Integrated Distance-Based Origami Paper Analytical Device for One

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Integrated Distance-based Origami Paper Analytical Device (ID-oPAD) for One-Step Visualized Analysis Tian Tian, Yuan An, Yiping Wu, Yanling Song, Zhi Zhu, and Chaoyong James Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09717 • Publication Date (Web): 17 Aug 2017 Downloaded from http://pubs.acs.org on August 18, 2017

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ACS Applied Materials & Interfaces

Integrated Distance-based Origami Paper Analytical Device (ID-oPAD) for One-Step Visualized Analysis Tian Tiana, Yuan Ana, Yiping Wua, Yanling Songa,b*, Zhi Zhua, Chaoyong 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 for Analytical Science of Food Safety and Biology, State Key Laboratory of Photocatalysis on Energy and Environment, College of Biological Science and Engineering, Fuzhou University, Fuzhou, 350002, China

* To whom correspondence should be addressed. Tel: (+86) 592-218-7601; Fax: (+86) 592-218-9959. E-mail: [email protected]; or Tel: (+86) 591-22860973; E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT

An integrated distance-based origami paper analytical device (ID-oPAD) is developed for simple, user-friendly and visual detection of targets of interest. The platform enables complete integration of target recognition, signal amplification and visual signal output, based on aptamer/invertase functionalized sepharose beads, cascaded enzymatic reactions and a 3D microfluidic paper-based analytical device with distance-based readout, respectively. The invertase-DNA conjugate is released upon target addition, after which it permeates through the cellulose and flows down into the bottom detection zone, while sepharose beads with larger size are excluded and stay in the upper zone. Finally, the released conjugate initiates cascaded enzymatic reactions and translates the target signal into a brown bar chart reading. By simply closing the device, the ID-oPAD enables sample-in-answer-out assay within 30 min with visual and quantitative readout. Importantly, bound probe/free probe separation is achieved by taking advantage of the size difference between sepharose beads and cellulose pores, and the downstream enzymatic amplification is realized based on the compatibility of multiple enzymes with corresponding substrates. Overall, with the advantages of low-cost, disposability, simple operation and visual quantitative readout, the ID-oPAD offers an ideal platform for point-of-care testing, especially in resource limited areas.

KEYWORDS: microfluidic paper-based devices, point-of-care (POC), distance-based detection, aptamer


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Introduction

Microfluidic paper-based analytical devices (µPADs), as a new class of platforms for point-of-care (POC) screening, have attracted a great deal of interest since they were first introduced by Whitesides’ group.1 With inherent merits such as low-cost, no instrument requirement, ease of mass production, disposability and desired biocompatibility, µPADs hold the potential to revolutionize healthcare diagnostics, environmental monitoring and food safety inspection, especially in resource-limited areas where inexpensive and user-friendly analytical devices are in urgent need.

2, 3

However, the growing need for point-of-care testing (POCT) has led

to additional demands for simpler, multiplex and portable analytical devices,4, 5 where integrated devices are highly desired. Ideal µPADs would integrate a large number of analysis elements with direct readout and would require minimal manipulation by users. In this regard, 3D µPADs, which are based on folding (origami)

6-8

or stacking layers of 2D µPADs,9, 10 have

aroused much interest since they enable increased functionality and multiplex analysis, while retaining small device size.11 Compared with the approach that relies on stacking multiple layers held together by double-sided tape, origami-based methods have unique merits, including one-step fabrication regardless of the number of layers, assembly by simple folding, and possible mass production.6,8 The design of 3D µPADs allows reagents to be stored in different zones. Once the reaction is initiated, liquids flow both vertically and laterally, enabling simultaneous parallel and multiplex assays, while eliminating cross contamination.12



