Paperfluidic Chip Device for Small RNA Extraction, Amplification, and

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Paperfluidic Chip Device for Small RNAs Extraction, Amplification and Multiplexed Analysis Huaping Deng, Xiaoming Zhou, Qianwen Liu, Bofan Li, Hongxing Liu, Ru Huang, and Da Xing ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12637 • Publication Date (Web): 08 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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ACS Applied Materials & Interfaces

Paperfluidic Chip Device for Small RNAs Extraction, Amplification and Multiplexed Analysis Huaping Deng a, Xiaoming Zhou a,*, Qianwen Liu b, Bofan Li a, Hongxing Liu a, Ru Huang a, Da Xing a,* a

MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China b

Department of Thoracic Surgery, Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center of Cancer Medicine ABSTRACT: Small RNAs have been considered as potential biomarkers of various human diseases. Sensitive and multiplexed determination of small RNAs with point-of-care (POC) assay would be of great significance. Herein, an integrated paperfluidic chip device for multiplexed small RNAs analysis was developed for the first time. In this system, the extraction and purification of small RNA was completed through polyethersulfone (PES) paper chip without the need for centrifugation. And subsequently, a newly designed hairpin probe-exponential amplification reaction (HP-EXPAR) was directly performed within the extraction paper chip. For realizing multiple detection simultaneously, multilayer paper chip was designed in foldable manner with more portability and usability. Quantum dots (QDs) were employed as signal labels, which endowed this assay with high optical detection efficiency. Moreover, magnetic sheets were introduced as an alternative method for layer stacking, not only guaranteeing adjacent layers contact but also facilitating the sample dispersion. With these outstanding characteristics, our platform obtained a satisfactory sensitivity range from 3×105 copies to 3×108 copies with a

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limit of 3×106 copies. Additionally, the multiplex small RNAs analysis from various cancer cells were in good agreement with the results of real-time polymerase chain reaction (qRT-PCR). More importantly, simultaneous analyzing two types of miRNAs from clinical tumor samples demonstrated the clinical applicability of the system. Therefore, the proposed paper-based device shows great promise of POC application in the future.

KEYWORDS: paperfluidic chip; quantum dots; small RNAs analysis; in situ amplification; multiplexed detection INTRODUCTION Small RNAs are also known as small non-coding regulatory RNAs, which play vital role in regulating different genes expression.1-2 There are three major types of small RNAs such as short interfering RNAs (siRNAs), PIWI-associated RNAs (piRNAs) and microRNAs (miRNAs), in line with the distinctions in biogenesis and cellular roles. MiRNAs, the best understood among the three classes, have been demonstrated to regulate at least 30% of the genes in humans.3 Furthermore, the miRNAs aberrant expressions are directly concerned to various diseases including neurodegenerative disorders, cardiovascular diseases and especially human cancer. Also, the cancer development and progression generally involve the expression alterations of several miRNAs species.4-6 With high diagnostic value, these specific-expression miRNAs have been considered as promising biomarkers in the cancer diagnostic. Therefore, reliable detection of multiplex miRNAs may be an effective strategy in early cancer diagnostic. Conventional approaches such as the real-time polymerase chain reaction (qRT-PCR), microarray technology and northern blotting have made great contributions to small RNAs analysis.7-8 While the tedious procedures and relying on kinds of laboratory-oriented sophisticated apparatus, may impede


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further practical applications, particularly in low-resource settings. Consequently, it is of great importance to develop convenient and sensitive scheme for small RNAs analysis at point-of-care (POC) level. Paper-based analytical platform has received increasing concerns for the development of POC diagnostics, owing to speedy assay, small sample volume, cost-effectiveness and user-friendly operation.9-12 As nucleic acid testing mainly involves sample preparation, amplification and detection procedures, attentions are being turned to develop paper-based analytical techniques that integrated extraction and amplification steps to the detection system.13 In recent years, much progress have been made in the field of paper-based device for DNA determination.14 Whitesides et al. developed a “paper machine”, incorporating Fast Technology Analysis (FTA card-based) DNA extraction, then in situ amplification and fluorescent detection by sliding the device.15 Xu's group has also reported an paper-based biosensor for DNA extraction, then amplification and lateral flow assay (LFA) detection.16 While only one study to date, from Klapperich's group has proved that integrated paper-based extraction and amplification for RNA detection.17 Nonetheless, the RNA was extraction and purification from virus, whose structural elements were much simpler than the complex cell components. To our knowledge, there is not yet paperbased device that integrated the extraction and amplification steps for small RNAs analysis. The challenges lie in that on one hand, paper-based detection platform is generally confronted with unsatisfactory sensitivity.9 On the other hand, the inherent characteristics of small RNAs include: (i) the short lengths and low abundance in samples,18-21 which raise the difficulty in extraction and purification; (ii) highly homologous sequences, accordingly a higher detection specificity and sensitivity must be required for the multiplex analysis.22-25


