Multiplex Paper-Based Colorimetric DNA Sensor Using Pyrrolidinyl

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Multiplex paper-based colorimetric DNA sensor using pyrrolidinyl peptide nucleic acid-induced AgNPs aggregation for detecting MERS-CoV, MTB and HPV oligonucleotides Prinjaporn Tee-ngam, Weena Siangproh, Adisorn Tuantranont, Tirayut Vilaivan, Orawon Chailapakul, and Charles S. Henry Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00255 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

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Analytical Chemistry

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Multiplex paper-based colorimetric DNA sensor using pyrrolidinyl peptide nucleic acid-

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induced AgNPs aggregation for detecting MERS-CoV, MTB and HPVoligonucleotides

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Prinjaporn Teengam†, Weena Siangproh‡, Adisorn Tuantranont§,Tirayut Vilaivan∥, Orawon

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Chailapakul#, ¶, *and Charles S. Henry¥,*

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†

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Bangkok, 10330, Thailand

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‡

Program in Petrochemistry, Faculty of Science, Chulalongkorn University, Pathumwan,

Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110,

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Thailand

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§

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Center, Pathumthani, 12120, Thailand

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∥Organic

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University, Pathumwan, Bangkok, 10330, Thailand

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#

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Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand

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¶

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Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand

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¥

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University, Fort Collins, Colorado 80523

Nanoelectronics and MEMS Laboratory, National Electronics and Computer Technology

Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn

Electrochemistry and Optical Spectroscopy Research Unit, Department of Chemistry,

National Center of Excellence for Petroleum, Petrochemicals, and Advanced Materials,

Departments of Chemistry and Chemical and Biological Engineering, Colorado State

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*Corresponding authors E-mail address:

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Charles Henry, [email protected]; Orawon Chailapakul, [email protected]

ACS Paragon Plus Environment


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ABSTRACT: The development of simple fluorescent and colorimetric assays that enable point-

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of-care DNA and RNA detection has been a topic of significant research because of the utility of

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such assays in resource limited settings. The most common motifs utilize hybridization to a

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complementary detection strand coupled with a sensitive reporter molecule. Here, apaper-based

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colorimetric assay for DNA detection based on pyrrolidinyl peptide nucleic acid (acpcPNA)-

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induced nanoparticle aggregationis reported as an alternative to traditional colorimetric

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approaches. PNA probes are an attractive alternative to DNA and RNA probes because they are

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chemically and biologically stable, easily synthesized, and hybridize efficiently with the

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complementary DNA strands. The acpcPNA probe contains a single positive charge from the

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lysine at C-terminus and causes aggregation of citrate anion-stabilized silver nanoparticles

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(AgNPs) in the absence of complementary DNA. In the presence of target DNA, formation of

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the anionic DNA-acpcPNA duplex results in dispersion of the AgNPs as a result of electrostatic

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repulsion, giving rise to a detectable color change. Factors affecting the sensitivity and

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selectivity of this assay were investigated, including ionic strength, AgNP concentration, PNA

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concentration, and DNA strand mismatches. The method was used for screening of synthetic

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Middle East respiratory syndrome coronavirus (MERS-CoV), mycobacterium tuberculosis

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(MTB) and human papillomavirus (HPV)DNA based on a colorimetric paper-based analytical

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device developed using the aforementioned principle. The oligonucleotide targets were detected

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by measuring the color change of AgNPs, giving detection limits of 1.53 nM (MERS-CoV), 1.27

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nM (MTB) and 1.03 nM (HPV).The acpcPNA probe exhibited high selectivity for the

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complementary oligonucleotides over single-base-mismatch, two-base-mismatch and non-

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complementary DNA targets. The proposed paper-based colorimetric DNA sensor has potential

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to be an alternative approach for simple, rapid, sensitive and selective DNA detection.

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INTRODUCTION

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Infectious diseases represent a major threat to human health in developed and developing

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countries alike. DNA alterations contribute to different types of diseases; therefore, the detection

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of specific DNA sequences plays a crucial role in the development method for early stage

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treatment and monitoring of genetic-related diseases. DNA diagnostics can provide sequence-

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specific detection, especially for single-nucleotide polymorphisms (SNPs),1 which critical for a

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range of applications including the diagnosis of human diseases and bacterial/viral infections.

