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Paper Capillary Enables Effective Sampling for Microfluidic Paper Analytical Devices Jin-Wen Shangguan, Yu Liu, Sha Wang, Yun-Xuan Hou, Bi-Yi Xu, Jing-Juan Xu, and Hong-Yuan Chen ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00335 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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Paper Capillary Enables Effective Sampling for Microfluidic Paper Analytical Devices Jin-Wen Shangguan‡, Yu Liu‡, Sha Wang, Yun-Xuan Hou, Bi-Yi Xu*, Jing-Juan Xu* and Hong-Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, People’s Republic of China. KEYWORDS: Microfluidics, paper analytical devices, paper capillary, sampling, POCT, multiplex bioassay. ABSTRACT: Paper capillary is introduced to enable effective sampling on microfluidic paper analytical devices. By coupling macroscale capillary force of paper capillary and microscale capillary forces of native paper, fluid transport can be flexibly tailored with proper design. Subsequently, a hybrid-fluid-mode paper capillary device was proposed, which enables fast and reliable sampling in an arrayed form, with less surface adsorption and bias for different components. The resulting device thus well supports high throughput, quantitative, and repeatable assays by hands operation. With all these merits, multiplex analysis of ions, proteins, and microbe have all been realized on this platform, which has paved the way to level-up analysis on μPADs.

Microfluidic paper analytical devices were (μPADs) first introduced by Whitesides’ group in 20071 to meet the fast-growing needs for point of care tests (POCT) in the field of clinical diagnostics, environmental monitoring and food industry. They have well lowered the threshold for installing merits of microfluidics for POCTs because they are simple for fabrication, cheap in price, easy for usage, portable and disposable for in-situ applications.2-4 So there’s no surprise that μPADs are experiencing booming development in recent years as one of the most promising candidates in microfluidics for commercialization.5-9 Fundamentally, it is the porous nature of paper that has distinguished μPADs from the rest of microfluidic devices, and endowed them with special advantages: reagent immobilization on porous media can be more straightforward, and fluid can be passively motivated merely by capillary force, namely liquid wicking.10 However, the porous nature is also a doubleedged sword. Liquid wicking through porous paper is more difficult to control than in smooth microfluidic channels. The subsequent slow flow speed is disastrous for sample transportation: it usually leads to longer time for surface adsorption of the samples on the paper substrate.11,12 Also, in many cases, this can also result in the biased transportation of differently sized components. All these characters have restricted the throughput and quality of sample analysis on μPADs. However, most research for sample analysis on μPADs focus on developing novel analytical methods,13-17 while that for hydrodynamic fluidic control is not fully exploited.

Addressed the fundamental hydrodynamic restrictions, many pioneering efforts were made by allowing fluid flow not only within the paper but also on the surface, which well accelerated flow speed on μPADs.18 Typically, they include hollow channel,19 multilayer structure,11,12,20 open channel21,22 and hybrid PDMS-paper channel23 schemes. And recently, multilayer μPAD has further increased the flow speed to an unprecedented level.24 Besides, electronic parts have also been integrated to break the limit of liquid wicking by introducing active liquid control on μPADs. 25 All these contributions help to lay solid foundations for high-quality sample analysis on μPADs. To make further progress, we are facing challenges from fluid control to sample analysis. For fluid control, in most cases, fluids on μPADs were treated as uniform samples, ignoring the fact that they might be composed of differently sized ingredients including small ions, large molecules, micro/ nanoparticles and even cells. They behave differently from each other in porous media. Also, we still lack enough flexibility in tuning the hydrodynamic behavior of liquid on μPADs. Thus combinations of multiple flow modes were not well explored. Advancement of fluid hydrodynamic control might provide access to rich fluidic behaviors and thus novel functions. For sample analysis, it is still demanding to achieve easy handling,26 low surface adsorption, reduced bias of sample transportation,27 viable functions for high throughput,28 high quality29,30 and multiplex analysis31,32 on μPADs. In this paper, we are introducing a novel paper capillary empowered μPAD (PCap-μPAD) as a prototype device which has