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To achieve high performance, many integrated paper devices have been developed with different types of readout.13-18 One typical example is the integration with a personal glucose meter (PGM), which allows rapid and digital analysis of targets. For example, Whitesides’ group proposed a PGM-integrated µPAD to detect glucose, cholesterol, alcohol and lactate, based on their enzymatic reactions with respective substrates.19 Recently, they upgraded the device into a “pop-up” 3D µPAD for detection of β-hydroxybutyrate (BHB).20 Although the method is powerful, targets of interest are to some extent restricted to the pairing of enzyme with corresponding substrates. More recently, Lu’s group integrated the PGM with a lateral flow device for analysis of both small molecules and protein (streptavidin) in a single step.21 In addition to PGMs, smartphones have also been integrated with µPADs.22-24 In this case, results are recorded by the smartphone, converted by the included software and finally displayed. The use of these electronic devices indeed offers precise detection results with high resolution and little need for user interpretation,25 but the additional operation introduces potential error. Moreover, the accompanying cost increase due to incorporation of these electronic readers will somehow preclude their application especially in resource-limited regions. Therefore, integrated POCT devices with simple operation, yet no instrumentation requirement, is highly desired. Distance-based measurements, which rely on establishing the relationship between the length of the visual signal and target concentration, have been reported as a novel class of quantitative methods.26-31 Elimination of external electronic readers, simple user interpretation, and ease of integration endow distance-based methods with


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great potential in applications such as biomedical diagnosis, environmental analysis and food security monitoring.32 Our group has developed a distance-based µPAD integrated with a target-responsive hydrogel for POCT.27 Although the system is versatile and equipment-free, one limitation is that target-induced collapse of the hydrogel requires controlled conditions and multiple washing steps. As a result, the target recognition and separation cannot be performed directly on the µPAD, limiting the full integration of target recognition with downstream analysis. Thus a fully integrated sample-in-answer-out system is highly desirable. Herein, we propose a fully integrated distance-based origami paper analytical device (ID-oPAD) for one-step quantitative POCT. To achieve fully integrated analysis in µPADs, the key point is implementation of target recognition with downstream signal amplification and output. To achieve probe separation, we take advantage of the size difference between cellulose pores and functionalized sepharose beads, using µPAD cellulose as a filter. And the downstream enzymatic amplification is realized based on the compatibility of multiple enzymes with corresponding substrates. In this case, target recognition is achieved via competitive binding of aptamer with the target, and separation is performed directly on the µPAD, where released probes permeate through the cellulose, while functionalized sepharose beads are excluded. Moreover, our previously developed µPAD devices have involved cascaded enzymatic reactions in two steps, because partial operations could only be performed in tubes.27,33 Here, a one-step initiated tri-enzyme cascaded reaction is achieved in the ID-oPAD, since it enables target-triggered probe release without



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external operation. Functionalized sepharose beads are prepared by immobilization of biotin-DNA via streptavidin-biotin binding and sandwich structure construction of biotin-DNA/aptamer/invertase-DNA, and the beads are then deposited on the upper zone of the ID-oPAD. When target is introduced, it binds preferentially with the aptamer, leading to a conformational change of the aptamer and thus release of the invertase-DNA conjugate from the sepharose beads. By “closing” the ID-oPAD, the released conjugate permeates through the cellulose and flows down into the bottom detection zone by gravity, while sepharose beads with size larger than the cellulose pores are excluded by the cellulose and stay in the upper zone. After the separation via this “open-to-close” process, the released invertase-DNA conjugate initiates a cascaded Invertase-GOx-HRP enzymatic amplification, and the target signal is finally translated into a brown bar chart reading. With increasing target concentration, more invertase-DNA conjugate is released, leading to increased length of the brown strip and establishing visual and quantitative target detection. Overall, by taking advantage of the size difference between the cellulose pores and functionalized sepharose beads, combined with a tri-enzyme cascaded system, the proposed ID-oPAD enables one-step analysis of targets. With the merits of low-cost, disposability, simplicity, user friendliness, ease of mass production, and visual quantitative readout, the ID-oPAD offers an ideal visual quantitative platform for point-of-care testing, especially in resource limited areas.