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Herein, we sought to develop an integrated paper-based device that capable of performing small RNAs extraction, isothermal amplification and multiplex detection. To circumvent the aforementioned limitations, our paperfluidic chip system was designed in multilayer format, fabricated by simple paper folding, which exhibited unique properties: reduced fabrication time and with more portability in contrast to the layer-by-layer stacked method;26-28 the ability to unfold the device realizing individual layer analysis; and the most of special interest was achieving multiplexed assays enabled sample dispersion to individual layer simultaneously. In addition, it is a key challenge to maintain contact between adjacent layers, enabling sample permeation through the device completely.29 Some reports utilized binder clips or adhesive tapes, which required precise alignment and were cumbersome.30-31 Two magnetic sheets with opposite polarities were employed in this assay, being an alternative to conventional methods. The use of magnetic sheets not only could guarantee the layers contact, but also facilitate the fluidic dispersion. For acquisition of high sensitivities, integrated molecular amplification technology is often a concern.32 Eliminating the need for the precise and repeated heating cycles, isothermal amplification strategies such as rolling circle amplification (RCA), the loop-mediated isothermal amplification (LAMP) and the exponential amplification reaction (EXPAR) have garnered of growing attentions.33-34 Nevertheless, RCA often need lengthy detection times (over 6 h) and heat denaturation leading double-stranded DNA to single stranded, which is complex.35 Although LAMP has proved to be available for paper-based assay, the requirement for complicated design of 4 different primers to each target, may limit its application for multiplex small RNAs detection.36 In contrast, EXPAR strategy with the higher amplification efficiency, is adaptive for the amplification of any short oligonucleotide and amenable to multiplexing.37-39 In


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this assay, a hairpin structure was introduced to be designed as EXPAR amplification template, which rendered it with preferable specificity for multiplex small RNAs determination. In this study, paperfluidic chip device integrated small RNA extraction, isothermal amplification, and optical detection was constructed. MiRNA 155 (miR-155) and miR-21, the potential cancer biomarkers that have been confirmed associated with lung cancer,40 were selected to test the performance of our platform. Extraction and purification of small RNA was performed through polyethersulfone (PES) filtration, which is adaptive for the extraction and purification eliminating the requirement of centrifuges or other sample treatment equipment, owing to its properties of low protein adsorption, high mechanical strength, thermal and chemical resistances. Subsequently, the newly designed hairpin probe-based EXPAR (HPEXPAR) was conducted directly within the same paper extraction chip via a handheld batterypowered heater. The downstream detection was applied on foldable multilayer paper chip. Quantum dots (QDs), the promising fluorescent nanomaterial with unique optical properties,41-42 were employed as signal labels. And the detection results consistented with real-time polymerase chain reaction (qRT-PCR) demonstrated that, this method could be potential candidate for small RNA determination in POC application. EXPERIMENTAL SECTION Materials and reagents. Polyethersulfone (PES) filter paper, cellulose absorption pads and glass fiber were purchased from Millipore (Billerica, MA). Quantum dots with streptavidin were synthesized in Wuhan Jiayuan Quantum Dots Co., Ltd. (Wuhan, China). Klenow Fragment (3'→ 5' exo-) polymerase, nicking endonuclease Nb.BbvCI, CutSmart buffer and NEBuffer 2 were the products of NEB (New England Biolabs, USA). The RNAase inhibitor and RNAiso for small RNA were purchased from Takara Biotechnology (Dalian) Co., Ltd. Streptavidin and bovine


