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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Multiplex Paper-Based Colorimetric DNA Sensor Using
Pyrrolidinyl Peptide Nucleic Acid-Induced AgNPs Aggregation for Detecting
MERS-CoV, MTB, and HPV Oligonucleotides</title><author><name sortKey="Teengam, Prinjaporn" sort="Teengam, Prinjaporn" uniqKey="Teengam P" first="Prinjaporn" last="Teengam">Prinjaporn Teengam</name><affiliation><nlm:aff id="aff1a">Program in Petrochemistry, Faculty of Science,</nlm:aff></affiliation></author><author><name sortKey="Siangproh, Weena" sort="Siangproh, Weena" uniqKey="Siangproh W" first="Weena" last="Siangproh">Weena Siangproh</name><affiliation><nlm:aff id="aff2">Department of Chemistry, Faculty of Science,<institution>Srinakharinwirot University</institution>, Bangkok, 10110,<country>Thailand</country></nlm:aff></affiliation></author><author><name sortKey="Tuantranont, Adisorn" sort="Tuantranont, Adisorn" uniqKey="Tuantranont A" first="Adisorn" last="Tuantranont">Adisorn Tuantranont</name><affiliation><nlm:aff id="aff3">Nanoelectronics and MEMS Laboratory,<institution>National Electronics and Computer Technology Center</institution>, Pathumthani 12120,<country>Thailand</country></nlm:aff></affiliation></author><author><name sortKey="Vilaivan, Tirayut" sort="Vilaivan, Tirayut" uniqKey="Vilaivan T" first="Tirayut" last="Vilaivan">Tirayut Vilaivan</name><affiliation><nlm:aff id="aff1a">Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science,</nlm:aff></affiliation></author><author><name sortKey="Chailapakul, Orawon" sort="Chailapakul, Orawon" uniqKey="Chailapakul O" first="Orawon" last="Chailapakul">Orawon Chailapakul</name><affiliation><nlm:aff wicri:cut=", and" id="aff1a">Electrochemistry and Optical Spectroscopy Research Unit, Department of Chemistry</nlm:aff></affiliation><affiliation><nlm:aff id="aff1a">National Center of Excellence for Petroleum, Petrochemicals, and Advanced Materials,<institution>Chulalongkorn University</institution>, Pathumwan, Bangkok, 10330,<country>Thailand</country></nlm:aff></affiliation></author><author><name sortKey="Henry, Charles S" sort="Henry, Charles S" uniqKey="Henry C" first="Charles S." last="Henry">Charles S. Henry</name><affiliation><nlm:aff id="aff7">Departments of Chemistry and Chemical and Biological Engineering,<institution>Colorado State University</institution>, Fort Collins, Colorado 80523,<country>United States</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">28394582</idno><idno type="pmc">7077925</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077925</idno><idno type="RBID">PMC:7077925</idno><idno type="doi">10.1021/acs.analchem.7b00255</idno><date when="2017">2017</date><idno type="wicri:Area/Pmc/Corpus">000126</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000126</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Multiplex Paper-Based Colorimetric DNA Sensor Using
Pyrrolidinyl Peptide Nucleic Acid-Induced AgNPs Aggregation for Detecting
MERS-CoV, MTB, and HPV Oligonucleotides</title><author><name sortKey="Teengam, Prinjaporn" sort="Teengam, Prinjaporn" uniqKey="Teengam P" first="Prinjaporn" last="Teengam">Prinjaporn Teengam</name><affiliation><nlm:aff id="aff1a">Program in Petrochemistry, Faculty of Science,</nlm:aff></affiliation></author><author><name sortKey="Siangproh, Weena" sort="Siangproh, Weena" uniqKey="Siangproh W" first="Weena" last="Siangproh">Weena Siangproh</name><affiliation><nlm:aff id="aff2">Department of Chemistry, Faculty of Science,<institution>Srinakharinwirot University</institution>, Bangkok, 10110,<country>Thailand</country></nlm:aff></affiliation></author><author><name sortKey="Tuantranont, Adisorn" sort="Tuantranont, Adisorn" uniqKey="Tuantranont A" first="Adisorn" last="Tuantranont">Adisorn Tuantranont</name><affiliation><nlm:aff id="aff3">Nanoelectronics and MEMS Laboratory,<institution>National Electronics and Computer Technology Center</institution>, Pathumthani 12120,<country>Thailand</country></nlm:aff></affiliation></author><author><name sortKey="Vilaivan, Tirayut" sort="Vilaivan, Tirayut" uniqKey="Vilaivan T" first="Tirayut" last="Vilaivan">Tirayut Vilaivan</name><affiliation><nlm:aff id="aff1a">Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science,</nlm:aff></affiliation></author><author><name sortKey="Chailapakul, Orawon" sort="Chailapakul, Orawon" uniqKey="Chailapakul O" first="Orawon" last="Chailapakul">Orawon Chailapakul</name><affiliation><nlm:aff wicri:cut=", and" id="aff1a">Electrochemistry and Optical Spectroscopy Research Unit, Department of Chemistry</nlm:aff></affiliation><affiliation><nlm:aff id="aff1a">National Center of Excellence for Petroleum, Petrochemicals, and Advanced Materials,<institution>Chulalongkorn University</institution>, Pathumwan, Bangkok, 10330,<country>Thailand</country></nlm:aff></affiliation></author><author><name sortKey="Henry, Charles S" sort="Henry, Charles S" uniqKey="Henry C" first="Charles S." last="Henry">Charles S. Henry</name><affiliation><nlm:aff id="aff7">Departments of Chemistry and Chemical and Biological Engineering,<institution>Colorado State University</institution>, Fort Collins, Colorado 80523,<country>United States</country></nlm:aff></affiliation></author></analytic><series><title level="j">Analytical Chemistry</title><idno type="ISSN">0003-2700</idno><idno type="eISSN">1520-6882</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="ac7b00255_0009" id="ab-d30e205"></graphic></p><p>The development of simple fluorescent
and colorimetric assays that
enable point-of-care DNA and RNA detection has been a topic of significant
research because of the utility of such assays in resource limited
settings. The most common motifs utilize hybridization to a complementary
detection strand coupled with a sensitive reporter molecule. Here,
a paper-based colorimetric assay for DNA detection based on pyrrolidinyl
peptide nucleic acid (acpcPNA)-induced nanoparticle aggregation is
reported as an alternative to traditional colorimetric approaches.
