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Microfluidic Distance Readout Sweet Hydrogel Integrated Paper-based Analytical Device (µDiSH-PAD) for Visual Quantitative Point-of-Care Testing Xiaofeng Wei, Tian Tian, Shasha Jia, Zhi Zhu, Yanli Ma, Jian-Jun Sun, Zhenyu Lin, and Chaoyong James Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04294 • Publication Date (Web): 14 Jan 2016 Downloaded from http://pubs.acs.org on January 19, 2016

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Analytical Chemistry

Microfluidic Distance Readout Sweet Hydrogel Integrated Paper-based Analytical Device (µDiSH-PAD) for Visual Quantitative Point-of-Care Testing Xiaofeng Weiab, Tian Tiana, Shasha Jiaa, Zhi Zhua, Yanli Maa, Jianjun Sunb, Zhenyu Linb*, Chaoyong James Yanga*

a: MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Collaborative Innovation Center of Chemistry for Energy Materials, Key Laboratory for Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University Xiamen 361005 (China)

b: MOE Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116, China

* To whom correspondence should be addressed. Tel: (+86) 592-218-7601, E-mail: [email protected]; or Tel: (+86) 591-22866135, E-mail: [email protected]

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Abstract: A disposable, equipment-free, versatile point-of-care testing platform, microfluidic Distance Readout Sweet Hydrogel Integrated Paper-based Analytical Device (µDiSH-PAD), was developed for portable quantitative detection of different types of targets. The platform relies on a targetresponsive aptamer crosslinked hydrogel for target recognition, cascade enzymatic reactions for signal amplification, and microfluidic paper-based analytic devices (µPADs) for visual distancebased quantitative readout. A “Sweet” hydrogel with trapped glucoamylase (GA) was synthesized using an aptamer as a cross-linker. When target is present in the sample, the “Sweet” hydrogel

collapses and releases enzyme GA into the sample, generating glucose by amylolysis. A hydrophilic channel on the µPADs is modified with glucose oxidase (GOx) and colorless 3, 3’diaminobenzidine (DAB) as the substrate. When glucose travels along the channel by capillary action, it is converted to H2O2 by GOx. In addition, DAB is converted into brown insoluble poly-3, 3’-diaminobenzidine [poly(DAB)] by horseradish peroxidase (HRP), producing a visible brown bar, whose length is positively correlated to the concentration of targets. The distance-based visual quantitative platform can detect cocaine in urine with high selectivity, sensitivity and accuracy. Because the target-induced cascade reaction is triggered by aptamer/target recognition, this method is widely suitable for different kinds of targets. With the advantages of low cost, ease of operation, general applicability, and disposability with quantitative readout, the µDiSH-PAD holds great potential for portable detection of trace targets in environmental monitoring, security inspection, personalized healthcare and clinical diagnostics.

Keywords: hydrogel, µPAD, aptamer, point-of-care, cocaine


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Introduction Paper-based devices, as excellent platforms for point-of-care tests (POCTs)1-5, have attracted extensive attention with potential advantages of low-cost, portability, low sample consumption, ease of use, and disposability in many fields, such as clinical diagnostics6-11, environmental monitoring12, drug abuse testing13, 14, food safety inspection15, 16, and biodefense17-21. Since the groundbreaking work22 of Whitesides group, multiple classical detection techniques, like colorimetry23, 24 electrochemistry25, 26, electrochemiluminescence (ECL)27, 28, chemiluminescence (CL)29,

30

and fluorescence31-33, have been applied to paper-based devices with simple readout

based on handheld instruments for rapid diagnostics. However, because most of these methods rely on laboratory or portable instrumentation for quantitative analysis, the associated cost of these devices has precluded their application especially in developing areas. Therefore, new signal readout methods that do not require instrumentation for POCTs are needed. Visual distance-based methods are desirable for POCT devices. Distance-based visual quantitative detection methods stand out as a new class of quantitative readout due to the characteristics of ease of use and low cost. Such methods require users only to read visual signal bars which are correlated with target concentrations, thereby eliminating the need for readout devices or instruments and can be easily integrated into portable analytical devices. Moreover, compared with conventional intensity-based colorimetric approaches, distance-based methods are less susceptible to user interpretation, which reduces personal errors. Up to now, many innovative devices with distance-based visual quantitative readout have been reported for detection of various targets. Qin et al.34-36 demonstrated visual distance-based quantitative readout with their original slipchip for the detection of diverse protein biomarkers and target nucleic acids. Woolley et al. 37, 38

also reported the “Flow Valve” microfluidic device for quantitative response of streptavidin

and DNA. Shin et al.39 developed a platelet function analysis method on a distance-based microfluidic system. The strategies above realize visual quantitative readout without assistant devices, but still require tedious micromachining of glass/polydimethylsiloxane channels. Therefore, Henry et al.40 used a simple, distance-based measurements for three kinds of paper devices. Stickl et al.41 screened blood coagulation and quantitatively monitored Ca2+ concentration in whole blood by the distance-based paper device. It is believed that distance-based paper visual quantitative methods, with minimal professional requirements, low cost and simple operation, are