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Middle East respiratory syndrome (MERS), tuberculosis (TB) and cervical cancers

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related to human papilloma virus (HPV) are examples of infectious diseases caused by bacterial

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and viral infections that benefit greatly from DNA detection. TB is an infectious disease caused

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by mycobacteria, usually M. tuberculosis (MTB) in humans.2HPV has been shown to be a major

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cause of cervical cancer.3 Middle East Respiratory Syndrome coronavirus (MERS-CoV) has

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recently emerged as an infectious disease with a high fatality rate in humans.4 Diagnostic

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methods developed for these infectious diseases include reverse transcription polymerase chain

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reaction (RT-PCR) for MERS-CoV,5 sputum smear microscopy, culture of bacilli and molecular

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species diagnostics for MTB6-11 and Digene Hybrid Capture assay (HC2) and Pap smear test for

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HPV.12,13 While these techniques have been used for successful detection, they are difficult to

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implement in point-of-care clinical diagnostics particularly in developing countries lacking

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specialized medical facilities and skilled personnel. Therefore, simple, rapid, low-cost and highly

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accurate on-site diagnostic platforms amenable to nucleic acid detection remain a challenge for

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early detection of infectious diseases for better patient management and infection control.

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Although DNA amplification is still needed with the current method to provide high sensitivity,

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we seek to further improve selectivity and assay simplicity to give immediate and quantitative

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responses in resource limited settings.



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Paper-based analytical devices (PADs) are a point-of-use technology that recently

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received renewed interest because they are simple, inexpensive, portable and disposable.14-16 To

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date, PADs have been extensively used for applications ranging from environmental analysis to

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clinical diagnostic assays.15,17,18 Colorimetric assays are particularly attractive when coupled

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with PADs due to their ease-of-use, lack of complicated external equipment and ability to

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provide semi-quantitative results.19-21 Moreover, quantitative analysis of colorimetric assays can

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be accomplished using simple optical technologies such as digital cameras22-24 and office

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scanners20,25 combined with image processing software to carry out color, hue, and/or intensity

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measurements. In the field of clinical diagnostics, the advantages of simplicity, sensitivity and

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low-cost are key reasons that make PADs coupled with colorimetric detection an effective

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diagnostic tool relative to traditional methods.

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Colorimetric assays based on the aggregation of silver (AgNPs) and gold nanoparticles

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(AuNPs) have attracted increasing attention in biomedical applications. The optical properties of

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these nanomaterials depend on their size and shape.26-31 AgNPs are known to have a higher

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extinction coefficient compared to AuNPs,32-34 leading to improved optical sensitivity. Chemical

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reduction of silver salts is frequently used to synthesize AgNPs; while specific control of shape

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and size distribution is achieved by varying the reducing agents and stabilizers.35-37 Among

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stabilizing agents, negatively-charged citrate has been widely used.38,39 Recently, colorimetric

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assays based on AgNPs aggregation for DNA detection has been reported.34 Colorimetric DNA

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detection using AgNPs usually involves modifying the particles with a DNA probe and mixing

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them with the DNA target containing the complementary sequence. When the hybridization of

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probe and target DNA occurs, the AgNPs aggregate and change color.33,34 The assay principal

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has been further adopted using charge-neutral peptide nucleic acids (PNA)40,41 as the


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hybridization agent. PNA causes aggregation of metal nanoparticles in solution without

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immobilization, thus simplifying the assay.42,43 Finally, PNA-based nanoparticle aggregation

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assays also provide a high hybridization efficiency of PNA-DNA duplexes leading to a rapid

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color change.43

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Recently, Vilaivan's group proposed a new conformationally constrained pyrrolidinyl

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PNA system which possesses an α,β-peptide backbone derived from D-proline/2-

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aminocyclopentanecarboxylic acid (known as acpcPNA).44,45 Compared to Nielsen’s PNA,40

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acpcPNA exhibits a stronger affinity and higher sequence specificity binding to DNA. acpcPNA

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exhibits the characteristic selectivity of antiparallel binding to the target DNA and low tendency

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to self-hybridize. Moreover, the nucleobases and/or backbone of acpcPNA can be modified to

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increase molecular functionality. These combined properties make acpcPNA an attractive

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candidate as a probe for biological applications.46-48

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Here, the multiplex colorimetric PAD for DNA detection based on the aggregation of

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AgNPs induced by acpcPNA is reported. acpcPNA bearing a positively-charged lysine

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modification at C-terminus was designed as the probe. The cationic PNA probe can interact with

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the negatively-charged AgNPs leading to nanoparticle aggregation and a significant color

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change. This proposed sensor was used for simultaneous detection of MERS-CoV, MTB and

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HPV. The developed paper-based DNA sensor has potential as an alternative diagnostic device

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for simple, rapid, sensitive and selective DNA/RNA detection.