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serum albumin (BSA) were purchased in Sigma-Aldrich (St. Louis, USA). Tween-20, Trisbuffered saline (TBS), borate buffer saline (BBS), the electrophoresis regents and phosphate buffered saline (PBS) were obtained in Shanghai Sangon Biotechnology Co. Ltd. All of the DNA probes including qRT-PCR primers and biotinylated probes were synthesized by Sangon Biotechnology Co. (Shanghai, China). And synthetic miRNAs were purchased in Invitrogen. The oligonucleotides sequences were all listed in the Table S1 and S2. Preparation of the QDs signal probes. The two specific DNA probes were modified with biotin firstly. QDs with streptavidin (QDs-525, QDs-605) and biotinylated DNA probes were mixed at the molar ratio of 1:30, respectively. Then the mixture solutions were stirred for 30 min guaranteeing the biotin and streptavidin combined completely. For removing unbound biotinylated probes, the mixture solutions were centrifuged at 2000 rpm on tubular ultrafiltration membrane of Nanosep 10K (Pall Corporation, NY). Then washed with PBS buffer (1×). QDs signal probes should be resuspended in the solution with 0.25% Tween-20, 5% BSA, 10% sucrose and 20 mM Na3PO4, stored at 4 °C for the future use. Characterizations of the two QDs signal probes were all presented in Figure S1. Fabrication of the paper-based device. The integrated paper-based device contains paperbased extraction setup and foldable paper detection chip. Extraction setup includes two layers of plastic magnetic sheets, polyethersulfone (PES) filter paper in the pore size of 0.2 µm and absorbent pad (see Figure S2). The upper layer magnetic sheet was punched with 3 mm diameter holes by puncher. The PES filter paper was also cut with 3 mm diameter pieces, then inlayed into the holes of the magnetic sheet. An absorbent pad was placed on the other plastic magnetic sheet. To construct foldable paper detection chip, paper (Whatman cellulose chromatography papers, grade 2) was cut into a suitable size (120 mm × 60 mm), then a wax-screen printing process was


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served to fabricate hydrophilic paper channels. As shown in Figure S3, the screen mesh openings with 2 mm pore size was putted on the paper, and the solid wax was pressed into the screen, baked for 10s in oven at 100 °C and cooled to room temperature, the wax diffused through the screen onto the paper. As specified hydrophobic and hydrophilic regions were patterned on the paper, the 1st layer was replaced with glass-fiber discs, onto which the QDs labels were dispensed and dried at room temperature. The streptavidin-biotinylated capture probe1 and capture probe 2 were dispensed onto other layers as test zones, respectively. Subsequently, the test zones were blocked by blocking buffer with 1% (w/v) BSA in PBS. Paper-based extraction procedure. The suspension cultured cells were mixed with 100 µL lysis buffer (RNAiso for Small RNA, (TakaRa, China)) and incubation for 5 min, then 20 µL chloroform was added to the lysate making the aqueous and organic phase separation. The supernatant needed to be transferred into another tube, then added with equal volume of isopropyl alcohol, 1× RNASecure (Ambion) and a final concentration of 50 µg/mL Glycoblue coprecipitant (Life Technologies). And pipetted the mixture onto that PES membrane. Then capillary forces generated from absorbent pad and the attractive force produced by the two magnetic sheets quickly made the liquid phase flow away, remaining the solid phase of the small RNA-Glycoblue precipitate. And then PES membrane was washed by 200 µL of 80% ethanol and subsequently with 100 µL 100% ethanol. After washing with the 100% ethanol, the PES membrane was dried for 2 min to prevent the interference from ethanol to subsequent amplification. When it needed quantification, PES membrane was putted into tube with appropriate nuclease-free water dissolving the complexes of RNA-Glycoblue, centrifuged to make the extracted small RNA completely out of the PES. The extractions were measured the OD260 by Nano-Drop ND2000c (Thermo Scientific).


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On-chip amplification and detection. Various concentrations of miRNA were added to the final volume of 5 µL, containing 150 nM hairpin probe, 0.5 units of Klenow Fragment, 1 units of Nb.BbvCI, 5 units of RNase inhibitor, 1×NEBuffer 2, 0.1×CutSmart buffer and 0.5 mM dNTPs. Subsequently, the solutions pipetted directly onto the PES membrane, sealed by an acetate film to prevent evaporation. And the amplification was conducted at 37 °C for 20 min in the portable heating block. Following amplification, the PES membrane containing HP-EXPAR reaction solution was placed onto the foldable paper chip, and aligned with the inlet port, capillary forces made the liquid flow to bottom layer, and then washed twice by the washing buffer. Unfold the paper device, and excitation with 365 nm UV light. The fluorescent images were photographed by cell phone or digital camera then analyzed with ImageJ software. The fluorescence intensity measurements was by calculating the mean fluorescence intensity of the paper chip, and divided the background. RESULTS AND DISCUSSION Principle for this paper-based device for small RNAs analysis. An overview of the integrated paperfluidic chip analysis system was schematically illustrated in Figure 1. Such a system mainly consists of paper extraction setup, specially designed handheld heating block and foldable multilayer detection chip. The paper extraction setup includes two layers magnetic sheets, PES filter paper chip and absorbent pad. The upper magnetic sheet with punched holes was used to inlay the PES, which is low protein adsorption and adoptable for small RNA extraction and filtration. The absorbent pad on the bottom magnetic sheet is placed under the PES disk for waste absorption. The detection paper chip was printed with wax to fabricate the hydrophilic paper channels, then folded to be multilayer contains conjugate layer dispensed with