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Figure 1. Characterization of the paper capillary. A. Hydrodynamic comparison of different μPAD structures. A1. Planar paper, A2. Corrugated paper, A3. Paper with tape, A4. Corrugated paper with tape, A5. Double layered paper, A6. Corrugated paper with a second paper layer. The band is 5 mm in width, 50 mm in length, dipped into the sample ink for 2 mm depth. The paper capillary is 50 mm in length. B. Paper capillary with force analysis. C. Tailoring geometrical factors for flow control. C1. Diameter of the paper capillary, capillary diameter: 0.1 mm, 0.2 mm, 0.5 mm, 0.75 mm, 1 mm; capillary length: 5 cm; C2. The density of the paper capillary, number of capillaries: 1, 2, 3; with capillary diameter: 0.5 mm, capillary length: 5 cm; C3. Length of paper capillary, with capillary diameter: 0.5 mm, capillary length: 0 cm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm.

well attached the benefit of robust flow control to high-quality sample analysis on μPADs. By coupling three modes of fluidic transportation on PCap-μPADs, fast sampling with low surface adsorption and homogenous sample distribution have all been realized. These specialties have facilitated repeatable and reliable sample analysis for ions, large molecules, and even microbes.

Results and discussion To understand the hydrodynamic behaviors of fluid transportation in paper devices and determine the influential geometric factors, we carried out tests over six different types of μPAD structures (figure 1A). They include planar paper (A1), corrugated paper (A2), paper sealed with tape (A3), corrugated paper sealed with tape to form paper capillary structure (A4), double layered paper directly put together (A5) and corrugated paper directly put together with a second paper layer (A6). Sampling was carried out by dipping the sampling inlet (figure 1B) to the liquid sample for 2 mm in depth (imaged in figure S1). Over the same sampling time, single-layer paper either planar (A1) or corrugated (A2) allows for very slow flow speed over limited distances. In comparison, within the same period, liquid flowed more than double the distance in all the double-layer based structures (A3-6). These four structures can be further categorized to those sealed with tape (A3, A4) and those formed by putting two paper layers together (A5, A6). Within each group, it is also observable the one with paper capillary allows for faster fluid transportation. Thus, paper capillary-based devices including A4 and A6 were compared finally. Considering paper capillary formed from tape-sealed paper capillary structure (A4) allows for more repeatable and reliable sampling than paper capillary formed

by binding corrugated paper with another piece of paper together (A6) (confirmed in figure S1), the former structure (A4) was adopted in the following experiments. Figure 1B further illustrates the structure and flow transportation in paper capillary. Here, fluid is transported mainly through the strong capillary pulling force of the paper capillary. We further carried out experiment to quantify the relationship of hydrodynamic behavior of paper capillary with capillary diameters (figure. 1C1), capillary densities (figure. 1C2) and capillary lengths (figure. 1C3). Figure 1C1 proved the contribution of paper capillary diminishes after reaching the maximum 𝑅𝑐𝑎𝑝𝑖𝑙𝑙𝑎𝑟𝑦 , with optimized capillary diameter reading 0.5 mm. This phenomenon is understandable. Increasing the capillary diameter can efficiently increase the flow speed when the capillary diameter is relatively small. However, since gravitational force grows at a fast speed with the increment of capillary diameter, it cancels out the positive effect of macroscale capillary force eventually. Besides diameter, a higher density of the paper capillaries can also contribute to faster flow speed (figure 1C2). Aside from achieving faster flow speed, we can also switch flowing mode from macroscale paper capillary dominant to microscale native paper capillary dominant by tailoring distribution of the paper capillary (figure 1C3). Flow speed is fast where the paper capillary present, but quickly slowdown in the planar paper region. The observed behavior is in good consistency with results deducible from Washburn equation,33 which has dominated the field of passive liquid flow in porous media for nearly a hundred years:

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d𝑙 ∑ 𝑃 2 (𝑟 ) = dt 8𝜂𝑙

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Figure 2. PCap-μPADs characterization. A. Structure of PCap-μPADs. B. Velocity field distribution, red arrows are velocity vectors proportional to the speed of flow and in parallel to the fluidic field. C. BSA colorimetric analysis. Control: planar paper regionally deposited with BSA colorimetry analytical reagent; μPAD: single-layer paper with test-pads array; μPAD with tape: single layer of paper sealed in the central lane with tape; PCap-μPAD: corrugated paper sealed with tape, paper capillary of 50 mm in length, 0.5 mm in diameter; 5 mm × 50 mm for tape covered region, 3.5 mm in diameter for test pads loaded with BSA colorimetry reagent. Sampling for 5 min by dipping in 10 mg/ml BSA solution.

