Supporting Information for. Multiple semi-quantitative colorimetric assays in compact embeddable microfluidic

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1 Supporting Information for Multiple semi-quantitative colorimetric assays in compact embeddable microfluidic cloth-based analytical device (µcad) for effective Point of Care Diagnostic Azadeh Nilghaz a,, Saeedeh Bagherbaigi a, Chee Leong Lam a, Sayed Mahdi Mousavi a, Emma P. Cόrcoles a, and Dedy H.B. Wicaksono a,b, * a Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, UTM, Skudai, Johor, Malaysia. b Bio-inspired Medical Devices Lab., Medical Devices and Technology Group (MediTeg), Materials and Manufacturing Research Alliance (MM-RA), Universiti Teknologi Malaysia, UTM, Skudai, 81310, Johor, Malaysia Corresponding Author *Dedy H.B. Wicaksono * dedy.wicaksono@biomedical.utm.my; dedy@utm.my; Tel: ; Fax: Present Address Dept. of Chemical Engineering, Australian Pulp and Paper Institute, Monash University, Clayton Campus, Clayton, VIC 3800, Australia. Page S-1

Performance of the Designs In order to prove the performance of µcads and quantify the amount of used reagents and samples, we utilized colored dyes solution.

2 Performance of the Designs In order to prove the performance of µcads and quantify the amount of used reagents and samples, we utilized colored dyes solution. Three different aqueous colored dyes solutions were localized at the reaction zones. Then, sample inlet points were dipped into yellow dye solution. To fill the 2D and 3D devices, we used 2 and 5 μl of colored dye solutions, respectively. The yellow dye was wicked from wet to dry region along the hydrophilic parts. Colored dye solution was mixed with the dried dyes in the arrays of detection zones to produce green and orange colors without any cross-mixing between the contents of the different reaction zones (Fig. S1A-F). The benefit of these designs is that they can perform multiple-assays using only a minimum amount of analyte solution. This feature is necessary especially in cases where the sample source is limited. Further, we tested the double inlet design for multiple assays with simultaneous onchip calibration. 20 μl of water and 3 μl of blue dye solution could be absorbed by the device in less than ten minutes. The different intensity of blue coloration could be observed in the reaction zones (Fig. S1I). This design demonstrates the low-cost and easy fabrication of 3D μcads for quantifying various concentrations of analytes in parallel. Fig. S1 Photographs of µcads performance evaluated by colored dyes solution. Two- (A, B) and three-dimensional (C-F) µcads designed for multiple assays: before (A), (C-E) and after reaction (B, F). Double inlet µcad (G-I) designed for simultaneous on-chip calibration: Yellow dye solution was stored into bottom layer (reaction zones) as reagent and serially diluted blue dye was dried into stock zone patterned in the middle layer (G). Bottom layer of the device before (H) and after reaction (I). After reaction, serial intensity of green color can be seen in the detection reaction zones (I). Page S-2

3 ANALYTES QUANTIFICATION WITH ON-CHIP CALIBRATION The original detail data shown in Fig. 3 is listed in the following table S1 to table S3. Table S1 Mean intensity of colour as a function of glucose concentration (mm) in Test 1 Detection Zones (The mean intensity of yellow in CMYK format in Adobe Photoshop ) Glucose (mm) Test 1 sample 1 sample 2 sample 3 MEAN S.D. 0* * The mean intensity at 0 level was actually measured for seven samples. The mean and S.D. were calculated from these seven samples accordingly. Page S-3

4 Table S2 Mean intensity of colour as a function of protein concentration (mg/ml) in Test 1 Detection Zones (The mean intensity of cyan in CMYK format in Adobe Photoshop ) Protein (mg/ml) Test 1 sample 1 sample 2 sample 3 MEAN S.D. 0* x x x x x * The mean intensity at 0 level was actually measured for seven samples. The mean and S.D. were calculated from these seven samples accordingly. Table S3 Mean intensity of colour as a function of Nitrite concentration (µm) in Test 1 Detection Zones (The mean intensity of magenta in CMYK format in Adobe Photoshop ) Nitrite (µm) Test 1 sample 1 sample 2 sample 3 MEAN S.D. 0* * The mean intensity at 0 level was actually measured for seven samples. The mean and S.D. were calculated from these seven samples accordingly. Page S-

5 The data were fitted using sigmoid equation as follows Glucose equation R 2 =0.98 Protein Equation R 2 =0.99 Nitrite Equation R 2 =0.99 The graph is shown in figure S2. Calculation of Limit of Detection (LoD) LOD is the x that gives 3*Blank (Standard Deviation at 0) Glucose: 3*Blank = LOD = x = mm Protein: 3* Blank = LOD = x = mg/ml Nitrite: 3* Blank = 13.5 LOD = x = µm. Page S-5

6 Fig. S2 Colorimetric data fitted with sigmoid equations. Page S-6

INTERFERENCE TESTING Analyte Sample Glucose

7 INTERFERENCE TESTING Analyte Sample Glucose (mm) Protein (mg/ml) Nitrite (µm) Figures Glucose Protein Nitrite Test Refer to Fig. 2J sample site no. Test Test Test Test Test Test Fig. S3 Evaluation of the Influence of glucose, protein and nitrite assays to each other. Page S-7

