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Inkjet-Printed Paper-Based Colorimetric Polyion Sensor Using Smartphone as a Detector Xuewei Wang, Mollie Mahoney, and Mark E. Meyerhoff Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03352 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 5, 2017

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Analytical Chemistry

Inkjet-Printed Paper-Based Colorimetric Polyion Sensor Using Smartphone as a Detector Xuewei Wang,* Mollie Mahoney, and Mark E. Meyerhoff* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States ABSTRACT: The first paper-based polyion-sensitive optodes are reported. Dinonylnaphthalene sulfonic acid (a cation exchanger) and chromoionophore I (a lipophilic optical pH indicator) are printed on filter paper in the absence of any plasticizer and/or additional hydrophobic polymeric phase. The resulting optodes exhibit sensitive colorimetric response to polycations such as protamine but not to small inorganic cations because only polycations are able to form cooperative ion pairs with dinonylnaphthalene sulfonate adsorbed to the cellulose paper. The color change of the optode is recorded via an iPhone camera and analyzed by an iPhone App. The protamine sensing optode platform is used to indirectly detect protease activity (trypsin) based on proteolytic digestion of protamine, and polyanions (pentosan polysulfate and heparin) based on the strong binding reaction of polyanions with protamine. The indirect sensing system is further simplified on a multilayer membrane device that consists of an optode paper site modified with buffer to prevent optode dependence on sample pH, and an underlying cellulose acetate filter membrane coated with protamine to eliminate addition of the indicator polycation into the sample. The detection of pentosan polysulfate concentrations in an undiluted urine sample is successfully demonstrated via this approach. Lastly, it is shown that plasticizer-free polyanion-sensitive optodes based on an adsorbed layer of quaternary ammonium type anion exchanger and a phenolic azo type proton chromoionophore can also be fabricated directly on cellulose paper strips.

Polyions are polymers with multiple positive or negative charges. Examples of important polyions include biological molecules such as nucleic acids and arginine/lysine-rich polypeptides, pharmaceutical molecules such as pentosan polysulfate, heparin, chondroitin sulfate, and fucoidan, food additives such as carrageenan and alginate, and flocculating agents/cosmetic ingredients such as polyquaterniums. Direct quantification of polyions is known to be challenging because a large number of polyions don’t have intrinsic visible absorption, fluorescent, or redox active units. Rather, oppositely charged dyes or nanoparticle dyes, oppositely charged redox active molecules, oppositely charged polymers, or oppositely charged surfaces are often used to indirectly indicate the levels of polyions present in given samples via optical methods (e.g., spectrophotometry, fluorimetry, surface plasmon resonance spectroscopy, Raman spectroscopy),1-5 electrochemical methods (e.g., voltammetry, impedimetry)6-10 and other techniques such as quartz crystal microgravimetry.6-7 However, because of tedious protocols, required instrumentation, and interference from sample matrix components, these methods have had limited applications. Ionophore-based plasticized polymeric membrane electrodes are best known as highly selective and reliable sensors for electrolytes (e.g., Na+, K+, Ca2+, Cl-) in complex physiological samples.11,12 Our group discovered that the same configuration with appropriate ion-exchanger ionophores could be used for potentiometric determination of polyions such as protamine and heparin.13-15 In such sensors, polyions are selectively extracted into polymeric electrode membrane via cooperative ion-pairing interaction with the ion-exchanger. Such phase transfer process induces a quasi-steady-state nonequilibrium phase boundary potential change that is proportional to the concentration of target polyions in aqueous

phase.14 More recently, fully reversible polyion sensing methods with polymeric membranes or water-immiscible liquid phases have been proposed by using electrochemically controlled polyion transfer strategies.16-25 Polyion-sensitive optodes, the optical counterpart of polyion-sensitive electrodes, have also been developed based on plasticized polymeric membranes doped with a lipophilic ion-exchanger and a proton-selective chromoionophore.26-30 Extraction of polyions into the membrane triggers ejection of protons from the membrane (for polycations) or extraction of protons into the membrane (for polyanions), that changes fluorescence or visible absorption properties of the polymeric film. Compared to electrode techniques relying on two- or three-electrode systems, optodes are more flexible in configuration, easier to miniaturize, and more compatible with ultralow-cost strip type sensing, high throughput analysis, and imaging.31,32 Recently, we discovered that ionophore-based optodes for small ions like Na+ and F- could be fabricated on cellulose paper without using plasticizers or hydrophobic polymers.33,34 The adsorption layer formed on the surface and in the bulk of the cellulose fibers by the hydrophobic sensing ingredients (ionophore, chromoionophore, ion-exchanger) functions as a water-immiscible ion sensing phase, analogous to traditional plasticized polymer-based optode membranes. Apart from elimination of toxic plasticizers and polymers, the paper-based optodes have common advantages of paper-based analytical devices (PADs) such as ultralow cost, ease to package, transport, and carry, and high compatibility with modern printing techniques and pump-free microfluidics.35-37 By combining optical sensing paper with chemically modified paper that enables separation/decolorization of the sample or elimination of other interference species, chemical analysis in complex matrices could be potentially achieved in an integrated paper-