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EXPERIMENTAL SECTION

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Chemicals and Materials. Analytical grade reagents, including AgNO3, NaBH4 and

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sodium citrate from Sigma-Aldrich, KH2PO4 and KCl from Fisher Scientific, Na2HPO4 from



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Mallinckrodt and NaCl from Macron, were used without further purification. 18 M

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Ωcm−1resistance water was obtained from a Millipore Milli-Q water system. Synthetic DNA

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oligonucleotides were obtained from Biosearch Technologies. The sequences of DNA

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oligonucleotides are shown in Table 1.

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Table 1. List of oligonucleotide used in this study Oligonucleotide

Sequence (5'-3')

MERS-CoV Complementary DNA Two-base-mismatch Non-complementary DNA

5′-CGATTATGTGAAGAG -3′ 5′-CGATTATCTGAGGAG -3′ 5′-TTCGCACAGTGGTCA -3′

MTB Complementary DNA Single-base-mismatch Non-complementary DNA 1 Non-complementary DNA 2

5′-ATAACGTGTTTCTTG -3′ 5′-ATAACGTCTTTCTTG -3′ 5′-TGGCTAGCCGCTCCT-3′ 5′-CACTTGCCTACACCA -3′

HPV Complementary DNA (HPV type 16) Non-complementary DNA 1 (HPV type 18) Non-complementary DNA 2 (HPV type 31) Non-complementary DNA 3 (HPV type 33)

5'-GCTGGAGGTGTATG-3' 5'-GGATGCTGCACCGG-3' 5'-CCAAAAGCCCAAGG-3' 5'-CACATCCACCCGCA-3'

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Synthesis of AgNPs. The AgNPs were synthesized using the citrate-stabilization

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method.49 Briefly, 4 mL of 12.6 mM sodium citrate and 50 mL of 0.3 mM AgNO3was mixed

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together. Then, 1 mL of 37 mM NaBH4 was added to the mixture under vigorous stirring and the

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solution turned yellow. The formation of AgNPs and their size distribution were verified by

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dynamic light scattering measurement, and the average size of AgNPs was found to be 19 nm

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(Fig. S1.).


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Synthesis of acpcPNA probes.The acpcPNA probeswere designed to detect the

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synthetic oligonucleotide targets with sequences corresponding to MERS-CoV, MTB and HPV

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type 16. The sequences of acpcPNA probes are as follows:

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MERS-CoV: CTCTTCACATAATCG-LysNH2

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MTB: CAAGAAACACGTTAT-LysNH2

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HPV type 16: CATACACCTCCAGC-LysNH2

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*(written in the N→C direction)

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The acpcPNA probe was synthesized by solid-phase peptide synthesis using Fmoc

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chemistry, as previously described.44 At the C-terminus, lysinamide was included as a positively

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charged group that could induce nanoparticle aggregation. All PNA were purified by reverse-

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phase HPLC (C18 column, 0.1% (v/v) trifluoroacetic acid(TFA) in H2O-MeOH gradient). The

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identity of the acpcPNA was verified by MALDI-TOF MS analysis (Fig. S2), and the purity was

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confirmed to be >90% by reverse-phase HPLC.

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Design and operation of paper-based multiplex DNA sensor. A wax-printing

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technique was used to create PADs.50 The sensor was designed using Adobe Illustrator. The wax

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colors were selected to be complementary to the colorimetric reactions to enhance visualization.