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powerful tools for POCTs, especially in resource-limited regions. In this manuscript, we propose a disposable, equipment-free, versatile point-of-care testing platform, microfluidic Distance Readout Sweet Hydrogel Integrated Paper-based Analytical Device (µDiSH-PAD), for portable quantitative detection of targets. The platform relies on a target-responsive aptamer-crosslinked hydrogel for target recognition, cascade enzymatic reactions for signal amplification, and a microfluidic paper-based analytic device (µPAD) for visual distance-based quantitative readout. Aptamers, isolated by systematic evolution of ligands by exponential enrichment (SELEX), can specifically recognize various targets, including small molecules, metal ions, proteins or protein complexes, viruses, whole cells and so on42, 43. For sensitive detection of targets, we synthesized an enzyme-embedded hydrogel using an aptamer as crosslinker, in which target binding results in enzymatic signal amplification. Using this characteristic, several POCT methods have been developed by our group, including colorimetric readout using gold nanoparticles44, multiple target detection by gel-sol phase switching45, slip-chip readout based on platinum catalysis46 and glucometer readout by enzyme catalytic reaction47. Herein, we use a target-responsive “Sweet” hydrogel to regulate the release of enzyme and subsequent cascade enzymatic reaction, which can achieve quantitative detection of targets with a distance signal readout in the µPAD. When target presents in the sample, the “Sweet” hydrogel collapses and releases GA into the sample. This leads to generation of glucose by a GA-catalyzed amylolysis reaction, thus inducing a cascade reaction on the enzyme/substratemodified µPAD to produce a distance signal as a stained stripe. In the absence of targets, no GA is released because of the encapsulation of hydrogel, resulting in no cascade reaction and no distance signal. The concentration of targets is determined by the length of the stained stripe to achieve the final quantitative detection by naked eye. The device is inexpensive, user-friendly, easy to use, and disposable with quantitative readout. Because the target-induced cascade reaction is triggered by aptamer/target recognition，this method is widely suitable for different types of targets. Moreover, the method can be applied in complex biological matrices such as urine. With the advantages of inexpensiveness, ease of use, general applicability, and disposability with quantitative readout, the µDiSH-PAD holds great potential for portable detection of trace targets in environmental monitoring, security inspection, personalized healthcare and clinical diagnostics.


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Experimental Reagents and Materials. All reagents for DNA synthesis were purchased from Glen Research (Sterling, VA, USA). As shown in Table 1, the oligonucleotides applied in the experiment were synthesized on a 12-Column DNA Synthesizer (PolyGen GmbH) in-house and purified with a reversed-phase C18 column by an Agilent (Santa Clara, CA, USA) 1100 series HPLC system. 2Cyanoethyl diisopropyl chlorophosphoramidite was received from ChemGenes (Wilmington, MA, USA). Acrylamide, ammonium persulfate (APS) and N, N, N’, N’-tetramethylethylenediamine (TEMED) were purchased from Sigma (Shanghai, China). The target samples, cocaine and adenosine were respectively obtained from the National Institutes for Food and Drug Control (Beijing, China) and Sigma-Aldrich (St. Louis, MO, USA). The cocaine buffer contained 77 mM Na2HPO4, 23 mM NaH2PO4, 50 mM NaCl, 5 mM MgCl2 (pH 7.3). The adenosine buffer contained 10 mM Tris-HCl buffer, 100 mM NaCl, 10 mM MgCl2 (pH 8.0). Horseradish peroxidase (HRP), glucose oxidase (GOx) and Glucoamylase (GA) were purchased from SigmaAldrich (St. Louis, MO, USA). Amylose and D-(+)-glucose were purchased from Sinopharm Chemical Reagent (Shanghai, China). Hydrogen peroxide (30%) was purchased from Guanghua Sci-Tech Co., Ltd (Guangdong, China). Other reagents were obtained from Sinopharm Chemical Reagent (Shanghai, China). Whatman No. 1 chromatography filter paper was purchased from GE Healthcare UK Limited. Hydrophobic wax pattern was printed on filter paper by the wax printer (Xerox Colorqube 8570).

