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In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/methods Page 1 of 11 Analytical Methods Analytical Methods RSC Publishing 1 2 3 ARTICLE 4 5 sCite this: DOI: 6 10.1039/x0xx00000x 7 www.rsc.org/ 8 9 10 11 12 The development of paper microfluidic devices for presumptive drug 13 detection. 14 Giacomo Musile, 1,2 Ling Wang 1, Jashaun Bottoms 1, Franco Tagliaro 2 and Bruce 15 1 16 McCord 17 A paper microfluidic device has been developed for the presumptive testing of 18 seized drugs in forensic casework. The procedure involves creating hydrophilic 19 channels on chromatographic paper using wax printing and thermal lamination. 20 The channels are connected to a single stem that draws an unknown analyte 21 Manuscript 22 solution up into 6 different lanes. A different colorimetric reaction occurs within 23 each lane, permitting the multiplexed detection of a variety of different 24 compounds, including cocaine, opiates, ketamine, and various phenethyl amines. 25 Furthermore, the linear orientation of the lanes permits series of reactants to be 26 placed in each channel, enhancing stability and permitting sequential interaction 27 with the analyte as the solvent front passes through each individual reagent. The 28 resultant device was characterized for sensitivity and tested with a variety of 29 common interferences and drug diluents. It should prove a useful device for 30 screening seized drugs. 31 Accepted 32 3 a single integrated process. A variety of presumptive 33 Introduction methods have been developed, including spot tests, 34 35 chemical microscopy, TLC, GC and IR but many of In spite of an increasing awareness of the need to 4 36 these tests require skilled handlers, or like spot tests reduce the worldwide spread of illicit drugs, the abuse 37 are incapable of simultaneously determining a wide 38 of psychotropic substances continues to be a rising variety of compounds in a single tube analysis step. A 39 phenomenon. Seizures of cocaine, heroin and illicit cheaper, simpler on-site test capable of performing morphine and cannabis have all increased from 2003 to 40 multiplex sample analysis is needed. Methods 41 2012. Up until 2010 the rate of seizures of amphetamine Microfluidic paper-based analytical devices 42 type stimulants rose at a similar rate; however, recently (µPADs ) with colorimetric detection are a potential 43 the rate has tripled. Although half of all amphetamine- solution to this problem. These devices involve 44 type stimulants were seized in North America, the chemical or enzymatic tests, which can be segregated 45 number of seizures of these compounds continues to and multiplexed by placing them within wax channels 46 increase in the Middle East, while Africa and Asia are printed on chromatographic paper. The sample solution 47 1 emerging as new markets for these drugs. moves in an area delineated by hydrophobic barriers, 48 In most forensic laboratories the detection of seized 49 toward the immobilized reagent via capillary action. drugs involves a two-step process in which a rapid 50 These systems rely on simple chemical reactions, and 51 presumptive test is used for screening followed by a produce visible results that can be interpreted with the Analytical 52 more accurate, identifying step using spectrometric naked-eye. Analytical response is obtained in a few 2 53 instrumentation. Typically, presumptive tests are much minutes, and the measurement can be performed on- 54 less inexpensive, and permit on-site measurements by site. 55 unskilled handlers. The response from these tests must µPADs were originally developed for applications 56 be rapid and the devices performing these tests need to involving medical testing in third-world countries 5 and 57 merge chemistry, signal recognition and processing into have been used for a variety of applications including 58 59 60 This journal is © The Royal Society of Chemistry 2013 J. Name ., 2013, 00 , 1-3 | 1 ARTICLE Analytical Methods Analytical Methods Page 2 of 11 1 the estimation of glucose and protein 5, uric acid 6, Dimethylsulfone, lactose, mannitol, inositol were 2 7 8 9 10 3 ketones , lactate , total iron and pathogenic bacteria . obtained from Fisher (Pittsburgh, PA, United States). 