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based device.38 Furthermore, highly porous paper sheets allow modification with buffers or reactants, which can help address the sample pH dependence issue of ion-selective optodes or enable on-site chemical reactions for indirect sensing applications. Similar to optodes for small ions, previously reported polyion optodes have all been based on plasticized polymers containing appropriate sensing ingredients, and have been fabricated as films on inert substrates such as glass slides and microtiter plate wells, or as suspended nanospheres.26-30 However, recognition of polyions is based on their cooperative electrostatic interaction with ionic sites rather than the typical host-guest supramolecular chemistry for small ions. Therefore, it is interesting to explore if such polyion recognition and sensing chemistry could be adapted onto the surface of paper strips, without need for a separate bulk plasticizer or polymer phase. Moreover, any new sensing scheme for polyions can enable applications for enzyme activity testing,39-44 enzyme inhibitor screening,41,42,45 non-separation immunoassay,46 and label-free aptamer-based sensing.23,47,48 Therefore, herein, we examine the feasibility of preparing paper-based optical polyion sensors without added plasticizer/polymer as a new costeffective platform for various polyion sensing applications, especially for potential use in resource-poor settings.

EXPERIMENTAL SECTION Reagents and Materials. Chromoionophore I, chromoionophore IV, protamine sulfate salt from salmon, trypsin from bovine pancreas (Type I, ~10,000 BAEE units/mg protein), heparin sodium salt from porcine intestinal mucosa, 2nitrophenyloctyl ether (NPOE), bis(2-ethylhexyl) sebacate (DOS), and tridodecylmethylammonium (TDMA) chloride were purchased from Sigma-Aldrich. Dinonylnaphthalene sulfonic acid (DNNS) was a gift from King Industries. Pentosan polysulfate sodium was kindly provided by Dr. Peter Ghosh (Royal North Shore Hospital, Sydney, Australia). WhatmanTM qualitative filter paper circles (grade 5; diameter: 18 cm; thickness: 200 µm) and WhatmanTM 1 Chr Chromatography paper sheets (25 cm×25 cm, thickness: 180 µm) were purchased from Fisher Scientific. Cellulose acetate (CA) filter membranes (pore size: 0.45 µm; thickness: 120 µm) were a product of Sartorius AG. Optode Fabrication. For polycation sensing optodes, 1.16 mg of chromoionophore I and 0.92 mg of DNNS (for plasticizer-free optode) or 1.16 mg of chromoionophore I, 1.56 mg DNNS, and 50 mg DOS (for DOS-based optode) or 1.16 mg of chromoionophore I, 0.92 mg of DNNS, and 50 mg NPOE (for NPOE-based optode) were dissolved in 0.58 mL of cyclohexanone. The resulting cocktail ink was filled into a Dimatix Materials Cartridge (10 pL drop size) and printed onto Whatman qualitative filter paper by a Dimatix MP-2831 Inkjet Printer. Drop spacing was 25 µm and the number of printed layers was 1. Both the cartridge nozzle and platen were at room temperature. Maximum jetting frequency was set to 5k Hz. For preparation of polyanion sensing optodes, 1.7 mg of TDMA chloride and 1.6 mg of chromoionophore IV were dissolved in 0.9 mL of cyclohexanone and 0.1 mL of tetrahydrofuran as the ink. Drop spacing was set to 15 µm and the number of printed layers is 3. Other conditions are the same as for the polycation optode fabrication. All optodes were stored in the refrigerator in the presence of desiccant. Colorimetric