Synthesis of Acrylic Phosphoramidite. As shown in Figure 1, acrylic phosphoramidite was synthesized to conjugate DNA strands onto the linear polyacrylamide. Detailed procedures can be found in our previous report 45.



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Figure 1 Synthesis of acrylic phosphoramidite

DNA Synthesis and Purification. Based on the standard DNA synthesis protocol, the oligonucleotides employed in the experiment were synthesized in-house on the 12-Column DNA Synthesizer. The acrylic phosphoramidite were modified at the 5’-end of Strand A and strand B. The product was cleaved using the solid support, deprotected with ammonia treatment, and purified by a reversed-phase C18 column in a LC3000 semi-preparative HPLC system (Chuang Xin Tong Heng, Beijing, China). The solution of (0.1 M, pH 6.5) triethylamine acetate and (Fisher) HPLC-grade acetonitrile were respectively applied as buffer A and buffer B in HPLC. After detritylation, the DNA were desalted with NAP-5 column (GE, Healthcare), quantified by UV-Vis spectrometer, and stored at -20ºC for future use. Table 1 DNA sequences used for the experiment a

a

Underlined letters represent aptamer sequence, and Italic and bold letters represent complementary sequences.


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Preparation of “Sweet” Hydrogel. The desired concentrations of purified strand A and B solutions were mixed separately with 4% acrylamide in centrifuge tubes, then placed in an electric vacuum drying oven to remove air at 37 oC in 10 min. Later, 1.4% (v/v) freshly prepared initiator (0.1 g/mL APS, 0.5 mL) and accelerator (5%, v/v TEMED, 0.5 mL) were quickly added to both solutions. The centrifuge tubes were returned to the electric vacuum drying oven at 37 oC for 15 min to obtain linear-chain P-SA and P-SB. For forming hydrogel, P-SA, P-SB and linker-Apt (LApt) were mixed at a 1:1:0.4 molar ratio of with GA (final concentration, 1 µg/µL in buffer). After a brief centrifugation to spin down the solutions into the bottom of the centrifuge tube, the mixture was incubated in a thermostat at 65 oC for 5 min. By slowly cooling to room temperature, the aptamer cross-linked “Sweet” hydrogel with GA trapped was produced. It should be noted that the “sweet” hydrogel could be prepared in a large quantity beforehand, end-users will not need to follow the above steps thus a centrifuge would not be needed during sample detection.

Design and Fabrication of the µPAD. µPADs were produced by a wax printing method. The pattern of device was designed with CorelDraw12 software. Paper patterns were fabricated on Whatman No. 1 filter paper using the wax printer, and then placed in a thermostat at 150°C for 2 min. The final paper devices were obtained by natural cooling to ambient temperature. As shown in Figure 2, the wax patterning paper device was composed of a circular reservoir (6 mm diameter) as sample reservoir, a straight channel (2 × 40 mm) and several scale bars marked at 5mm intervals for conveniently observing the chromogenic reaction by naked eye. The area of the circular reservoir was designed for the storage of sample (20 µL), and the width of channel was optimized as narrow as possible for a stable flow and clear color bar. Then, the mixed solution (0.7 µL) of 714 U/µL GOx and 714 U/µL HRP was dropped on the sample reservoir and (10 mg/mL) 3, 3’-diaminobenzidine (DAB) was dipped onto the reaction straight channel by a writing brush to ensure uniform diffusion in all channels. Finally, the treated µPADs were dried in air and stored in a dark place.

Analytical Procedure. Different concentrations of targets were prepared. The final concentrations for preparation of the GA-doped hydrogel were 95 µM P-SA, 95 µM P-SB and 38 µM L-Apt. Fifteen µL GA (1 mg/mL) -doped hydrogel was washed three times with 20 µL ultrapure water,



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and 20 µL targets including 6 mg/mL amylose were added on top of the gel in the centrifuge tube. The tube was then incubated at 25oC with gentle shaking at 150 rpm for full reaction. The reaction time for cocaine testing was 1 hr. and for adenosine testing it was 2 hr. Then, the supernatant (20 µL) was removed as the sample solution and spotted onto the sample reservoir of the treated µPAD for distance-based visual readout. The experimental result was observed by naked eye and collected by a cellular phone camera after 30 min in ambient environment.