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QDs, and test layers immobilized with different capture probes. After the amplification reaction, the amplification products vertically flow through each layer, realizing multiple samples analysis. The magnetic sheets employed in this assay, not only could guarantee contact between adjacent layers, but also facilitate the sample wicking flow completely.

Figure 1. The schematic illustration of this paperfluidic chip analysis platform. (A) The paperfluidic chip analysis system contains paper extraction setup, foldable multilayer detection chip and handheld battery-powered heating block. (B) Schematic diagram of the procedure for small RNAs extraction, in situ HP-EXPAR amplification and multiplexed analysis. To amply the detection signal, a small RNAs amplification procedure is needed. We anticipated that the small RNAs amplification procedure should meet the following requirements for paperfluidic chip: (i) it should be isothermal reaction thus facilitate the paper based


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amplification; (ii) it should hold high amplification efficiency for sensitive detection and singlebase discrimination; (iii) the amplification products should be short and single-stranded for facilitating hybridization reaction at room temperature. However, currently there isn’t existed amplification procedure that paperfluidic chip can adopt. EXPAR can meets these requirements at a large extent but the amplification specificity is usually not enough. For these reasons, we designed a hairpin probe based EXPAR (HP-EXPAR) to improve the specificity. As for the HPEXPAR, a designed partial hairpin probe containing miRNA-binding domain (X), two repeat Y domains as template sequences for EXPAR amplification, and two specific recognition sites for the Nb.BbvCI nicking endonuclease, one is in the middle of the two Y sequences and another is between X and Y sequences. The 3' termini was phosphate labeled, which could avoid nonspecific extension. Particularly, owing to the stem-loop structural constraint of the hairpin probe, a higher specificity for target recognition would be obtained compared with linear template.39 Once the target miRNA hybridizes with X and the hairpin probe would be unfolded, miRNA could extend with the help of Klenow Fragment polymerase. And subsequently, nicking endonuclease Nb.BbvCI specifically recognizes the nicking sites, resulting in single-strand DNA nicking in the newly extended strand then generating another new replication site for the polymerase. Due to the strand displacement of Klenow Fragment DNA polymerase, the short single stranded DNA (Y') will be displaced and released. In turn, the released Y' could hybridize with the Y section of another amplification template, leading the initiation of a new exponential amplification cycle. The repeated extension, cleavage and strand displacement can yield large amounts of the amplification product single-stranded Y', which subsequently applied on the paper detection chip and specifically hybridized with signal probes and capture probes for optical detection with UV light excitation.


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Optimization of experimental parameters. Prior to perform the HP-EXPAR reaction on paper chip, of which the feasibility has been confirmed by tube-based amplification and analyzed with polyacrylamide gel electrophoresis (PAGE) (see Figure S4). As triggered by miR-21, HPEXPAR reaction was conducted. And the well-defined band of the amplification product singlestranded Y' (24 nt) was obvious. In contrast, the negative control without miR-21 showed a negligible band, which demonstrated this amplification strategy was feasible. To improve the analytical performance of the platform, different experimental parameters were investigated with signal-to-noise ratio as evaluation criterion. Blocking buffer with BSA could reduce nonspecific adsorption thus plays key role in the performance of paper platform. However, BSA with high concentration possibly hinders reaction binding sites. In the present study, BSA with different concentrations were added in PBS as the blocking buffer to be optimized. The result was recorded in Figure 2A. PBS buffer with 1% (w/v) BSA achieved the highest signal-to-noise ratio, while 2% BSA got the lower signal-to-noise ratio. Therefore, the buffer PBS with 1% BSA is selected as blocking buffer. Furthermore, to explore the effect of washing buffer, three types of buffer: PBS with 0.05% Tween-20 (PBST), Tris-buffered saline with 0.05% Tween-20 (TBST) and borate buffer saline with 0.05% Tween-20 (BBST, pH 8.4) were compared (Figure 2B), and a maximum value was achieved with BBST. Therefore, BBST was employed as the optimum washing buffer. For optimization of HP-EXPAR reaction on paper chip, the template concentration is crucially on the amplification efficiency of HP-EXPAR. To optimize the template concentration, we performed HP-EXPAR amplification on chip with a range of 50 nM to 200 nM template. As depicted in Figure 2C, with the increasing concentration of template from 50 nM to 150 nM, the signal-to-noise ratio was synchronously improved while decreased beyond the concentration of