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tests and other characterizations were performed after at least one day of storage. A hole punch (Sparco Adjustable 2-Hole Punch) was used to cut a piece of circular optode (1/4’’ in diameter) from the large filter paper and the small optode was adhered onto a piece of Parafilm by using the adhesive property of the Parafilm. For the optode when used in a layered membrane device, an array of black wax circles (1/4’’ in diameter, unfilled, two layers) was printed onto the chromatography paper by a Xerox ColorQube 8580DN Solid Ink Printer. By heating this paper through a ScotchTM thermal laminator 5 times, the wax melts through the paper and forms as a hydrophobic 3D barrier for aqueous solutions. Then, optode ingredients were printed within this wax circle by using the Dimatix Inkjet Printer. Thirty µL of 0.1 M HEPES buffer at pH 7.4 was added onto the optode area and dried under vacuum to function as a sample pH adjuster. Plasticized PVC-based optodes for water contact angle tests were prepared by spin-coating of tetrahydrofuran cocktails on glass coverslips. The cocktail contained 0.42 mg chromoionophore I, 0.33 mg DNNS, 11 mg PVC, and 22 mg DOS or NPOE. Fabrication of Multilayer Membrane Device. CA filter membranes were hydrated with DI water containing 1000 µg/mL protamine for 1 min and then sandwiched between two pieces of filter paper for seconds to remove excess solution. The membrane was then dried in a 37˚C oven for 30 min. A piece of circular membrane (1/4’’ in diameter) was cut from the protamine-coated CA membrane by a hole punch and pressed onto a piece of Parafilm. A larger circle (¾’’ in diameter) with the optode/wax area in the middle was then cut from the chromatography paper by a Fiskars Lever Punch. Finally, this paper circle was firmly adhered onto the prepared CA membrane-Parafilm with the optode area right above the CA membrane. Physical Characterization. XPS spectra were collected by a Kratos Ultra AXIS photoelectron spectrometer with a monochromatic Al-Ka radiation source. Depth profiling analysis was performed using an argon ion sputtering gun operating at 4 keV and a raster size of 2 mm. Survey scans were collected with a pass energy of 160 eV and a scan step of 1eV. Confocal fluorescence imaging was performed on an ALBA TimeResolved Confocal Microscope. A 630 nm laser was used as the excitation light and an APD detector was used to collect fluorescence in a wavelength range of 700±37.5 nm. The employed objective lens was a 60× water immersion lens. The spatial resolution of all images was 512 × 512 pixels. Forty layers of 2D images with a vertical step width of 1 µm were reconstructed to a z-stack image by using ImageJ software. Water contact angles were measured using a Cam 100 optical contact angle goniometer. Photography. The prepared Parafilm with a single layer of optode paper or layered optode-CA membrane on the top was further adhered on the back side of a transparent 96 well microtiter plate. Pictures of optodes were taken in a homemade black box by an iPhone 5S and the LED flash of the smartphone was used as the sole light source.

RESULTS AND DISCUSSION Colorimetric Responses of Plasticizer-Free and PlasticizerBased Optodes on Paper toward Protamine


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Figure 1. Schematic illustration of response principle of the polycation optode based on chromoionophore I and DNNS.

DNNS has been found previously to be an excellent polycation ionophore in plasticized polymers and organic liquids because of its cooperative ion-pairing interaction with polycations.49 To visibly indicate this electrostatic interaction, chromoionophore I, a lipophilic proton-selective dye derived from Nile blue was co-doped into the sensing phase. This type of pH indicator (chromoionophore) has been widely used previously as the optical read-out element of various ion-selective optodes.11,32 Figure 1 illustrates the mechanism of a polycation sensing optode based on DNNS and chromoionophore I. In an aqueous buffer solution at near neutral pH, chromoionophore I’s amine group is protonated because of the presence of DNNS as a counterion. When there are polycations in aqueous solution, they will transfer into the sensing phase and form ionic complexes with multiple DNNS anions. To maintain phase electroneutrality, chromoionophore I becomes deprotonated and protons are expelled into the aqueous phase. Chro-