Results and Discussion Working Principle of Microfluidic Distance-readout “Sweet” Hydrogel Integrated Paperbased Analytical Device (µDiSH-PAD).

Figure 2 Schematic diagram of µDiSH-PAD for vsiual quantitative POCT. A: target recognition induces dissolution of the hydrogel to release gluoamylase to catalyze the generation of a large amount of glucose. B: Conversion of glucose to gluconic acid catalyzed by GOx generates H2O2, which reacts with DAB with the catalysis of HRP yieldinga brown poly(DAB) stripe in µPAD for signal readout.

The design and working principle of the µDiSH-PAD are schematically illustrated in Figure 2. Target responsive “sweet” hydrogel doped with GA is formed by crosslinking the pendant DNA strands on linear polymer chains (PS-A and PS-B) with a linker aptamer chain L-Apt via WatsonCrick base pairing. In the presence of targets, aptamer sequence L-Apt dissociates from P-SA and P-SB to bind with target, which causes the hydrogel to collapse and release GA into the solution. Amylose in the solution is then hydrolyzed by the GA to generate a large amount of glucose (Figure 2A). The resulting solution is then spotted onto the µPAD, where capillary action causes the glucose to flow with the solution in the straight channel. The glucose is then catalytically


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converted into gluconic acid and H2O2 by GOx pre-deposited on the µPAD. The resulting H2O2 immediately oxidizes colorless DAB into brown poly(DAB) catalyzed by HRP (Figure 2B). Since poly(DAB) is polymer, the brown mark does not migrate with the flow. The length of the brown color poly(DAB) grows until all the glucose in the solution is consumed. As a result, the higher the target concentration in the sample, the more GA is released, and the more glucose is produced, lending to a longer brown channel. The positive correlation of the length of the brown stripe, as a result of targets recognition and the induced cascade enzymatic reaction, with the target concentration forms the basis of the visual distance-base quantitative detection. The µDiSH-PAD is useful for point of care testing because of several important features. First, the target-responsive hydrogel using an aptamer as the recognition element can be generally applied for the detection of various targets. By simply changing the aptamer, different target responsive hydrogels can be synthesized for sensing the respective target. Second, the use of paper as the substrate significantly reduces the cost of fabrication (~$0.003 per paper device) and makes the device disposable. Third, from target recognition to signal readout, the method involves several cascade enzymatic steps, offering potential for highly sensitive detection. More importantly, the signal readout is based on distance-based measurement, which is easy to implement, less susceptible to user interpretation, and capable of quantitative detection without the need for complicated devices or instruments. The universal applicability, low cost, ease of use, high sensitivity, and quantitative detection characteristics make µDiSH-PAD an attractive tool for POCT applications.

Feasibility of Converting Target Concentration into Distance Signal. A series of experiments were designed and carried out to demonstrate the feasibility of our strategy of converting target concentration into a visually detectable distance signal for quantitative readout. We chose cocaineresponsive hydrogel as a model to detect cocaine as proof-of-concept experiment. According to the principle, our µDiSH-PAD involves three enzymatic (GA, GOx, and HRP) reactions. These three enzymatic steps were systematically tested step-by-step using the µPAD with HRP and GOx pre-deposited on the circular inlet and DAB in the straight channel. First, different concentrations of H2O2 solutions were introduced into the µPAD. As shown in Figure 3 (A), a brown stripe appeared with the addition of H2O2, while no visible change was observed with blank sample.