Experimental


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Materials and methods. DNA sequences used in the experiment (Table S1) were purchased from Shenggong (Shanghai, China). Streptavidin Sepharose High Performance beads and Whatman No. 1 chromatography filter paper was obtained from

GE

Healthcare

(Pennsylvania,

USA).

4-(N-Maleimidomethyl)

cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC), 3,3’-diaminobenzidine (DAB), horseradish peroxidase (HRP), glucose oxidase (GOx), tris(2-carboxyethyl)phosphine (TCEP), invertase from baker’s yeast (grade VII) and acrylamide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sucrose and other reagents were obtained from Sinopharm Chemical Reagent (Shanghai, China).

Invertase buffer contained 0.1 M NaCl and

0.1 M sodium phosphate (pH 7.3). All the µPADs were fabricated by a wax printer (Xerox Colorqube 8580). Design and fabrication of µPADs. ID-oPADs were designed by the software CorelDraw and fabricated by wax printing.34 First, designed patterns were printed on Whatman No.1 filter paper via a wax printer. Then, patterned papers were kept in an oven at 120°C for 1 min to ensure wax permeation and produce µPADs with double-sided wax-defined channels. The dimensions and design of 2D and 3D µPADs (ID-oPAD) are shown in Figure 1. Green wax is printed on the surrounding of paper area to enhance the mechanical strength of µPADs and the hydrophobicity of wax reduces sample leakage when µPADs are folded. Fabrication of the ID-oPAD was inspired by a pop-up electrochemical paper-based analytical device (EPAD).20 Briefly, patterned paper was cut along printed lines and then folded to form a 3D structure.



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Additional reaction zones with certain thickness were fabricated by cutting paper with multilayers, which were then stuck to µPADs to avoid sample leakage. The ID-oPAD consisted of a sample well, reaction zone and detection channel. This 3D structure allowed precise control of fluidic flow. The ID-oPAD changed from “open” to “closed” by mechanical pressure (we used a 20 g weight). Thus the upper and lower zones were connected, allowing liquid transfer to the bottom reaction zone (Figure S1). Before analysis, 3,3’-diaminobenzidine (DAB) was spread evenly onto the straight channels, and 0.7 µL or 1.0 µL of GOx (714 U/mL) /HRP (714 U/mL) was added onto the circular sample zone for 2D µPAD or ID-oPAD, respectively.

Figure 1. Designs of (A) 2D µPADs and (B) 3D µPADs (ID-oPADs). (C) Spatial structure of ID-oPAD in an open format. Target recognition was achieved on the sample zone upon sample introduction. The enzymes and substrates were pre-deposited on the reaction zone and the distance readout was finally displayed along the channel.

Conjugation of DNA and invertase. The conjugation of DNA and invertase was performed according to a previous report.35 Briefly, 60 µL of 1 mM thiol-DNA, 4 µL


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of sodium phosphate buffer (pH=5.5) and 4 µL of 30 mM TCEP were mixed. Meanwhile, 800 µL of 20 mg/mL invertase in 0.1 M potassium phosphate buffer was mixed with 1 mg sulfo-SMCC. Both reactions were rotated at room temperature for 1 h. After the removal of excess insoluble sulfo-SMCC by centrifugation (14000 rpm, 10 min), the activated thiol-DNA and invertase were purified 8 times by Amicon-3K and Amicon-30K ultrafiltration, respectively, using PBS as the solvent. Subsequently, the purified thiol-DNA was mixed with the activated invertase and incubated for 48 h at room temperature. Finally, the DNA-invertase conjugate was purified 8 times by Amicon-30K to remove unreacted DNA. Preparation of DNA-invertase functionalized sepharose beads. For each concentration, 10 pmol cocaine aptamer, 10 pmol biotin-DNA and 100 pmol DNA-invertase conjugate were mixed at room temperature for 10 min, followed by another 10 min incubation with 1 µL streptavidin sepharose beads at room temperature. Afterwards, the obtained DNA-invertase functionalized beads were washed 3 times using PBS to remove excess DNA-invertase. Finally, functionalized beads were dispersed in 5 µL invertase buffer for subsequent analysis. Analysis of invertase on 2D µPADs. To demonstrate the feasibility of one-step cascaded enzymatic reactions, 0.7 µL GOx (714 U/mL) /HRP (714 U/mL) and 10 µL 1 M sucrose were added to the circular sample zone of 2D µPADs, followed by the addition of 10 µL of invertase with various concentrations. Subsequently, µPADs were placed in the oven at 37°C. After 20 min, the detection result was observed by naked eye for quantitation.