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For paper-based device fabrication, the wax design was printed onto Whatman Grade 1 filter

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paper (VWR) using a wax printer (Xerox Phaser 8860). The wax pattern was subsequently

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melted at 175 °C for 50 s to generate the hydrophobic barriers and hydrophilic channels. The

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sensor was based on Origami concept consisting of two layers.51,52 As shown in Scheme 1A, the

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base layer contains four wax-defined channels extending outward from the sample reservoir (6

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mm i.d.) and the top layer contains four detection and control zones (4 mm i.d.). Scheme 1B

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illustrates operation of the multiplex sensor. First, the sample reservoir of the top layer was



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punched to provide a solution connection directly from the top to the bottom layer, and then the

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device was assembled by folding the top layer over the base layer to create the three-dimension

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origami paper-based device. A polydimethylsiloxane (PDMS) lid was used for holding the two

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layers together. The lid consisting of one 6 mm-diameter hole over the sample reservoir and

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eight 4 mm-diameter holes over the colorimetric detection and control zones was aligned over

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the device to provide consistent pressure across the surface of the device. Next, the acpcPNA

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probe and AgNPs solution were added onto the detection and control zones. Finally, the sample

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solution was added onto the sample reservoir and flow through the channels to wet the

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colorimetric detection zones.

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Scheme 1. (A) Design and (B) operation of multiplex paper-based colorimetric device.

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Colorimetric detection of MERS-CoV, MTB and HPV DNA target. According to the

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concept of PNA-induced AgNPs aggregation,42,43 acpcPNA was designed as a specific probe for

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quantitative detection of synthetic MERS-CoV, MTB and HPV DNA targets. For colorimetric

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detection, the detection zone was prepared by adding 10 µL of AgNPs in 0.1 M phosphate buffer

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saline (PBS) pH 7.4 in a ratio of 5:1 (AgNPs: PBS), followed by 1 µL of specific acpcPNA

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probe. Control zones were prepared using the same conditions as the colorimetric detection

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zones. Next, 25 µL of DNA target was added to the open sample reservoir. Upon sample

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addition, solution moved outward through the channels to wet the colorimetric detection zone of

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the top layer. Finally, the AgNPs aggregation occurred and the color intensity was measured.

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Image processing. The detection images were recorded using a scanner (XEROX

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DocuMate 3220) and saved in JPEG format at 600 dpi. ImageJ software (National Institutes of

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Health) was used to analyze the mean intensity of the color for each colorimetric reaction zone

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by applying a color threshold window for removing the blue background. Images were then

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inverted and the mean intensity was measured.20,53

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RESULTS AND DISCUSSION

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acpcPNA-induced AgNPs aggregation. The process of acpcPNA-induced AgNPs

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aggregation is shown in Scheme 2. The anionic AgNPs are initially well dispersed due to

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electrostatic repulsion. On addition of the cationic acpcPNA, the electrostatic repulsion is

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shielded resulting in nanoparticle aggregation. When complementary DNA (DNAcom) is present,

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the specific PNA-DNA interaction outcompetes the less specific PNA-AgNPs interaction

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resulting in a negatively charged PNA-DNAcom duplex and de-aggregation of the anionic

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nanoparticles. Upon addition of non-complementary DNA (DNAnc), the acpcPNA should remain



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bound to the AgNPs and no color change occurs. To prove the concept, we designed and

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synthesized acpcPNA probes to detect synthetic oligonucleotide targets with sequences

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corresponding to MERS-CoV, MTB and HPV type 16. The photographs of the results are shown

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in Fig. 1. The yellow AgNPs turned red when the acpcPNA was added. When the solution

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contained of the acpcPNA and DNAnc, the color also changed to red due to aggregation of the

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AgNPs. On the other hand, the color changed from red (aggregated) to yellow (non-aggregated)

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in the presence of DNAcom, with the intensity dependent on the DNA concentration. Next, the

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sequence of adding the PNA probe and DNA target was investigated. As shown in Fig.S3, when

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equimolar DNAcom was added either before or after the addition of acpcPNA probe into the

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AgNPs, the same color intensities were obtained indicating that the sequence of adding acpcPNA

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and DNAcom did not impact the final signal.

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200


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Scheme 2. The process of acpcPNA-induced AgNP aggregation in the presence of DNAcom and

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DNAnc.

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Fig. 1. Photograph of visual color changes obtained from detection of MERS-CoV, MTB and

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HPV in the presence of DNAcom.