8 a) b) c) Fig. S: Standard curve made from the data in Test 2, collected by a camera for a) glucose assay; b) protein assay; and c) nitrite assay. a) b) c) Fig. S5 Standard curve made from the data in Test 3, collected by a camera for a) glucose assay; b) Protein assay; and c) Nitrite assay. a) b) c) Fig. S6 Standard curve made from the data in Test, collected by a camera for a) glucose assay; b) Protein assay; and c) Nitrite assay. Page S-8

9 a) b) c) Fig. S7 Standard curve made from the data in Test 5, collected by a camera for a) glucose assay; b) Protein assay; and c) Nitrite assay. a) b) c) Fig. S8 Standard curve made from the data in Test 6, collected by a camera for a) glucose assay; b) Protein assay; and c) Nitrite assay. a) b) c) Fig. S9 Standard curve made from the data in Test 7, collected by a camera for a) glucose assay; b) Protein assay; and c) Nitrite assay. Page S-9

10 (a) Control box (b) Fig. S10 Insignificant influence of urine s yellow background colour in 3D µcad (a) of four experiments, as can be seen by comparing the colour in control box with the 0 mm box of the calibration zone; (b) the yellow background colour only affect significantly in conventional 3D volume-based analysis. Page S-10

11 Fig. S11 Red copper stamp used for patterning microfluidic channels on cotton cloth. Fig. S12 2D microfluidic channels patterned by copper stamp on cotton cloth using wax. Page S-11

12 Fig. S13 2D µcad patterned with wax by copper stamp to evaluate the stability of the devices. Detection zones are intended for glucose (reagent is colorless) and protein detection (reagent is yellow color). Reagents was stored and dried into the detection zones. Page S-12

13 Percentages of the Correct Results 120 Stability of the Devices Glucose Protein ᵒ ᵒ ᵒ ᵒ ᵒ ᵒ -18 First Second Third Number of Weeks Fig. S1 Percentages of the correct results after comparisons of glucose and protein assays with artificial urine diluted by BSA (1 mg/ml) and glucose (1 mg/ml) and stored in three different conditions. Page S-13

Fig. S15 (A)-(F) µcad embedded in sanitary napkin; Top (A) and bottom layer (B) of the embedded device in napkin before applying the colored dye solution; Top (C) and bottom layer (D) of the device

14 Fig. S15 (A)-(F) µcad embedded in sanitary napkin; Top (A) and bottom layer (B) of the embedded device in napkin before applying the colored dye solution; Top (C) and bottom layer (D) of the device after applying 300 µl of the colored dye solution; top (E) and bottom layer (F) of the device after applying 10 ml of yellow dye solution. (G)-(L) µcad embedded in baby diaper; Overall view (G) and bottom layer view (H) of the embedded device in diaper before applying the colored dye solution; top (I) and bottom layer (J) of the device after applying 500 µl of the colored dye solution; top (K) and bottom layer (L) of the device after applying 25 ml of yellow dye solution. Page S-1

15 Some comments related to the fabrication technique being implemented in this work In terms of the fabrication technique used to make our device, in accordance to the aforementioned aim of our research, we use simple and low-cost fabrication technique available in even resource poor regions. Most microfluidic which are made of silicon (Gravesen, 1993), glass (Rodriguez, 2003), polymers (Zhang et al. 2009; Gong et al. 2010; Cerdeira Ferreira et al. 2013) and even papers (Carrilho et al. 2009b) are still made using photolithography technique which requires special equipments, consumables and expertise. Some paper-based device is made using wax printing machine (Carrilho et al. 2009a) which even though more straightforward, yet still requires a special wax printing machine. We have proposed fabrication method for microfluidic cloth-based analytical device (µcad), which is inspired from batik processing technique (Nilghaz et al. 2011; Nilghaz et al. 2012; Belfer 1992). More complex device can be made using folding technique (origami) which was proposed almost at the same time by us (Nilghaz et al. 2012) and others (Liu and Crooks 2011). While the fabricated device using our batik-inspired method may not be as accurate and as miniaturized as other devices made by more sophisticated machines, our device is easy to fabricate at prototyping level using simple batik drawing, or for mass production using batik printing technique (Belfer 1992). This will enable its rapid delivery to people in need at an affordable price due to its low development cost. REFERENCES Belfer N (1992) Batik and Tie Dye Techniques. Dover Publications, Carrilho E, Martinez AW, Whitesides GM (2009a) Understanding Wax Printing: A Simple Micropatterning Process for Paper-Based Microfluidics. Analytical Chemistry 81 (16): doi: /ac901071p Carrilho E, Phillips ST, Vella SJ, Martinez AW, Whitesides GM (2009b) Paper Microzone Plates. Analytical Chemistry 81 (15): doi: /ac90087g Cerdeira Ferreira LM, da Costa ET, do Lago CL, Angnes L (2013) Miniaturized flow system based on enzyme modified PMMA microreactor for amperometric determination of glucose. Biosensors and Bioelectronics 7 (0): doi: Gong X, Yi X, Xiao K, Li S, Kodzius R, Qin J, Wen W (2010) Wax-bonding 3D microfluidic chips. Lab on a Chip 10 (19): Gravesen P, Branebjerg J, Jensen OS (1993) Microfluidics-a review. Journal of Micromechanics and Microengineering 3 (): Liu H, Crooks RM (2011) Three-Dimensional Paper Microfluidic Devices Assembled Using the Principles of Origami. Journal of the American Chemical Society 133 (): doi: /ja Nilghaz A, Wicaksono DHB, Majid FAA Batik-inspired wax patterning for cloth-based microfluidic device. In: Instrumentation Control and Automation (ICA), nd International Conference on, Nov pp doi: /ica Nilghaz A, Wicaksono DHB, Gustiono D, Abdul Majid FA, Supriyanto E, Abdul Kadir MR (2012) Flexible microfluidic cloth-based analytical devices using a low-cost wax patterning technique. Lab on a Chip 12 (1): Rodriguez I, Spicar-Mihalic P, Kuyper CL, Fiorini GS, Chiu DT (2003) Rapid prototyping of glass microchannels. Analytica Chimica Acta 96 (1 2): doi: Zhang W, Lin S, Wang C, Hu J, Li C, Zhuang Z, Zhou Y, Mathies RA, Yang CJ (2009) PMMA/PDMS valves and pumps for disposable microfluidics. Lab on a Chip 9 (21): Page S-15