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Comparison of one-step and two-step methods. The one-step method was carried out as described in the analysis of invertase on 2D µPADs. For the two-step method, 10 µL 1 M sucrose and 10 µL invertase were mixed in Eppendorf tubes at 37°C for 5 min. Afterwards the mixture was transferred onto the sample zone of 2D µPADs and reacted at 37°C for 20 min. The experimental results were compared with those obtained by the one-step method. Feasibility of ID-oPAD and comparison with the glucose meter. The feasibility of the ID-oPAD was demonstrated by invertase analysis. First, 2.5 µL 6 M sucrose was preloaded on the bottom circular zone, and 20 µL invertase with different concentrations were added to each sample zone. After 1 min permeation, the ID-oPAD was folded and pressed using a 20 g weight to ensure stable pressure. When the upper and bottom zones were connected and the cascaded enzymatic reaction was triggered, the device was held at 37°C for 15 min. For glucose meter readout, 2.5 µL 6 M sucrose and 20 µL invertase with different concentrations were mixed and incubated at 55°C (optimum temperate for invertase according to the product information) for 15 min. Finally, the glucose concentration was recorded by a glucose meter. Target detection on ID-oPAD. The ID-oPAD was preloaded with DAB (10 mg/mL), 1 µL GOx/HRP (714 U/mL) and 2.5 µL sucrose (6 M). For cocaine and adenosine analysis on ID-oPAD, 5 µL of functionalized beads were spotted onto the upper sample zone, followed by the addition of 20 µL of cocaine with various concentrations. The ID-oPAD was inverted for 5 min to allow cocaine binding with


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aptamers, which caused decomposition of the DNA sandwich structure and release of DNA-invertase. Due to the surface tension, liquids formed a hemispherical shape and would not drop from the sample zone. After incubation, the ID-oPAD was inverted again and held for 1 min. At this time, liquids containing released DNA-invertase permeated through the upper sample zone due to gravity, while functionalized sepharose beads, whose sizes (30-50 µm) were larger than the paper pore size (11 µm), stayed on the surface of the upper zone. By “closing” the ID-oPAD with a 20 g weight, the upper zone was connected to the bottom zone, transferring the liquid into the bottom detection zone and initiating the cascaded enzyme amplification. After 10 min reaction at 37°C, 20 µL invertase buffer was added to the upper zone to elute the non-transferred released conjugate and enhance the signal. After another 10 min reaction, the detection result was observed by naked eye and distance was measured for quantitation.

Results and Discussion

Working Principle of Integrated Distance-based Origami Paper Analytical Device (ID-oPAD). As schematically illustrated in Figure 2, the ID-oPAD consists of an upper sample zone for sample loading and target recognition, and a bottom reaction zone for signal transduction and amplification. Functionalized beads were obtained by immobilization of biotin-DNA via streptavidin-biotin binding and the sandwich structure construction of biotin-DNA/aptamer/invertase-DNA conjugate. The upper zone is pre-deposited with invertase functionalized beads, while the bottom



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zone is preloaded with GOx/HRP, sucrose in the circular zone and DAB in the straight channel. When target is introduced, it binds preferentially with the aptamer, causing de-hybridization of the DNA sandwich structure and release of DNA-invertase. The released invertase permeates through the paper, while functionalized beads, with sizes larger than the paper pores, are excluded and stay on the surface of the upper zone. By a simple folding, the upper and bottom zones are connected, transferring the released invertase into the detection zone. At this time, sucrose is hydrolyzed into glucose by invertase, while the produced glucose is immediately converted into gluconic acid and H2O2 by GOx, and the resulting H2O2 oxidizes the colorless DAB into brown poly (DAB), a precipitate that does not migrate with the flow. Since the length of poly (DAB) is positively correlated with the amount of glucose, which depends on the amount of the released invertase, the target concentration can be quantified by this visual and distance-based method.