moionophore I changes its color from blue to purple upon deprotonation, which forms the basis of optical polyion analysis. Kim et al. previously reported protamine sensors based on PVC-DOS membrane on microtiter plate by using this sensing principle.27 In this work, three types of polycation optodes were fabricated on cellulose paper by using an inkjet printing technique. The plasticizer-free optode has only DNNS and chromoionophore on paper and the other two optodes, for comparison purposes, also have DOS or NPOE as a plasticizer. Figure 2A shows pictures of these optodes in pH 7.4 Tris-HCl buffer containing different concentrations of protamine. By using a color analysis App “Color Mate - Convert and Analyze Colors”,50 the hue of each picture was extracted and used to plot calibration curves toward protamine (Figure 2B, left). Interestingly, the plasticizer-free optode shows a detection limit, dynamic range, and selectivity over small inorganic cations that are quite similar to the plasticizer-based optodes. In the presence of plasticizer, sensing ingredients are dissolved in the plasticizer phase supported by paper and the sensing principle is essentially the same as the traditional plasticizer-based optodes with PVC as the membrane backbone. However, the plasticizer-free optode can only rely on the chromoionophore I and DNNS molecules adsorbed on/in the cellulose fibers of the paper. Like any other sensing elements in ion-selective electrodes/optodes, these two chemicals are also highly hydrophobic. Indeed, LogD values of protonated chromoionophore I

Figure 3. Water contact angle results of polycation optodes using cellulose paper and plasticized PVC. All pictures were taken ~ 1s after addition of the water drop. Water fully spreads on paper optode within a few seconds, but doesn’t spread on PVC-based optodes.

Figure 2. A: pictures of different paper-based polycation-sensitive optodes in 30 µL of 0.1 M Tris-HCl buffer (pH 7.4) containing different amounts of protamine; B: hue-based calibration curves toward protamine sulfate, NaCl, and KCl on different paper-based optodes.

and DNNS anion are predicted to be 7.7 and 7.9, respectively, by MarvinSketch (ChemAxon). Also, a negligible difference in hydrophobicity has been observed between the plasticizerfree optode and the plasticizer-based optode via water contact angle measurements, although all porous cellulose paperbased surfaces show much lower hydrophobicity than the plasticized PVC-based optodes (Figure 3). Therefore, the two hydrophobic chemicals are likely to form a water-immiscible adsorbed phase that mimics the plasticizer-based layer/membrane. Protamine molecules interact with such an adsorption phase via their electrostatic interactions with DNNS, and chromoionophore I becomes deprotonated, which is responsible for the observed color change of the plasticizer-free optode. To confirm the DNNS-based response mechanism, various tetraphenylborate type cation exchangers were used to replace the DNNS in the plasticizer-free optodes. This type of anion exchanger does not form strong ion pairs with the polycationic protamine because of sterically hindered anionic charge, and thereby a lower sensitivity is expected.49 Indeed, optodes based on chromoionophore I and tetrakis[3,5bis(trifluoromethyl)phenyl]borate or tetrakis(4-



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chlorophenyl)borate show negligible responses toward protamine at a concentration of 100 µg/mL or lower (see Figure S1 for an exemplary response). The smaller response of the tetraphenylborate type ion-exchanger-based optodes also indicates that the observed protamine response on plasticizer-free optodes is not due to any non-specific adsorption of protamine with the underlying cellulose paper. Cyclohexanone (viscosity: 2.02 cp, surface tension: 34.4 dynes/cm) was used here as the solvent of all cocktail inks, which is an alternative to the commonly used casting solvent, tetrahydrofuran, for fabrication of ion-selective sensors. This is because the viscosity of tetrahydrofuran (0.46 cp) is much lower than the recommended viscosity (2 to 30 cp) for inkjet printing, and fast evaporation of tetrahydrofuran causes the cartridge nozzle to become clogged easily. However, considering the relatively high boiling point of cyclohexanone (156 °C vs. 66 °C of tetrahydrofuran), there may be residual cyclohexanone on the paper after printing. In contrast to traditional plasticizers having very low solubility in water (< 0.1 g/L for DOS and NPOE), cyclohexanone has a much higher water solubility (25 g/L) but is not fully water-miscible. Therefore, one question is whether the residual cyclohexanone functions as a “pseudo-plasticizer” in the plasticizer-free optode. We tested the amount of cyclohexanone on the optode paper by extracting chemicals from the paper optodes into tetrahydrofuran and analyzing the tetrahydrofuran solution by GC-MS (Shimadzu QP-2010). Residual cyclohexanone in one plasticizer-free polycation optode (1/4’’ in diameter) was found to be only 0.19 µg. The weight ratio of cyclohexanone to chromoionophore I on this optode is only 0.19:1, which is ~2 orders of magnitude lower than the typical plasticizer to chromoionophore ratio of traditional optodes for small ions and polyions.27 Moreover, manually fabricated polycation optodes prepared by using a tetrahydrofuran-based cocktail containing the same sensing ingredients exhibit a color change similar to the printed optode toward protamine (pictures not shown). Tetrahydrofuran is easy to evaporate and also fully miscible with water, and thus it is impossible to function like plasticizer. Overall, there is only a very low amount of residual cyclohexanone on the plasticizer-free optode and this solvent does not seem to be critical for the observed optical response. The response time of the paper-based optodes was examined by recording the hue change of the sensor over time after addition of 50 µg/mL protamine (Figure S2). Similar to previously reported plasticized PVC-based polyion optode films that have a response time of >10 min,27 the DOS and NPOE-based optodes on paper exhibit slow responses as well. The hue increase of the DOS and NPOE-based optodes from 5 min to 20 min after sample addition is as large as 15.5° and 14.0°, respectively. In contrast, the plasticizer-free optode on paper shows a drift of only 8.8° over the same period of time, indicating a faster response time. However, this response is much slower than that of protamine optode based on nanospheres with an average dimeter of 0.1 M of Na+, the colorimetric response of protamine in