Where 𝑙 is the distance of the liquid transported, t is the time, 𝑃 is the total driving pressure on the fluid, η is the dynamic viscosity of the liquid, r is the equivalent diameter of the capillary for the media. Details for the force analysis for sampling in paper capillary was attached in the SI. The tailorable fluidic behavior reminds us of the possibility to combine different fluidic modes on the same μPAD for novel functions. Subsequently, a prototype PCap-μPAD as shown in figure 2A was designed, where one macroscale paper capillary is installed in the center, and one piece of tape covers all the center lane but leaves the regions of branched test pads uncovered. The prototype PCap-μPAD show well-regulated fluidic behavior in the movie. S1, from which we can deduce three distinct regions on the chip, where:

𝑭𝒑𝒂𝒑𝒆𝒓 𝒄𝒂𝒑𝒊𝒍𝒍𝒂𝒓𝒚 > 𝑭𝒊𝒏𝒕𝒆𝒓𝒍𝒂𝒚𝒆𝒓 > 𝑭𝒏𝒂𝒕𝒊𝒗𝒆 𝒑𝒂𝒑𝒆𝒓 Here, 𝐹𝑝𝑎𝑝𝑒𝑟 𝑐𝑎𝑝𝑖𝑙𝑙𝑎𝑟𝑦 is the force generated by macroscalepaper capillary (blue coloured region in figure 2A); 𝐹𝑖𝑛𝑡𝑒𝑟𝑙𝑎𝑦𝑒𝑟 is the force generated by paper and tape dual layer structure (yellow coloured region in figure 2A), and 𝐹𝑛𝑎𝑡𝑖𝑣𝑒 𝑝𝑎𝑝𝑒𝑟 is the microscale capillary force of porous paper (white coloured regions in figure 2A). Figure 2B further illustrates the velocity field, where the sample flows fastest in the macroscale paper capillary but slows down when penetrating the branched regions and spreads throughout the testing pad region with even more slower speed. This has led to a well-directed sample transportation process. The movie also demonstrates that filling 14 test pads took less than 3 minutes. The fast sampling speed is especially valuable for high throughput and time-sensitive analysis.11 First, the side effect of sample evaporation is diminished, which otherwise can change the concentration of the sample, lead to concentration difference on each test pads or even dehydrate the sample before they can reach all the test pads. Second, the fast sampling also contributes to synchronize the reactions on the pads, allowing for simultaneous reading of the reaction results on test pads with lest error for timing. Beside fast speed, paper capillary-based sampling also improves sampling quality. In the paper capillary, since most of the sample would not touch the peripheral surface, sample adsorption can be significantly reduced. Even