Figure 2. Working principle of the integrated distance-based origami paper analytical device (ID-oPAD). (A) When target is present, it binds preferentially with the aptamer, leading to the release of invertase-DNA conjugate. (B) By “closing” the ID-oPAD,


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the released conjugate permeates through the cellulose and flows down into the bottom detection zone, while sepharose beads with larger size remain in the upper zone. (C) The released invertase-conjugate initiates cascaded enzymatic reactions, and finally translates the target signal into a brown bar chart movement, whose length is correlated with the target concentration. Performance of the one-step initiated cascaded enzymatic reactions. Different from previous methods,27,33 where cascaded reactions occurred step-by-step, we combined invertase, GOx and HRP, together with sucrose as the substrate, for one-step initiated cascaded enzymatic reactions. To test the feasibility of the one-step initiated cascaded enzymatic reactions, a 2D µPAD was used. GOx, HRP and sucrose were pre-deposited on the circular zone, and DAB was applied evenly along the channel and dried before the assay. During the analysis, different concentrations of invertase were added to each circular zone. As shown in Figure 3A, the length of the distance signal was correlated with the concentration of invertase via cascaded enzymatic amplification of invertase, GOx and HRP. The result shown in Figure 3B demonstrated a positive correlation between the invertase concentration and the distance signal, indicating that one-step initiated cascaded enzymatic reactions have the capability for quantitative detection of targets. Such one-step initiated cascaded enzymatic reactions eliminates the need for repeated addition of reagents. Moreover, by mixing all reagents together, the method takes less time and simplifies the analysis process.



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Figure 3. Feasibility of the one-step method using invertase with different concentrations. (A) Pictures of invertase detection results on 2D µPADs. (B) Working curve of the brown bar chart length versus invertase concentration. Each point is the average of three repeated analyses.

To further investigate whether the one-step method would retain the sensitivity of the two-step method (Figure 4A), in which sucrose was hydrolyzed into glucose by invertase in a tube, followed by adding the resulting glucose product to µPADs to initiate the subsequent catalytic reaction, detection of invertase by both methods was carried out on 2D µPADs. In Figure 4B, both methods showed comparable sensitivity, while the one-step method showed a wider dynamic range, possibly because in the two-step method, all glucose generated in the first step is introduced into 2D µPADs. This results in a large amount of poly (DAB) produced in a short time, thus causing early retention of the liquid due to the capillary effect influenced by the formation of poly (DAB). Conversely, for the one-step method, the generation and consumption of glucose remains balanced, allowing the liquid to move farther. Thus, the one-step


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method produces a larger distance signal than the two-step method for the same concentration of invertase.

Figure 4. Comparison of the one-step and two-step methods. (A) For the one step method, invertase, GOx, HRP and sucrose are mixed together. For the two-step method, sucrose was hydrolyzed into glucose by invertase in solution, followed by adding the resulting glucose product to the µPAD to initiate the subsequent catalytic reaction. (B) Working curve of the brown bar chart length versus invertase concentration using one-step and two step method, respectively. Each point is the average of three repeated analyses.