16 SUPPLEMENTARY VIDEOS Supplementary Video 1 contains step by step procedures of preparing the 3-D microfluidic cloth-based analytical device for multiple assays with on-chip calibration. The steps illustrated in the video are the following: 1. Spotting Bromophenol Blue (BPB) into the detection zones of the protein assay part of the device (long rectangular channels) 2. Spotting nitrite assay reagent into the detection zones of the nitrite assay part of the device (box-shaped hydrophilic containers) 3. Spotting glucose assay reagent into the detection zones of the glucose assay part of the device (box-shaped hydrophilic containers). Spotting serially-diluted nitrite sample solutions into the stock zones of the nitrite assay part of the device (long rectangular channel) 5. Spotting serially-diluted Bovine Serum Albumin (BSA) sample solutions into the stock zones of the protein assay part of the device (box-shaped hydrophilic containers) 6. Folding the 2-dimensional CMD along the folding lines to make 3-dimensional CMD. 7. Gluing the side lines to form a compact 3-dimensional CMD and pressing it under a mass. 8. Optionally, further pressure can be exerted to the folded device using pressing machine. The steps of spotting the reagents and serially-diluted analytes may be varied according to the situation. In the current video, we do not show the step of spotting the serially-diluted glucose sample solutions into the stock zones of the glucose assay part of the device. Supplementary Video 2 contains step by step procedures of using the 3-D microfluidic cloth-based analytical device for multiple assays with on-chip calibration. The steps illustrated in the video are the following: 1. Dipping one of the inlet point (i.e. the water inlet point) to wick water for eluting the serially diluted samples into the detection zones for mixing with chemical analytical reagents. 2. Water is wicked from the inlet point and elutes the diluted samples in the stock zones branches. 3. Incubation: mixing the diluted samples with the analytical reagents stored at the detection zones, forming a calibration colour pattern.. Turning the CMD 180, dipping the analyte inlet point to a sample containing various analytes to be assayed. 5. Incubation: reaction between the analytes with the respective colourimetric reagents. 6. Cutting the sides of the CMD for unfolding the device to see the results of the multiple assays. The cut away parts are the isolator layers. Supplementary Video 3 (Part 1 to 3) contain step by step fabrication procedures of embedding µcad into baby diapers. Supplementary Video (Part 1 to 3) contain step by step fabrication procedures of embedding µcad into sanitary napkin. Page S-16

17 Supplementary Video 5 contains a test of embedded µcad in baby diapers, where 500 µl of artificial urine containing unknown arbitrary concentration of glucose and protein was poured onto diaper. Supplementary Video 6 contains a test of embedded µcad in baby diapers, where 500 µl of control artificial urine sample with no glucose nor protein was poured onto the diaper. Supplementary Video 7 contains a test of embedded µcad in sanitary napkin, where 300 µl of artificial urine containing an unknown arbitrary concentration of glucose and protein was poured onto the sanitary napkin. Supplementary Video 8 contains a test of embedded µcad in sanitary napkin, where 300 µl of control artificial urine sample containing neither glucose nor protein was poured onto the napkin. Page S-17

ELECTRONIC SUPPLEMENTARY INFORMATION (ESI) Flexible Microfluidic Cloth-based Analytical Devices Using Low-Cost Wax Patterning Technique

ELECTRONIC SUPPLEMENTARY INFORMATION (ESI) Flexible Microfluidic Cloth-based Analytical Devices Using Low-Cost Wax Patterning Technique Azadeh Nilghaz, a,b Dedy H.B. Wicaksono,* a Dwi Gustiono Riban, c