this reason the use of an interleaving sheet was unnecessary. When these µPAD were stored in a refrigerator or freezer their lifetime was prologed to 5 and 8 months, respectively. Storage of the µPADs in the dark did not improve their stability.

Analysis of natural samples The results obtained in the evaluation of the 3-nitrophenol and bromothymol blue µPADs for the determination of ammonia in sewage and soil water samples are summarized in Tables S1 (Supporting Information). Comparison plots presented in Fig. 2 show very good correlation between results obtained using both 3-nitrophenol (Eq. (3)) and bromothymol blue (Eq. (4)) µPADs and the batch and flow injection reference methods, described in the Supporting Information. C (3-nitrophenol) = (1.000 ± 0.003) C (Reference methods) - (0.180 ± 0.098)

(R2 = 0.9999) (3)

C (bromothymol blue) = (0.995 ± 0.003) C (Reference methods) + (0.020 ± 0.085)

(R2 = 0.9999) (4)



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The errors in the slopes and intercepts of Eqs (3) and (4) are the standard errors. The standard errors for the determination of ammonia by the proposed 3-nitrophenol and bromothymol blue µPADs are 0.24 and 0.17 mg N L-1, respectively. The performance of the 3-nitrophneol and bromothymol blue µPADs when applied to water samples of high salinity was studied by spiking seawater samples with NH4Cl. The excellent recovery obtained (i.e. 98-103% and 98-101% for 3-nitrophneol and bromothymol blue, respectivily), as shown in Table S2 (in the Supporting Information), demonstrates that the µPADs, developed as part of this study, are applicable to water samples of high salinity.

A

B

Figure 2: Comparison between results for ammonia determined by the bromothymol blue (A) and 3-nitrophneol (B) µPADs and the batch and flow injection reference methods. Error bars are ± standard deviation for 1σn-1 (n ≥ 10). The main characteristics of the proposed µPAD are compared in Table 3 with commercially available ammonia test strips. The data presented in this table show that ammonia determination based on the proposed µPAD is cheaper than the commercial test strips. In addition, it does not require the use of liquid reagents thus simplifying the analytical procedure and minimizing the potential for contamination. Limit of detection values have not been compared due to the lack of the relevant information provided by the manufactures of the test strips.


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Table 3: Comparison of the proposed µPAD with commercially available test strips. MQuantTM NH4+ test

ReflectoquantR ReflectoquantR Insta-Test NH4+ test 2 NH4+ test 1 Analytic

Indigo NH3 test

Supplier Catalogue #

Merck 1.100024.0001

Merck 1.16977.0001

Merck 1.16899.0001

LaMotte 3023-G

Indigo 33817-AMM

0.49 Cost per analysis (AUD$)

0.82

2.94

2.94

1.80

1.14

Working range 10-100 (mg L-1 NH4-N)

8-310

15.5-140

3.9-15.5

0.4-5.0

8-78

CN- , Fe2+, Mn2+> 10 Ca2+>100

Proposed µPAD

Interferences (mg L-1)

CH3NH2 a

CN-, Fe2+, Mn2+ > 10

Colorimetric reaction/reage nt involved

Acid-base indicator

Nessler reagent

Use of liquid reagents

No

Yes

Sample pretreatment Reaction time (s) a

CN- > 1 Not quoted Fe2+, H2O2 > 10 NO2- > 50 Cu2+, Mn2+, Fe3+ >100 Nessler reagent Indophenol Not quoted blue reaction

Not quoted

Yes

Yes

Yes

Not quoted

Not required Not required

Required

Not required

Not quoted

Not quoted

120

15

240

Not quoted

6

13

Acid-base indicator

Sensitivity is similar to that of ammonia.

Conclusions The µPAD described in this paper is a low cost, fast, and easy to use disposable sensor for quantitative analysis of ammonia in waste and soil waters using colorimetric detection with a desktop or portable scanner. The cost of the device presented here provides analysis at $0.03 per determination taking into account the cost of the paper, fabrication and reagents. In addition, there is no reagent manipulation or sample pre-treatment (neutralization, filtration) required. This is an economic option as well as an easy means of determining ammoniac nitrogen ≥ 5 mg N L-1 in soil waters and wastewaters. To the best of our knowledge, this is the first use of membrane-based gas-diffusion separation on a paper-based platform and the first application of this type of analytical devices for the determination of ammonia. The concept of gas-diffusion separation, which has been so successfully used in flow and sequential injection analysis, is expected to expand the analytical capabilities of the µPAD technology to the selective determination of volatile or semi-volatile analytes or analytes that can be converted to volatile chemical species. 13 ACS Paragon Plus Environment


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Acknowledgements The authors would like to thank the Australian Research Council for financial support of this research (Grant LP110200595) and Hercules Australia for providing the paper sizing agent. B. Manori Jayawardane is grateful for receiving a research scholarship from the University of Melbourne.

Supporting Information Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.


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