for sample near the boundary of the paper capillary, since the interacting time is short due to the fast sampling speed, surface adsorption by paper capillary can also be smaller than planar paper. Thus, before entering the branched test pads, the location-dependent difference of concentration along the test pad array can be well reduced. These advantages were confirmed by comparison between typical μPADs and the proposed PCap-μPADs (figure 2C), employing typical colorimetry of bovine serum albumin (BSA). The testing pads were preloaded with the reagent of colorimetry, which would turn from yellow to green in the presence of BSA. The “control” represented the state when no sample was supplied. Within 5 min of sampling, color changed only on 6 test pads in a 14 test-pad array for both single layer μPAD and tape sealed double layer μPAD. In contrast, all the 14 test pads turned green for PCap-μPAD, confirming that the PCap-μPADs can support effective sample transportation to all the test pads. Homogeneous and repeatable sampling was further quantified by statistical colorimetric light intensity analysis over all the 14 test pads (Figure S2). Reliability test over three devices indicates the errors are smaller than 5%. We also optimized the dipping depth for sampling (Figure S3), where 2 mm is the best, with small error and homogenous sample distribution. Measurement of sample consumption was also taken (figure S4). The result indicates the device with 14 test pads consumes approx. 46 uL of the sample each time, which is small enough for most applications. After confirmed the capability of the paper capillary chip for fast, repeatable and non-biased sampling, experiments were carried out to examine the versatility of PCap-μPADs for real sample analysis. Following analytical mechanisms proposed in pioneering works,34-36 typical colorimetric assays for contaminant metal ions, proteins, and pathogenic microbes are all testified. For all the test-pad based PCap-μPADs, the sampling time of 3 min was adopted. Figure 3 shows the colorimetry results for Fe (3A), Ni (3B), Cr (3C) and Cu (3D) ions. The reagents for metal ion colorimetry are all colorless and become visible after contacting with corresponding ions. The color typically turns from colorless to red for Fe ion, pink for Ni ion, purple

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Figure 3. Colorimetry of metal ions including A. Fe, B. Ni, C. Cr and D. Cu ions with corresponding standard curves. E. Image and system error analysis for multiplex metal ions colorimetry. Mixture sample contains 100 μg/mL for each kind of metal ion. The fitting equation is: y = A × exp(-x/B) + C, where A, B, and C are all constants, x is a variable, and y is dependent on the value of x. The corresponding values of A, B and C for different metal ions were attached in the supporting information (table S1).

Figure 4. Colorimetry of A. BSA and B. Aspartate transaminase (AST) with the corresponding standard curve; C. Multiplex protein colorimetry image and corresponding color analysis. Mixture sample contains 60 mg/mL BSA and 500 U/L AST.

for Cr ion and orange for Cu ion. Based on the Image-J color analysis, their standard curves for colorimetry were deduced, with the corresponding limit of detections (LOD) reads 5, 10, 5, 10 μg/mL. Test pads for multiplex analysis were preloaded with different reagents spatially separated from each other. Then a sample of all the mixture targets was applied. After

the sample enters the test pad region, the color change is triggered if the target is present. Up to 32 test pads were successfully integrated for 8 repetitive regions; each was composed of a group of test regions for the four types of metal ions. As expected, the final colorimetric profile followed the predefined spatial distribution. In addition, color

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Figure 5. Colorimetry of A. E. Coli; B. S. Typhimurium and C. L. monocytogenes with corresponding standard curves; D. Multiplex microbe colorimetry image and corr esponding light intensity analysis. Mixture sample contains 3.3×10 6 cfu/ml E.Coli, 3.3×107 cfu/mL S. Typhimurium and 3.3×108 cfu/mL L. monocytogenes.

Figure 6. Evaluation of error distribution along the PCap-μPAD. A. Mapping of errors for metal ion colorimetry; B. Mapping of errors for protein analysis; C. Mapping of errors for microbe analysis. Map from left to right corresponds to the positions of test pads on the device, where the position with test pads nearest to the sample inlet is noted as No.1, while the topmost is No.7.

intensity data were extracted from each pad to evaluate the quality of multiplex analysis (Figure 3E). Results well proved that the errors are satisfyingly small between test pads for the same targets even they are distant away from each other on the PCap-μPAD. Colorimetry assays for proteins shown in figure 4 were also carried out for BSA (4A) and aspartate transaminase (AST) (4B). BSA turns the colorimetric reagent from yellow to deep green, and AST changes the color from violet to magenta. Corresponding standard curve in indicate LOD of 5 mg/mL for BSA and 10 U/L for AST. Mixture colorimetry analysis shown in figure 4C confirmed the availability for high-quality multiplex protein analysis. Detections for three types of common microbes including E. coli (figure 5A), S. Typhimurium (5B) and L. monocytogenes (5C) were also realized. Color changed from yellow to orange in the presence of E. Coli with LOD of 105 colony-forming-unit (cfu)/ml, from colorless to pale purple for S. Typhimurium with LOD of 105 cfu/ml and from colorless to pale green for L. monocytogenes with LOD of 107 cfu/ml. Multiplex analysis over 24 test points together with the color intensity analysis (figure 5D) proved that the device could support reliable microbe analysis. With the above colorimetry data, we also mapped the distribution of error for all three types of analytes in figure 6. The Y-axis corresponds to the concentration of the sample, while the X-axis denotes the No. of the test pad along the paper device. Since in our case there are 7 ladders from the bottom to the top of the device, they are numbered 1 to 7 on the map. The results indicate the error distribution along the X-axis has