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150 nM. It could be attributed to the facts that high template concentration causes nonspecific amplification thus lead to high back-ground. However, low template concentration might lack enough hybridization sites with the available miRNAs. Hence, the template concentration of 150 nM is selected in the subsequent research. Additionally, amplification time is also crucial parameter of HP-EXPAR. Amplification time range from 15 min to 30 min was thus evaluated. The results were shown in Figure 2D. It is found that maximum signal-to-noise ratio was reached at the time of 20 min, which indicated the optimum time was 20 min.

Figure 2. Optimization of the experimental parameters. (A) The effect of BSA concentrations in blocking buffer on the paper-based platform; (B) The effect of different washing buffer solutions: PBST, TBST and BBST. (C) The effect of template concentrations in HP-EXPAR reaction on paper chip (50–200 nM); (D) The effect of amplification time in HP-EXPAR reaction on paper chip (15–30 min).


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Analytical performance. Under optimal experimental conditions, miR-21 with different amounts ranging from 3×105 copies to 3×108 copies were used to validate the sensitivity of our platform. As depicted in Figure 3A, the fluorescence photograph of paper chip could be observed obviously. And with the reduction of target amounts, QDs-605 fluorescence intensities decreased gradually. Through analyzing the interrelated intensities (see Figure 3B), detection limit of the approach was determined to be 3×106 copies. The inset was the linear relationship between the intensities of QDs fluorescence and the target amounts, and a correlation coefficient (R2) value of 0.9941 was obtained, which demonstrated this assay could be prominent analytical platform for sensitive miRNA detection.

Figure 3. (A) Fluorescence photographs of the paper chip for the quantification of miR-21 with different amounts from 3×105 copies to 3×108 copies; (B) Histogram indicating the fluorescence intensities of the paper chip (A). (C) Fluorescence photographs of the paper chip for the


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specificity assay of miR-21: complementary matched target (CM), single-base mismatched strand (SM) and non-complementary miRNA (miR-214, NM). (D) Histogram indicating the fluorescence intensities of the paper chip (C). The three spots of each row are identical. Specificity is a vital factor to evaluate the performance of the platform, as it is still great challenge to accurately recognizing specific miRNA with the interference of homologous miRNAs. To assess the specificity of our method for miRNAs detection, three types of miRNA sequences (listed in Table S1) including complementary matched target (CM), single-base mismatched strand (SM) and non-complementary miRNA (miR-214, NM) were tested in the same condition with the miR-21-specific templates. Corresponding results were obtained and shown in Figure 3C. The group of CM exhibited a strong QD fluorescence intensity. While for the SM, the fluorescence intensity was obviously decreased. And the NM gave out very weak fluorescence intensity. It is indicated that the proposed assay has good specificity to clearly discriminate complete matched miRNA from those even with single-base mismatched. Multiple miRNAs detection. Compared with single biomarker detection, a multiplexed miRNAs assay provides more reliable evidence for disease diagnosis. To demonstrate the multiplexing capability of our platform, the capture probe 1 and 2 that specific for miR-21 and miR-155 were immobilized on different layers of paper chip, respectively. Three different samples and control group were prepared: the first containing only miR-21, the second only miR-155, and the third is a combination of both. As illustrated in Figure 4A, as expected the amplification products of miR-21 hybridized with the QDs-605 labeled signal probe were bound to the layer immobilized of capture probe 1, the miR-155 amplification products hybridized with the QDs-525 labeled signal probe were bound to the layer immobilized of capture probe 2. And the mixed sample were captured in both layers, in which the fluorescence of QDs-605 and QDs-


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525 could be observed. These results demonstrated the feasibility of the platform for multiplexed miRNAs detection.