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Figure 5. Pictures of plasticizer-free optodes in 30 µL of Tris-HCl buffer containing 50 µg/mL protamine and various amounts of trypsin, and the corresponding calibration curve. Protamine and trypsin were incubated for 5 min for enzymatic digestion before the buffer was added onto the optode, and pictures were taken after another 5 min. Data points are average ± s.d. for n= 3 sensors.

Tris-HCl buffer containing 0.14 M NaCl was initially examined (Figure S5). Although the response toward a low concentration of protamine (10 µg/mL or below) becomes smaller because of competition from the Na+, the response slope over a concentration range of 10 to 500 µg/mL (10-5.7 to 10-4 M) is similar to that in buffer without NaCl (Figure 1, black squares). This optode is most sensitive at a protamine concentration around 50 µg/mL. Therefore, 50 µg/mL protamine in Tris-HCl buffer with 0.14 M NaCl has been used for the following indirect applications. The determination of trypsin activity in blood or urine is important for diagnosis of pancreatic diseases like pancreatitis and cystic fibrosis, and prognosis of pancreas transplant sur-

Figure 6. Pictures of plasticizer-free optodes in 30 µL of Tris-HCl buffer containing 50 µg/mL protamine and various amounts of PPS (A) or heparin (B), and the corresponding calibration curves. Pictures were taken at 5 min after sample addition. Data points are average ± s.d. for n= 3 sensors.