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not adopted any observable pattern, while in the Y-axis, errors increase with the decrement of the concentration of samples. The results imply PCap-μPAD allows for homogenous distribution of the sample, which pose little transportation distance related errors to the system. The presented colorimetry tests on PCap-μPAD hold detection sensitivity comparable with existing reports on μPADs as summarized in table S2, but extended the analyzing ability with higher throughput, excellent performance for multiplex analysis and strong controllability over fluidic velocity field. Through the proposed assays on PCap-μPADs, we can get a brief touch of its unprecedented power for sample analysis. PCap-μPAD can be easily strengthened in the future by integration with advanced analytical principles. Since the test pad regions are composed of merely planar paper, it is well compatible with most of the paper-based analytical mechanisms. It also deserves notice, that further increasing throughput requires prolonged sampling time, which sets the upper limit for high throughput analysis through with PCap-μPAD. Nevertheless, we still have a large room to improve the throughput by parallel sampling. Since only one inlet is needed for sampling on each PCap-μPAD, we can easily achieve higher throughput by assembling the units with arrayed inlets.

Conclusions In conclusion, we have successfully integrated paper capillary structure to empower the μPADs. The structure facilitates fast fluidic flow on μPAD, and thus renders us flexibility for sampling by modulating the paper capillary structure and distribution. More importantly, it brings us new opportunities for high-quality sample analysis by adopting hybrid fluidic flow modes. With such mechanism, fast, reliable, repeatable and homogenous sampling over 14 test pads has been achieved, while sample adsorption and size bias are significantly reduced. Multiplex colorimetric analysis for metal ions, proteins, and even microbes have all been achieved on this platform. We believe with the development of more well-designed PCap-μPADs in the near future, efficient and versatile fluidic control is in prospect and will, in turn, empower high-quality analysis on μPADs for POCT applications.

Experimental Materials and reagent Whatman 202 quantitative filter paper was used for μPAD fabrication. The transparent tape was bought from Deli Group Co., Ltd. Trehalose, ethylenediamine-tetraacetic acid (EDTA, AR), Tris, polyvinyl alcohol (PVA, AR), methyl green (CP), Triton X-100 (GR), sodium citrate (AR), citrate (AR), tetrabromophenol blue (TBPB, AR), ammonium dichromate (VI, AR), iron III chloride hexahydrate (AR), nickel II sulfate hexahydrate (AR), copper (II) sulfate pentahydrate (AR), hydroxylamine (AR), NaF (AR), Dimethylglyoxime (DMG, AR), ammonium hydroxide (AR) are from Sinopharm Chemical Reagent Corp. o-Phthalic anhydride, acetic acid (AR) is from