Figure 4. (A) Fluorescence photographs of the paper chip for multiplexed detection of miRNAs. Capture probe 1 and 2 which specific for miR-21 and miR-155 were immobilized on different layers of paper chip, respectively. Three different samples and control group were prepared: the first containing only miR-21, the second only miR-155, and the third is a combination of both. (B) Histogram indicating the fluorescence intensities from the paper chip (A). Real sample analysis. For further testing the validity of our assay in biological sample analysis, this platform was evaluated by direct detection of miR-21 and miR-155 from three human cell lines (A549, MCF7 and normal cell line LO2). Cell samples were treated via our paper extraction, then total small RNAs were quantified through measuring the OD260, and the total small RNA concentrations across all 3 strains prior to the HP-EXPAR assay were equalized. As the result depicted in Figure 5A, the QDs fluorescence intensities on the two layer paper chip of the two cancer cell lines were distinctly stronger than normal cell line LO2, which was


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served as the control group. To quantitative detection, intensities of the optical spot on paper chip were measured (see Figure 5B), the results not only showed the over-expression of miR-21 and miR-155 in cancer cells, but also demonstrated the different expression levels of miR-21 and miR-155 in different cancer cell lines, respectively. It was also worth noted that current results is consistent with the reports on up-regulated expressions of miR-155 and miR-21 in a particular tumor.43-44 What's more, the detection capability of this method in cell samples was also compared with qRT-PCR. And U6 the small nuclear RNA (snRNA) was selected as the universal endogenous control and relative expression, which was calculated through the equation: Fold change = 2−∆∆Ct. Table S2 and S3 were listed the sequences of probes and other details in qRT-PCR. As shown in Figure 5C and 5D, results from the two methods has a high accordance. Furthermore, clinical applicability of the system was also confirmed through analyzing two types of miRNAs (see Figure S6). All the experimental results indicated that this approach is capable of multiplex miRNAs analysis in the complicated biological samples and holds promise for POC diagnosis.


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Figure 5. (A) Fluorescence photographs of the paper chip for the quantification of miR-21 and miR-155 in various cell lines. (B) Histogram indicating the fluorescence intensities of the paper chip (A). (C) The relative expressions of miR-21 in various cell lines via this method and qRTPCR. (D) The relative expressions of miR-155 in various cell lines via this method and qRTPCR. CONCLUSION In summary, the integrated paperfluidic chip device with high portability and usability has been developed, which permitted extraction and the purification of small RNAs directly from cell samples, in situ HP-EXPAR amplification and optical detection. It is worth mentioning that HP-EXPAR amplification was performed on chip for the first time. We concluded that current analysis system exerted various advantages: the introduction of magnetic apparatus facilitated sample distribution; multilayer paper chip assembled in foldable manner realizing multiple


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detection simultaneously. Meanwhile, current system eliminating the need for sophisticated equipment except for a specially designed handheld heating block. This assay employed QDs as signal labels and took advantage of isothermal HP-EXPAR amplification to further improve the sensitivity and specificity. And a satisfactory detection limit of 3×106 copies was obtained in 90 min (the whole process). The ability for multiplex miRNAs analysis in various human cancer cells was also confirmed. With the excellent performance presented above, this platform exhibited its great potential for various POC applications in healthcare, environmental monitoring and food safety, which could be developed as rapid small RNAs detection kit for future commercialization; or extended to other different targets via changing template sequences in other nucleic acid assay. ASSOCIATED CONTENT The Supporting Information is available on the ACS Publications website. Further experimental details including characterization of QDs signal probes, fabrication of the foldable paper device, PAGE analysis of the amplification products, qRT-PCR experiment and detection of clinical tumor tissues. (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel.: +86-20 8521-0089. Fax: +86-20 8521-6052. *E-mail: [email protected]. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China [21475048], and the National Science Fund for Distinguished Young Scholars of Guangdong Province [2014A030306008], the Project of Guangzhou Science and Technology Plan [201508020003], and the Program of the Pearl River Young Talents of Science and Technology in Guangzhou [2013J2200021], the Special Support Program of Guangdong Province (2014TQ01R599), and the Outstanding Young Teacher Training Program of Guangdong Province (HS2015004).

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Paperfluidic Chip Device for Small RNA Extraction, Amplification, and

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