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More importantly, the length of the stripe was found to increase with H2O2 concentration from 0.1 µM to 100 µM. This result indicated that H2O2 can induce the generation of brown poly(DAB) with the help of HRP and that stripe length can be used for quantitation of H2O2.. Next, different concentrations of glucose were introduced. As shown in Figure 3 (B), no brown stripe was observed when blank sample was introduced, while brown stripes of increasing length appeared with the addition of increasing concentrations of glucose ranging from 0.7 mM to 10.5 mM, confirming that glucose can be quantitatively converted by GOx to generate H2O2 for subsequent production of poly(DAB) catalyzed by HRP. The sensitivity of the chip was found to be affected by the volume of sample added. As shown in Figure 3 (C), the sensitivity of µDiSHPAD increased remarkably with the increasing glucose volume from 5 µL to 20 µL. No significant sensitivity increase was observed when increasing sample volume from 20 µL to 25µL. More importantly, 25 µL solution was found to exceed the volume capacity of the channel design. As a result, 20 µL was selected as the optimal sample volume on our µDiSH-PAD. Finally, a linear positive correlation trend was observed for the length of brown stripes against increasing concentration of GA from 50 ng/µL to 0.5 µg/µL in 6 mg/mL amylose solution. As shown in Figure 3 (D), the visual distance signal can be regulated by the concentration of GA via a cascaded reaction mediated by GA, GOx and HRP for the production of poly(DAB). Finally, the feasibility of the system was tested by treating the hydrogel with cocaine solution. As shown in Figure 3 (E), a brown stripe was observed with cocaine solution of 200 µM, while no visible color change was observed for the control sample, indicating that cocaine can cause breakdown of hydrogel to release GA and trigger subsequent enzymatic reactions for generation of the brown stripe signal. Taken together, these experimental results demonstrated that the µDiSH-PAD can effectively and quantitatively translate target concentration into distance signal for visual quantitative detection.


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Figure 3 Response of µDiSH-PAD to different concentrations of H2O2 (A) and glucose (B); Influence of different sample volume to the sensitivity of the µDiSH-PAD (C) with the standard deviation obtained from three measurements; Response of µDiSH-PAD to different concentrations of GA (D); Response of µDiSH-PAD to cocaine (E).

Quantitative Detection for Cocaine. The proposed µDiSH-PAD was employed to quantitatively detect cocaine. Cocaine with various concentrations (0 ~ 400 µM) was tested with three measurements in parallel. As shown in Figure 4 (A), the microfluidic bars increased in length with increasing concentration of cocaine from 10 µM ~ 400 µM. As shown in Figure 4 (B), excellent linear relationship between the distance of microfluidic bar and the cocaine concentration in the range of 10 ~ 400 µM was obtained. A detection limit of 3.8 µM cocaine was achieved, based on 3σb/slope, where σb is the standard deviation of blank samples. It is worth noting that, even without the use of any electronic equipment, the detection limit of the µDiSH-PAD is comparable to that of our preview system with a commercial glucometer readout47. Furthermore, based on capillary effect of µDiSH-PAD, the flow will move slower and slower when the distance becomes longer. Maximum distance will not be proportional to the high concentration of the analyte.



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Several approaches such as changing the pore diameter of cellulose, optimizing the width of hydrophilic channel, and use of a special chip design could be applied to increase the dynamic range of analyte. The pore diameter of cellulose and the channel width can regulate capillary force thus the flow speed. Use of special chip designs such as hollow channel48 can minimized sample evaporation and increase capillary force thus the flow speed. Overall, the experimental results establish that the µDiSH-PAD can be used for visual quantitative detection of trace amounts of targets.

Figure 4 (A) Pictures: the detection of cocaine from 0 ~ 400 µM on µDiSH-PAD; (B) The relationship of microfluidic bar distance against cocaine concentration with the standard deviation obtained from three measurements.

Selectivity and Real Sample Analysis. To evaluate the selectivity of the µDiSH-PAD, we utilized the cocaine metabolites ecgonine methyl ester and benzoyl ecgonine as negative controls. As seen in Figure 5 (B), an insignificant signal was observed for 1 mM cocaine metabolites. However, cocaine sample at only 50 µM reached 12 mm in length, which was almost 10-fold longer than that of 1 mM cocaine metabolites. This result strongly demonstrated the µDiSH-PAD maintained the favorable selectivity of the aptamer for its target. To test the feasibility of the µDiSH-PAD for sensitive target detection in complex biological matrixes, a series of concentrations of cocaine spiked in urine was evaluated. As shown in Figure 5 (A & C), a quantitative response to cocaine was observed in 50% urine and the detection limit was calculated to be 5.8 µM. This result in a real sample was similar to that obtained in buffer, indicating that the relevant components in urine hardly interfered with the performance of our method. The detection limit of cocaine by our system is comparable to reported electrochemistry (10 µM)49, colorimetric (2 µM)50 and


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fluorescence (10 µM)51 methods. And only our design can achieve the visual quantitative detection without any instrument. It has been reported that mean cocaine concentration in urine is about 75 µM within 24 hours after drug intake.52 Therefore, the hydrogel-combined µPAD possesses satisfactory applicability and the potential for applications in complex biological fluids.

Figure 5 (A) Pictures: the detection of cocaine in urine from 0 ~ 1 mM with the µDiSH-PAD; (B) Selective analysis of µDiSH-PAD system with cocaine and cocaine metabolite; (C) The relationship of microfluidic bar distance against cocaine concentration in urine with the standard deviations obtained from three measurements.