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Critical coagulation concentration (CCC). The influence of electrolyte solution on the

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aggregation behavior of citrate-stabilized AgNPs was investigated based on the CCC.54The CCC

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represents the electrolyte concentration required to cause aggregation of the nanoparticles in the

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absence of acpcPNA. In Fig.S4, the color intensity of citrate-stabilized AgNPs in the absence of

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acpcPNA probe is shown as a function of NaCl concentration. The intensity, and therefore the

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degree of aggregation, increased with the concentration of NaCl, indicating that increasing ionic

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strength led to enhanced aggregation.55 We believe that the ionic strength can decrease the

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electrostatic repulsion of citrate-stabilized AgNPs as a result of shielding, accelerating the

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AgNPs dissolution. The CCC was obtained when the degree of aggregation reached a maximum

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and became independent of NaCl concentration. In this experiment, the CCC of citrate-stabilized



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AgNPs was found to be 30 mM. Above this concentration, PNA-induced aggregation was not

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observed.

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Optimization of assay parameters. For a colorimetric assay based on acpcPNA-induced

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AgNPs aggregation, assay parameters including 0.1 M PBS (pH 7.4) ratio and acpcPNA

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concentration were optimized using a simple paper-based design. The degree of AgNPs

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aggregation was determined by measuring the color intensity of the resulting solution in the

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presence of acpcPNA without target DNA. First, the impact of the PBS concentration on AgNPs

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aggregation was measured. The differential color intensity (Δ intensity, ΔI) obtained before and

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after addition of acpcPNA as a function of AgNPs to PBS ratio is shown in Fig. 2A.ΔI increased

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until the ratio of AgNPs:PBS reached5:1 and then decreased until it plateaued at 5:2. Thus, the

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ratio of 5:1AgNPs:PBS was selected as the optimal condition because it gave the largest ΔI.

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Another important aspect for the DNA assay is probe concentration. The influence of acpcPNA

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probe concentration on absolute intensity was studied. As shown in Fig. 2B, the acpcPNA

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concentration was varied within a range of 0-2.5 µM and the highest aggregation was obtained at

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the concentration of 1.0 µM. At this concentration, the aggregation became independent of

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acpcPNA concentration, which was desirable for simplifying the assay. Higher concentrations of

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AgNPs were not tested in order to minimize reagent consumption. As a result, the optimal

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conditions consisting of AgNPs:PBS ratio of 5:1 and acpcPNA concentration of 1.0 µM were

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selected for further experiments.

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Fig. 2. Influence of (A) AgNP:PBS ratio and (B) acpcPNA probe concentration on color

239

intensity for MERS-CoV, MTB and HPV detection. The error bars represent one standard

240

deviation (SD) obtained from three independent measurements (n=3).

241 242

Selectivity ofMERS-CoV, MTB and HPV detection.To investigate the selectivity of

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this system, the color intensity obtained from the DNAcom of MERS-CoV, MTB and HPV was

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compared to that of single-base mismatch (DNAm1), two-base mismatch (DNAm2) and DNAnc



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sequences. The color intensity decreased significantly in the presence of DNAcom; whereas the

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intensity did not change for the mismatched and non-complementary targets (Fig. 3). We believe

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the affinity of PNA-DNA hybridization was reduced due to the contribution of one- and two-

248

base mismatches, leaving free PNA to aggregate the nanoparticles. PNA-DNAcom complex can

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retard the ability of PNA to induce AgNPs aggregation as discussed above and result in different

250

color intensities. These results suggest that the fully complementary DNA selectively hybridized

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the acpcPNA probe and yielded measurable signals. In addition, bovine serum albumin (BSA),

252

which is commonly used in cell culture protocols, was used to investigate the protein

253

interference of the proposed system. The DNA target was prepared in the presence of 3% BSA

254

solution. It was observed that the color intensities of the DNA targets for MERS-CoV, MTB and

255

HPV in 3% BSA solution were statistically identical to the ones without BSA (Fig. S5). Hence,

256

common proteins should not negatively affect the analysis of this system.


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257 258

Fig. 3. The color intensity of (A) MERS-CoV, (B) MTB and (C) HPV detection after

259

hybridization of DNAm1, DNAm2 and DNAnc. The error bars represent one standard deviation

260

(SD) obtained from three independent measurements (n=3).