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Shanghai Lingfeng Chemical Reagent Co., Ltd. NaCl (AR) is from Nanjing Chemical Reagent Co., Ltd. Disodium Bathocuproine disulfonate (AR) is from Tokyo Chemistry Industry (TCI). Cysteine sulfonic acid (CSA, 99%) ， βgalactosidase (99%), esterase (99%), 5-bromo-4-chloro-3indolyl-myoinositol phosphate (XInP, 99%), chlorophenyl red β-galactopyranoside (99%), and 5-bromo-6-chloro-3-indolyl caprylate (magenta caprylate, 99%) were purchased from Sigma-Aldrich Co., Ltd. Rhodamine B (CP) is from Tianjin Research Institute of Chemical Reagent. α-Ketoglutarate (BR), 1,10-phenanthroline (AR), poly (acrylic acid) (PAA, MW 30, 000), poly (diallyldimethylammonium chloride) (PDDA, AR), PEG 400 (CP), 1,5-diphenylcarbazide (AR) were provided by Aladdin Industrial Corporation. One Step Bacteria Active Protein Extraction Kit is from Sangon Biotech. Aspartate aminotransferase (AST, BR) is from Shanghai Yuanye Biotech Co., Ltd. Bovine serum albumin (BSA, BR) and phosphate buffer saline (X1 PBS) are from Keygen Biotech. E. coli, S. typhimurium, and L. monocytogenes are from Shanghai Luwei Technology Co., Ltd. Nutrient Agar is from Guangdong Huankai Microbial Sci. & Tech. Co., Ltd. Cysteine (BR) is from Huixing Biochem Reagent Co., Ltd. Equipment and settings The chips were designed by CorelDRAW X4 and carved by UNIVERSAL VLS2.3 laser engraver. HH-601 thermostatic water bath and THZ-82 constant temperature shaker were used for bacteria incubation. Movies were recorded by HUAWEI PLK-UL00 smartphone. Colorimetry data were collected by the scanner of Brothers DCP-1618W printer. Preparation of PCap-μPADs First, carve grooves with predefined width and depth on PMMA by laser engraving, which was later used as the mould for paper capillary preparation. The boundary of the filter paper was accurately defined using laser engraver. After generating corrugated paper with the help of PMMA template, it was sealed with a slice of well-aligned tape to form the paper capillary structure. Test pad preparation for metal ion colorimetry Colorimetry of metal ions followed a modified way of what has been reported before34,37. All the test pads were air dried at room temperature following each reagent addition. Fe ion For the preparation of Fe ion test pads, the detection zone was prepared by adding 0.8 μL of hydroxylamine (0.1 g/mL) in 3 M acetate buffer. Next, 0.6 μL of PAA (0.7 mg/mL) was added, followed by addition of 0.7 μL aliquots of 1,10-phenanthroline (8 mg/mL) in 3 M acetate buffer for twice. The chemical equation for Fe ion colorimetry is: Fe2+ + 3 phen → [Fe(phen)3 ]2+ Ni ion For Ni ion test pads, each pad was added twice with 0.7 μL aliquots of NaF in DI water (0.5 M), followed by 0.6 μL of acetic acid (3 M, pH 4.5). 0.5 μL aliquots of DMG in methanol were then added, repeated for five times, followed by two 0.7 μL aliquots of ammonium hydroxide (pH 9.5).

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ACS Sensors The chemical equation for Ni ion colorimetry is: Ni2+ + 2 C4 H8 N2 O2 → Ni(C4 H8 N2 O2 )2 + 2H+ Cr ion For Cr ion test pads, first, add two 0.7 μL of ceric IV ammonium nitrate (0.35 mM), followed by 0.7 μL cysteine (50 mg/mL) and 0.7 μL of PDDA (5% w/v). Two 0.5 μL aliquots of a 1,5-diphenylcarbazide (1,5-DPC) and phthalic anhydride (15 and 40 mg, respectively, in 1 mL acetone) mixture were also added to the test pads. The chemical equation for Cr ion colorimetry is: Cr(VI) + DPC → Cr(III)+DPCO → Cr(III)-DPCO complex Cu ion For Cu, add 0.8 μL aliquot of hydroxylamine (0.1 g/mL), followed by a 0.7 μL aliquot of acetic acid/NaCl buffer (10 mM, pH 4.5) was first added to the test pads. The Cu detection solution was prepared by adding 50 mg/mL bathocuproine and 40 mg/mL PEG 400 to chloroform. Twice addition of 0.7 μL aliquots of the bathocuproine/PEG detection solution was then taken. The chemical equation for Ni ion colorimetry is : Cu2+ + 2 C26 H20 N2 → Cu(C26 H20 N2 )2 Test pad preparation for protein colorimetry Colorimetry of protein followed a modified way of what has been reported before35. All the test pads were air dried at room temperature following each reagent addition. AST First, spot reagents in the following order onto the test pads: 0.7 μL solution of 10% trehalose in water; 0.7 μL of substrate solution containing CSA (306 mg/mL), α-ketoglutarate (34 mg/mL), EDTA (1.6 mg/mL) in TRIS buffer (400 mM/mL), and 0.7 μL of a reagent solution containing 1% PVA, 0.4% methyl green, 0.2% rhodamine B, zinc chloride (110 μg/mL), triton X100 (1 drop/25 mL). After that, spot 0.7 μL of a reagent solution containing 1% polyvinyl alcohol, 0.1 % methyl green, 0.1% rhodamine B, zinc chloride (2.75 mg/mL), triton X-100 (1 drop/ 25 mL) in ultrapure water. BSA First, spot reagents in the following order to the test pads: 1.2 μL of a 250 mM citrate buffer solution (pH 1.8) containing Triton X-100 (1 drop/ 25 mL), repeat for twice; then add 0.7 μL of a 6.0 mM TBP solution in 95% ethanol.