gery.56,57 A trypsin assay could also be used for screening of trypsin inhibitors as drugs.41,42,45 Therefore, trypsin sensing was chosen as an initial example of an enzyme activity assay using the new polycation optode system. Trypsin is able to digest arginine-rich protamine into small fragments via cleaving the carboxyl side of arginine.39 A reduced concentration of intact protamine after digestion by trypsin will induce a smaller colorimetric response on the optode. As shown in Figure 5, protamine solutions incubated with increasing activities of trypsin do indeed yield increasingly blue optodes. This method exhibits a linear range of 0 to 5 U/mL and a detection limit of 0.25 U/mL or 25 ng/mL (3σ/S). This detection limit is lower than or comparable to those obtained by potentiometric or chronopotentiometric polyion electrodes39-41 and some other colorimetric trypsin assay methods.58-60 This dynamic range is also potentially suitable for diagnosing pancreatitis since blood trypsin concentration has been found to be 0.25 ± 0.1 µg/mL in healthy individuals and increases to 1.4 ± 0.6 µg/mL in acute pancreatitis patients.61 Moreover, the smartphonebased optode technique relies on simple instrumentation, has ultralow cost per strip sensors, uses a very low amount of sample (a few µL to tens of µL), and doesn’t require any sample convection, which makes it an attractive protease assay strategy for potential screening purposes. The protamine optode was further used to detect polyanionic PPS (an anti-inflammatory and chondroprotective drug, an anticoagulant) and heparin (an anticoagulant) that could neutralize the positive charge of polycations. As shown in Figure 6, with a higher concentration of polyanion, the optode becomes bluer due to a reduced concentration of free protamine. The linear range of PPS detection is 0 to 25 µg/mL and the detection limit is 1.4 µg/mL (3σ/S). Human and animal experiments have demonstrated that the concentration of PPS in blood or urine may range from 0.5 µg/mL to 2000 µg/mL after drug administration, and most blood/urine samples have a PPS concentration higher than the detection limit of the proposed optode.62,63 Therefore, this optode may form the foundation of a new point-of-care PPS assay platform that is much simpler and faster than the current ELISA kit, which requires a total assay time of >5h.64 Further, the new polyion optode could benefit clinical studies of PPS pharmacokinetics and dose optimization for therapy of diseases such as interstitial cystitis, mucopolysaccharidosis and allergic rhinitis.65-67 Bedside monitoring of blood heparin levels is known to be critical for administration of an appropriate dose of heparin during extracorporeal procedures and an appropriate dose of heparin antidote (protamine) at the conclusion of the surgery.14,49 The linear range of heparin detection obtained on the paper-based optode is 0 to 8 U/mL, well covering the blood heparin concentration in extracorporeal procedures such as cardiopulmonary bypass surgery (2-5 U/mL).14 Multilayer Membrane Device for User-Friendly Polyanion Sensing Although the indirect sensing application of the proposed optode in buffered solutions was successful, testing of real samples has two challenges. First, the pH of any real sample is variable/unknown and the optode based on proton-selective chromoionophore has different polyion sensitivities at different sample pH values owing to the protonation equilibrium of the indicator chromoionophore adsorbed to the surface of cellulose chains. Second, the polycation reactant has to be manually added into the real sample, which makes the sensing pro-



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Figure 7. Schematic representation and picture of the Parafilmbased multilayer device for indirect sensing application of paperbased protamine optode.

tocol tedious and less suited for point-of-care or field use. To solve these problems, an integrated layered device was designed that is able to control pH of the sample and release polycations into sample solutions (Figure 7). A piece of filter paper with hydrophobic sensing ingredients and solid HEPES buffer (pH 7.4) within a 3D wax circle barrier is pressed onto a piece of Parafilm that has been partially covered by a protamine-modified CA membrane. The strong adhesive property of Parafilm enables tight stacking of the paper and membrane layers. Upon addition of an aqueous sample onto the optode area, buffer salt will be dissolved and thereby buffer the sample to the desired pH. The sample solution further flows into the underlying CA filter membrane and dissolves protamine, which eventually diffuses to the optode area right on top and makes the polycation optode purplish in color. In the presence of polyanions in the sample, they will neutralize protamine during the dissolution and diffusion processes. Consequently, less protamine is able to reach the optode and a bluer color is expected. Indeed, as shown in Figure 8, the optode becomes increasingly blue with an increasing concentration of PPS in non-buffered DI water and corresponding hue has been obtained. If there is no solid buffer deposited on the optode, the optode is less purple in pure DI water and the hue-based response to PPS is much smaller. This is because the presence of carboxylic acid sites on cellulose paper decreases the pH of the water samples and the polycation optode has a lower sensitivity under acidic conditions (Figure S6). Although the buffering capacity of the buffer salt may need to be optimized for different types of real samples, the approach described here is a facile way to adjust variable sample pH to a value that is constant and also yields

Figure 8. Pictures of the optode area of the multilayer membrane device after addition of 30 µL of PPS solution of different concentratios in DI water (incubation time: 15 min), and the corresponding hue-based response. Data points are average ± s.d. for n= 3 sensors.

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Figure 9. Pictures of the optode area of the multilayer membrane device in 30 µL of human urine spiked with different concentrations of PPS (incubation time: 15 min), and the corresponding hue-based response. Data points are average ± s.d. for n= 3 sensors.