261 262

Analytical performance. To assess the sensitivity of the proposed method for DNA

263

quantification, the intensity as a function of the target DNA concentration was determined. The

264

color intensity decreases with the target DNA concentration. The calibration curves for each

265

species are shown in Fig. 4 A, B and C for MERS-CoV, MTB and HPV, respectively. The linear

266

range for each DNA target using a logarithmic DNA concentration and color intensity

267

(Fig.4inset) was also obtained. The analytical performances for all three DNA targets are



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summarized in Table 2. It can be seen that a sufficiently low detection limit could be obtained for

269

MERS-CoV, MTB and HPV detection without the need for multiple PCR cycles. Moreover, this

270

multiplex system can provide sensitive and selective detection for simultaneous analysis of

271

multiple DNA targets in a single device, which simplifies the analysis compared to traditional

272

diagnostics.9,56-59

273 274

Fig. 4. The change of probe color intensity versus DNA target concentration (∆I) and calibration

275

graph between ∆I and log DNA target concentration (inset) for (A) MERS-CoV, (B) MTB and

276

(C) HPV detection. The error bars represent standard deviation (SD) obtained from three

277

independent measurement (n=3).

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Table 2. Summarized analytical performance of the Multiplexed 3DPAD for colorimetric DNA

281

assay DNA target MERS-CoV MTB HPV

Linearity 20-1000 nM 50-2500 nM 20-2500 nM

LOD 1.53 nM 1.27 nM 1.03 nM

%RSD (n=3) 0.17-0.50 0.12-0.67 0.43-0.93

282 283

Device design.Next, a multiplex device (Scheme 1) was designed for simultaneous

284

detection of MERS-CoV, MTB and HPV. The top layer contained four detection zones and four

285

control zones. Each zone contained AgNPs with a single acpcPNA probe to provide selectivity

286

for DNA. The base layer contained four wax-defined channels extending outward from a sample

287

inlet. After the device was folded and stacked together, the channels of the base layer were

288

connected to four detection zones of the top layer. Upon sample addition, the solution moved

289

outward through the channels of the base layer to wet the colorimetric detection zones of the top

290

layer and lead to color change. Fig. 5 illustrates the ability of the proposed sensor for detection of

291

100 nM MERS-CoV, MTB and HPV. Only the colorimetric detection zones that contained the

292

selective probes changed color compared to their control zones. This result indicated that the

293

multiplex paper-based colorimetric sensor is promising for simultaneous determination of

294

MERS-CoV, MTB and HPV.

295



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Page 18 of 27

296 297

Fig. 5. The selectivity of 100 nM MERS-CoV, MTB and HPV detection using multiplex

298

colorimetric PAD (1,C1= AgNPs+MERS-CoV acpcPNA probe; 2,C2= AgNPs+MTB acpcPNA

299

probe;

3,C3= AgNPs+HPV acpcPNA probe)

300 301

CONCLUSIONS

302

A multiplex colorimetric PAD was developed for simultaneous detection of DNA

303

associated with viral and bacterial infectious diseases, including Middle East respiratory

304

syndrome coronavirus (MERS-CoV), mycobacterium tuberculosis (MTB) and human

305

papillomavirus (HPV). AgNPs were used as a colorimetric reagent for DNA detection based on

306

acpcPNA-induced nanoparticle aggregation. This colorimetric DNA sensor exhibited high

307

selectivity against single-base mismatch, two-base mismatch and non-complementary target

308

DNA. Under the optimized condition, the limit of detection for MERS-CoV, MTB and HPV

309

were found to be 1.53, 1.27 and 1.03 nM, respectively. As a result, this developed multiplex


Page 19 of 27

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310

colorimetric PAD could be a low-cost and disposable alternative tool for rapid screening and

311

detecting in infectious diagnostics.

312 313

ACKNOWLEDGMENTS

314

PT gratefully appreciates the financial supports from Thailand Graduate Institute of

315

Science and Technology (TGIST 01-55-014) and The Thailand Research Fund (RTA5780005).

316

CH acknowledges financial support from Colorado State University and the United States

317

Department of Agriculture through the National Wildlife Research Center (1574000859CA). TV

318

acknowledges technical assistance of Ms. Chotima Vilaivan (Organic Synthesis Research Unit,

319

Chulalongkorn University) and the financial support from Thailand Research Fund

320

(DPG5780002, to TV) for the PNA synthesis. The authors thank Dr. Yuanyuan Yang for

321

assistance with manuscript editing. The authors also acknowledge important discussions with Dr.

322

Christopher Ackerson surrounding the critical coagulation concentration.

323 324

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Graphical abstract

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Multiplex Paper-Based Colorimetric DNA Sensor Using Pyrrolidinyl

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