4 Several fabrication methods have been used to obtain King Arthur gluten free flour, Arm & Hammer baking 5 5 microfluidic devices. The fabrication procedure soda, Publix granulated sugar, Rumford aluminium free 6 involves the use of a commercial wax printer and baking powder, Shower Bath Salt absorbent body 7 chromatographic paper to create channels in which powder, iodized salt, Publix antacid tablets, and Argo 8 different reagents are placed .11 Semi-quantitative 100% pure corn starch were purchased from 9 analysis can be performed through the use of digital supermarkets in Miami, FL, United States. Prior to 10 image scanners or cameras. 12 The resulting devices analysis a solution or slurry of each interferent was 11 permit a quick, inexpensive, determination of a wide prepared at a concentration of 100 mg/mL in 50% 12 variety of analytes. acetone/50% deionized water. 13 In this project we have developed a set of paper 14 microfluidic colorimetric tests for the analysis of seized µPAD 15 16 drugs. To the best of our knowledge this work presents A six-lane µPAD was designed as a test bed to 17 a unique process for running multiple assays detect the following psychotropic compounds: cocaine, 18 simultaneously. The test can detect a wide variety of codeine, thebaine, amphetamine, ephedrine, morphine, 19 analytes using only a few micrograms of sample ketamine, MDMA and methamphetamine (Figure 1). 20 solution. Semi-quantitative analysis is also possible The device was prepared using a commercial wax 21 using a smartphone and simple software. The proposed printer (Xerox Color Cube 8750, Xerox, US), which Manuscript 22 method doesn’t require highly qualified persons or was used to print the six-lane pattern on 23 expensive instrumentation, and it can be performed on- chromatographic paper Whatmann no.1 (GE 24 site enabling a prompt analytical response during police Healthcare, UK). Next the paper was placed into an 25 actions, border services, and airport security. 26 aluminum foil pouch and passed twice through a laminator at 160 °C, at a speed of 1.6 cm/sec (Tamerica 27 28 Tashin Industrial Corp, TCC-6000) to melt the wax 29 Experimental completely through the paper and create hydrophobic 30 barriers. 31 Chemicals Each lane was designed to detect a different set of Accepted 32 compounds, with certain channels producing a variety 33 All chemicals were analytical grade. Cobalt of colored responses depending on the analyte present. 34 thiocyanate was purchased from Aldrich; iron (III) Figure 1 illustrates the design of the device with 35 chloride and glycerol were purchased from Acros numbers identifying each channel and letters showing 36 Organics (Waltham, MA, United States); fast blue B, the potential locations for the placement of each 37 molybdic acid, ninhydrin were purchased from Sigma- reagent. Samples and reagents were placed in different 38 Aldrich (St. Louis, MO, Unites States); potassium 39 zones to optimize system stability and color generation. permanganate, sodium hydroxide, hydrochloric acid Lane 1 (L1 in Figure 1) was designed to detect 40 Methods and acetone were purchased from Fisher (Pittsburgh, ephedrine, MDMA and methamphetamine in unknown 41 PA, United States). 42 samples. In this lane, 0.5 µL of an aqueous solution Amphetamine (Amp), methamphetamine (MA), cocaine 43 containing 100 mg/mL sodium sulphate and 10 mg/mL. 44 (Coc), ketamine (Ket), ephderine (Eph) were purchased fast blue B were added to zone A. Lane 2 (L2 in Figure 45 from Sigma (StLouis, MO, United States), morphine 1) was designed to detect four compounds: cocaine, 46 (Morp), codeine (Cod) and thebaine (The) were codeine, thebaine and ketamine. Colorimetric detection 47 purchased from RBI (Natick, MA, United States). was performed using a mixture of 300 µL of 100 48 For each drug, a solution 100 mg/mL in 50% mg/mL cobalt thiocyanate and 200 µL of glycerol. This 49 acetone/50% deionized water was prepared before each reagent was added to L2 zone A (Figure 1). Lane 3 50 analysis. Analytical 51 (L3 in Figure 1) was designed to detect codeine, 52 Interference testing MDMA, morphine and methamphetamine. By using the 53 tip of a spatula, 1 mg of potassium permanganate was 54 Lidocaine and procaine were purchased from Acros pressed onto lane 2 zone A, (Figure 1), and 2 µL of 1 55 Organics (Waltham, MA, United States), quinine was mg/µL molybdic acid were placed at L3 zone B (Figure 56 purchased from J. T. Baker Inc. (Philipsburg, NJ, 1). Lane 4 (L4 in Figure 1) was designed to detect 57 United States) and caffeine was purchased from Sigma- 58 Aldrich (St. Louis, MO, United States).

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