To prepare the bacteria for the test, first, break bacteria by an ultrasonic treatment for 20 min in ice water followed by 5 min of sampling. Storage for 12 h in a humidified box under 37℃ before scanning for colorimetric data collection. One Step Bacteria Active Protein Extracting (Sangon Biotech) was carried out in addition before other steps for the sampling of L.monocytogenes. Sampling and Colorimetry analysis Sampling was carried out by dipping PCap-μPAD to the sample for 2 mm in depth for 5 min, where a 2 mL Eppendorf tube was applied as the reservoir for the sample. Both colorimetry for protein and microbe requires the paper devices be imaged before drying to prevent drying-related color-change, while that for metal ions were imaged after dry. The corresponding color value on the test pad was collected by integrating the data on the surface of the test pad for averaged reading using the Image-J software. For reagent immobilization, integration only takes the area with the reagent and leaves other regions neglected. There are two types of colorimetric analysis routes applied in the experiment, either RGB analysis for the components of red, green or blue color intensities or black and white color intensity was carried out, based on the character of colorimetric change. Details for the software image processing procedures are attached in supporting information.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Theoretical force analysis for sampling with paper capillary; comparison of sampling reliability between corrugated paper with tape and corrugated paper with paper (Figure S1); reliability of sampling (Figure S2); influence of dipping depth for the quality of colorimetry (Figure S3); quantification of consumed sample (Figure S4); constants for the fitting equation in figure 3 (Table S1); comparison of LOD in this work and the reference work (Table S2); liquid flowing process (Movie S1)

AUTHOR INFORMATION

Test pad preparation and colorimetry of bacteria

Corresponding Author

Colorimetry of bacteria followed a modified way of what has been reported before36. All the test pads were air dried at room temperature following each reagent addition. On each test pad, spot 0.8 μL CPRG testing reagent (1 mM) for E. Coli, 1 μL 10 mM magenta caprylate for S. Typhimurium and1 μL 80 mM XInP for L. monocytogenes. To enumerate the microbes, first, pick a few bacteria by a 1 μL inoculating loop, suspend it with solution contained 1 mL PBS and a drop of Triton-X 100, and stepwise dilute the bacterium solution to 1/10 concentration. After that, coat 100 μL bacterium solution with the two samples of lowest concentration on an agar plate. Observe and calculate the number of colonies after 12 h.

*Tel./Fax: +86-25-89687294, E-mail: [email protected], [email protected]

Author Contributions J.S. conducted most of the experiment, and Y.L analyzed the data, helped in the experiment and assisted in writing the paper. S.W. did part of the experiment for protein surface absorption. Y.H. carried out part of the calorimetry assay. J.X. and B.X. fostered the idea and wrote the paper. J.X. and H.C. guided the research. ‡These authors contributed equally.

Notes The authors declare no conflict of interest.

ACKNOWLEDGMENT

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This work was supported by the Ministry of Science and Technology Program of China (2016YFA0201200), the National Natural Science Foundation of China (21327902, 21505069).

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