optimal optode sensitivity. Unlike modification with buffer, modification of the optode with protamine from an aqueous solution prevents signal reversal by polyanions because of the irreversible nature of the passive polyion responses.14 Therefore, protamine was loaded onto a CA membrane underneath the optode. Other types of membranes/films are also possible to preload with protamine and use as a protamine-releasing layer. However, the use of filter paper for this layer generates a smaller protamine response with the optode (data not shown), which is presumably due to strong binding of protamine with the paper. CA filter membrane was therefore chosen here because it is known to have low binding ability toward proteins and may facilitate dissolution and release of the deposited protamine. Stacking multiple membrane layers onto adhesive Parafilm represents a new approach to fabrication of vertical flow analytical devices and potentially 3D microfluidics systems.68 Compared with previously used “origami” (paper folding) strategies for 3D microfluidics,69,70 the use of Parafilm enables use of different membrane materials for different layers and eliminates the bulky clamp to hold multiple paper layers together. To demonstrate a preliminary application of this device in real samples, urine was chosen since it represents a noninvasive type of sample that is easily obtained outside the hospital setting. As shown in Figure 9, PPS in an undiluted human urine sample with an original pH of 6.3 can be successfully indicated by this multilayer device. The response curve in urine is slightly different from that in water, but the trend is the same. The difference may originate from the yellowish color of urine. This color interference could be minimized by using a smaller sample volume which reduces the optical path. For example, this could be achieved by employing a laminated optode strip.34 One limitation of polyion optodes compared to polyion electrodes is the optical interference from blood, which prohibits its direct use in whole blood samples. However, various power-free microfluidics-based membranes/devices for separation of plasma from whole blood are commercially available (e.g., Vivid™ plasma separation membrane) or are about to be released (e.g., HemaXisTM DP passive plasma separation device). The unique wicking ability of our paper-based optodes facilitates the integration of the optode unit and the plasma


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separation unit into a single device. By using the integrated power-free device, more point-of-care applications such as heparin and trypsin tests in whole blood may become possible. These studies are currently underway in our laboratory. Preliminary Result for Paper-Based Polyanion-Sensitive Optode Another type of optode for polyions is a polyanion-sensitive optode, which typically uses TDMA as an ionophore in the hydrophobic sensing phase.26 Chromoionophore IV (ETH2412) has been used previously as a proton-sensitive indicator dye in TDMA-based polyanion optodes.26 With TDMA as a counterion, the hydroxyl group of chromoionophore IV is deprotonated. In the presence of polyanions in aqueous sample, they are extracted into the sensing phase via cooperative ion-pairing interaction with TDMA cations. To maintain phase electroneutrality, protons are co-extracted into the sensing phase from aqueous solution, which protonates chromoionophore IV and

The Supporting Information is available free of charge on the ACS Publications website. Figures showing comparison of different ion exchangers, optode response time, XPS depth profiling results, confocal images, and effect of sodium chloride on the optode response. (PDF)

AUTHOR INFORMATION Corresponding Author *Email: [email protected]; [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Institutes of Health (Grant EB-000784). Inkjet printing was performed at the University of Michigan Lurie Nanofabrication Facility. XPS test was performed in Michigan Center for Materials Characterization. We thank James Windak in Department of Chemistry at University of Michigan for GC-MS test. We also thank the Single Molecule Analysis in Real-Time (SMART) Center of the University of Michigan, seeded by NSF MRI-R2-ID award DBI-0959823 to Nils G. Walter, as well as Damon Hoff for training, technical advice and use of ALBA Time-Resolved Confocal Microscope.

REFERENCES

Figure 10. Pictures of polyanion-selective optodes in 0.1 M TrisHCl buffer containing different concentrations of PPS and the corresponding hue.

changes its color. Herein, TDMA chloride and chromoionophore IV were printed onto cellulose paper as a plasticizer-free sensing layer. As shown in Figure 10, this optode is indeed quite responsive to polyanionic PPS. Notably, this response was obtained in the presence of ~80 mM Cl- (in 0.1 M TrisHCl buffer at pH 7.4), indicating good selectivity of this optode over near physiological levels of chloride ion.

CONCLUSIONS Cellulose paper has been shown to be a new substrate for fabrication of both polycation and polyanion sensitive optodes even in the absence of any plasticizer and/or added polymer. The paper-based polyion optode coupled with a smartphone as a detector/analyzer allows direct and indirect colorimetric detection of polycations, polyanions, and digestive enzymes. Compared to plasticized PVC-based polyion optodes, the highly porous paper-based optode is compatible with inkjet printing technique, is easy to modify by hydrophilic buffer salt, and could be integrated into 2D or 3D pump-free multifunctional paper/membrane-based analytical devices. In future work, we will examine more polyion sensing chemistries (e.g., other indicators and ionophores) on paper, and explore applications for the detection of polyions in more real samples.

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