Performance of the ID-oPAD. To achieve one-step analysis of targets, competitive assays were introduced. Since the target binds preferentially with the aptamer, leading to the disassembly of the DNA sandwich structure and the release of the invertase-DNA conjugate, the key is integration of the separation process into the µPAD. In this work, we adopted a separation method based on the size difference between the cellulose pores (11 µm) and sepharose beads (30-50 µm). First, the feasibility of separating released DNA from bound DNA with the device was tested



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(Figure S2). When sample containing sepharose beads was added to the upper zone, the liquid on the front side remained turbid while the liquid permeating through the cellulose turned clear, indicating that sepharose beads with larger sizes than cellulose pores stayed on the surface. The idea was further confirmed by microscopic images of each side, where abundant sepharose beads were observed on the top surface and none were found on the back side and bottom zone. These results verified that efficient probe separation can be realized using cellulose as the filter to separate free from bound probes. In order to integrate the separation process into the µPAD, ID-oPAD, a reconfigurable 3D µPAD was developed. The reversible origami allows the transfer of released invertase-DNA conjugate from retained sepharose beads into the detection zone by “closing” the ID-oPAD. The performance of ID-oPAD was also investigated for invertase analysis (Figure S3A). Invertase with various concentrations was added to the upper sample zone. By folding the ID-oPAD, invertase was transferred to the bottom zone and initiated cascaded reactions. As a result, a positive correlation between invertase concentration from 22.4 nM to 1.12 µM and brown stripe length was displayed, similar to the result for the 2D µPAD shown in Figure 3, indicating the feasibility of ID-oPAD for target analysis. Furthermore, we used a personal glucose meter (PGM), one of the most prevalent POC devices, to test the accuracy of the proposed method. In Figure S3B, the trend of PGM signal was similar to the result of the ID-oPAD, demonstrating that the sensitivity of the proposed method is comparable to that of commercial personal glucose meters.


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Detection of cocaine. To evaluate the ability of the ID-oPAD to detect a variety of targets, we first investigated quantitative detection of cocaine using cocaine aptamer.36,

37

As a global illegal drug, cocaine has caused serious cases of drug

addiction. Thus the POCT of cocaine plays an important role in the prevention of drug abuse. In the experiment, cocaine with different concentrations (0-500 µM) was added to each sample zone. As shown in Figure 5, the distance signal increased in length in proportion to the cocaine concentration, with a limit of detection (LOD) of 1.8 µM. It is worth noting that this LOD is comparable to those of previously reported cocaine sensors developed by others and ourselves, as well as commercial cocaine test kits.27,33 Thus, the ID-oPAD retains sensitivity while simplifying and integrating all the analysis steps. Moreover, small volumes of samples (20 µL) are sufficient to initiate the assay simply by “closing” the device, and quantitative readout is easily obtained within 30 min. Overall, the proposed method allows single step sample-in-answer-out analysis while maintaining user-friendliness and simplicity.



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Figure 5. Detection of cocaine in buffer using the ID-oPAD. (A) Images of the ID-oPAD with different concentrations of cocaine in buffer. (B) Working curve of cocaine in buffer with standard deviations obtained from three measurements.

Selectivity of ID-oPAD for Cocaine Detection. Since the selectivity of the system is highly dependent on the specific recognition between the target and the corresponding aptamer, it is expected that the ID-oPAD should retain the high selectivity of the target. Herein, cocaine metabolites and blank sample were chosen as negative controls to demonstrate the selectivity. As shown in Figure 6, a distinct brown bar chart was observed for 50 µM cocaine, while negligible distance signal was observed for 500 µM cocaine metabolites, indicating that the ID-oPAD retains the high selectivity of the aptamer.

Figure 6. Selectivity of the ID-oPAD. (A) Images of the ID-oPAD in response to 50 µM cocaine and 500 µM cocaine metabolites. (B) Histograms demonstrating the selectivity of ID-oPAD.


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Detection of cocaine in real samples. To investigate the compatibility of the ID-oPAD in real samples, 50% raw urine samples spiked with cocaine were tested with our method. As shown in Figure 7, a growing trend of brown bar length versus cocaine concentration was observed, and cocaine concentrations down to 5 µM could be detected. This limit of detection is comparable to that obtained in buffer system, suggesting that the components in real samples hardly interfere with the performance of the ID-oPAD. In addition, it is reported that the normal urinary glucose levels are low38 and thus should hardly influence the performance of the ID-oPAD. However, for diabetic patients whose urinary glucose levels are high, GOx can be introduced prior to the analysis to remove endogenous glucose in samples.39 Overall, the successful analysis of cocaine in complex sample matrices endows our method with great promise for on-site and real-time screening.