Comparison with LC-MS. Generally, quantitative cocaine detection is performed by large-scale instruments and professional operators, including LC/MS53, GC54, and GC/MS55, although some commercial qualitative detectors for cocaine have been based on cheap and disposable chips56. To confirm the precision and credibility of the hydrogel-combined µPAD, LC/MS was used as a comparison for cocaine detection (Figure 6). We evaluated 13 samples in urine in both methods. Calibration curve in Figure 5C was used for the detection of cocaine in urine sample for the µDiSH-PAD. An obvious positive relationship between the two methods was found, with a slope of

1.04 ± 0.03 and a correlation coefficient of 0.98. Considering the experiment error, these results from two methods matched very well. The result adequately indicated that the accuracy of the hydrogel- combined µPAD method was comparable to that of the standard cocaine detection method, suggesting that our system is appropriate and dependable as an alternative test method with low-cost, easy accessibility and disposability.



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Figure 6 Comparison of the µDiSH-PAD method with the standard LC/MS method based on 13 samples in urine. The markers and error bars reflect the average and standard deviations of three measurements.

Detection for Adenosine. For the µDiSH-PAD method, the flexibility of the aptamer-hydrogel design ensures detection of a wide variety of non-glucose targets. By changing the aptamer sequence, our system can be adapted to detect a wide range of targets when corresponding aptamer sequences are used. To demonstrate such universal applicability, an adenosine hydrogel was prepared using ATP aptamer as the linker. Here, adenosine, a kind of nucleoside composed by adenine and ribose, possesses the signaling function in the peripheral and central nervous system. It is meaningful to detect adenosine in physiological level for the influence of brain function and behavior.57 Some biosensors about adenosine detection such as luminescent aptamer sensor58, colorimetric biosensor59 were developed for on-site or real-time analysis. Especially as a cancer biomarker, urinary adenosine can be applied to monitor the progress of diseases60-62. A simple POC method for adenosine detection is useful and meaningful in clinical diagnosis. This adenosine-hydrogel-combined µPAD method achieved the quantitative detection of adenosine with a linear range from 0 to 800 µM (Figure 7). Hence, the platform based on hydrogel-combined µPAD provides a general approach to realize a visual, disposable and quantitative detection of various targets using diverse aptamer-hydrogels.


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Figure 7 (A) Pictures: the detection of adenosine from 0 ~ 800 µM with µDiSH-PAD; (B) The distance of microfluidic bar plotted against the concentration of adenosine. The markers and error bars reflect the average and standard deviations of three measurements.

Conclusions In summary, a disposable, equipment-free, versatile point-of-care testing platform, microfluidic Distance Readout Sweet Hydrogel Integrated Paper-based Analytical Device (µDiSH-PAD), was developed for portable quantitative detection of targets. The platform relies on a target-responsive aptamer-crosslinked hydrogel for target recognition, cascade enzymatic reactions for signal amplification, and a microfluidic paper-based analytic device (µPAD) for visual distance-based quantitative readout. The µDiSH-PAD offers several advantages which are very attractive for POCT applications. First, the paper based analytical devices are cost-effective, disposable and easy to fabricate by printing. Second, the target-responsive aptamer hydrogel system enables sensitive and selective detection of a variety of targets. Moreover, the distance-based design provides equipment-free readout for user-friendly quantitation by naked eyes, with less influence of user interpretation. Further integration of µDiSH-PAD with sample introduction/enzymatic reaction will enable a device with user-friendly sample-in-answer out operation63. For example, as demonstrated in our recent work58, hydrogel with amylose solution can be integrated in a reaction chamber on a paper chip. Sample introduction would lead to dissolution of hydrogel to initial hydrolysis of amylose and production of glucose. A simple folding of the paper chip would allow the glucose solution to wick into the µDiSH-PAD by capillary action for simple distance readout. Based on low cost, ease



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of use, high versatility, and disposability with visual quantitative readout, the µDiSH-PAD holds great potential for portable detection of trace targets in environmental monitoring, security inspection, personalized healthcare and clinical diagnostics.

Acknowledgments We thank the National Science Foundation for Distinguished Young Scholars of China (21325522), National Science Foundation for Excellent Youth Scholars of China (21222506, 21422506), and the National Science Foundation of China (91313302, 21205100, 21275122) for financial support.

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