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Figure 7. Detection of cocaine in real samples. (A) Images of the ID-oPAD with different concentrations of cocaine in 50% raw urine. (B) Working curve of cocaine in 50% raw urine with standard deviations obtained from three measurements.

Generality of the ID-oPAD. Since the specificity of this system relies on the specific recognition of aptamers towards targets, it is convenient to adapt our system to the detection of a variety of targets. To demonstrate the universal applicability, adenosine, a crucial biological cofactor with vasodilator and antiarrhythmic activities, was chosen as the target of interest.40 Monitoring the level of adenosine in physiological conditions helps to evaluate the role it plays in brain function as well as behavior.41, 42 The DNA sequence for adenosine detection was described by Lu et al. (Table S1),35 who reported successful competition of the aptamer for adenosine


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over cDNA. As shown in Figure 8, with various concentrations of adenosine ranging from 0 to 200 µM, a corresponding increase of the length of bar charts was observed, indicating that the system had the capacity to quantitatively detect other targets simply by using the corresponding aptamer. As low as 20 µM adenosine can be detected by ID-oPAD, which is comparable to the LODs of reported methods.35, 39 Overall, the developed platform is versatile and provides a universal method for simple, visual and quantitative analysis of targets of interest with selected aptamers.

Figure 8. Generality of the ID-oPAD. (A) Images of the ID-oPAD with different concentrations of adenosine. (B) Working curve of adenosine with standard deviations obtained from three measurements.

Conclusions In conclusion, we have developed an integrated distance-based origami paper analytical device (ID-oPAD) for one-step visual analysis of targets of interest. The device enables the complete integration of target recognition, signal amplification and transduction, which are based on enzyme-functionalized sepharose beads, cascaded enzymatic reactions and a 3D µPAD with distance-based readout, respectively. Importantly, the probe separation is achieved by taking advantage of the



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size difference between sepharose beads and cellulose pores, and the downstream enzymatic amplification is realized based on the compatibility of multiple enzymes with corresponding substrates. The developed method has several merits. First, as a paper-based device, ID-oPAD is user-friendly, cost-effective (a simple µPAD costs less than $0.01 for paper and patterning),43 disposable and amenable to mass production. Second, the reconfigurable 3D design of the ID-oPAD enables precise control of fluidic flow path and timing during multi-step reactions. Compared with previous µPADs, ID-oPAD enables simple, visual and integrated detection of targets, where one-step analysis is initiated by the addition of sample. Third, small volumes of samples (20 µL) are sufficient to initiate the assay simply by “closing” the device, and the total sample-in-answer-out time is within 30 min. Fourth, because the ID-oPAD retains the specificity of aptamers which are currently available or can be selected by SELEX,44-47 a wide range or analytes can be detected simply by changing the recognition element of the ID-oPAD. Besides using aptamers, the proposed method can also be applied to Enzyme-Linked ImmunoSorbent Assay (ELISA), and the target signal can be transduced into invertase concentrations that are dependent on competitive reaction between the target and antibody. Moreover, compared to intensity-based or electrochemical methods, the distance-based method is resistant to user interpretation variance and eliminates the need for external electronic devices. Overall, with characteristics of low cost, disposability, simplicity, low-volume consumption, user friendliness and versatility, the ID-oPAD is expected to find wide


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application in personalized healthcare, clinical diagnostics and environmental analysis, especially in resource limited areas.

Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org Detailed information of DNA sequences used in the experiment, photographs of the ID-oPAD, microscope images of sepharose on paper, performance of the ID-oPAD and the comparison with a personal glucose meter. Acknowledgments We thank National Science Foundation of China (21325522, 21422506, 21435004, 21521004), National Basic Research Program of China (2013CB933703), Program for Changjiang Scholars and Innovative Research Teams in University (IRT13036), and National Found for Fostering Talents of Basic Science (NFFTBS, J1310024) for their financial support.

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