PNA probes are an attractive alternative to DNA and RNA probes because
they are chemically and biologically stable, easily synthesized, and
hybridize efficiently with the complementary DNA strands. The acpcPNA
probe contains a single positive charge from the lysine at C-terminus
and causes aggregation of citrate anion-stabilized silver nanoparticles
(AgNPs) in the absence of complementary DNA. In the presence of target
DNA, formation of the anionic DNA-acpcPNA duplex results in dispersion
of the AgNPs as a result of electrostatic repulsion, giving rise to
a detectable color change. Factors affecting the sensitivity and selectivity
of this assay were investigated, including ionic strength, AgNP concentration,
PNA concentration, and DNA strand mismatches. The method was used
for screening of synthetic Middle East respiratory syndrome coronavirus
(MERS-CoV), <italic>Mycobacterium tuberculosis</italic> (MTB), and human papillomavirus (HPV) DNA based on a colorimetric
paper-based analytical device developed using the aforementioned principle.
The oligonucleotide targets were detected by measuring the color change
of AgNPs, giving detection limits of 1.53 (MERS-CoV), 1.27 (MTB),
and 1.03 nM (HPV). The acpcPNA probe exhibited high selectivity for
the complementary oligonucleotides over single-base-mismatch, two-base-mismatch,
and noncomplementary DNA targets. The proposed paper-based colorimetric
DNA sensor has potential to be an alternative approach for simple,
rapid, sensitive, and selective DNA detection.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Wei, F" uniqKey="Wei F">F. Wei</name></author><author><name sortKey="Lillehoj, P B" uniqKey="Lillehoj P">P. B. Lillehoj</name></author><author><name sortKey="Ho, C M" uniqKey="Ho C">C.-M. Ho</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Smith, I" uniqKey="Smith I">I. Smith</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Burd, E M" uniqKey="Burd E">E. M. Burd</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De X0a Wit, E" uniqKey="De X0a Wit E">E. de
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<front><journal-meta><journal-id journal-id-type="nlm-ta">Anal Chem</journal-id><journal-id journal-id-type="iso-abbrev">Anal. Chem</journal-id><journal-id journal-id-type="publisher-id">ac</journal-id><journal-id journal-id-type="coden">ancham</journal-id><journal-title-group><journal-title>Analytical Chemistry</journal-title></journal-title-group><issn pub-type="ppub">0003-2700</issn><issn pub-type="epub">1520-6882</issn><publisher><publisher-name>American Chemical
Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28394582</article-id><article-id pub-id-type="pmc">7077925</article-id><article-id pub-id-type="doi">10.1021/acs.analchem.7b00255</article-id><article-categories><subj-group><subject>Article</subject></subj-group></article-categories><title-group><article-title>Multiplex Paper-Based Colorimetric DNA Sensor Using
Pyrrolidinyl Peptide Nucleic Acid-Induced AgNPs Aggregation for Detecting
MERS-CoV, MTB, and HPV Oligonucleotides</article-title></title-group><contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Teengam</surname><given-names>Prinjaporn</given-names></name><xref rid="aff1a" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath2"><name><surname>Siangproh</surname><given-names>Weena</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="ath3"><name><surname>Tuantranont</surname><given-names>Adisorn</given-names></name><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath4"><name><surname>Vilaivan</surname><given-names>Tirayut</given-names></name><xref rid="aff1a" ref-type="aff">∥</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath5"><name><surname>Chailapakul</surname><given-names>Orawon</given-names></name><xref rid="cor2" ref-type="other">*</xref><xref rid="aff1a" ref-type="aff">⊥</xref><xref rid="aff1a" ref-type="aff">#</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath6"><name><surname>Henry</surname><given-names>Charles S.</given-names></name><xref rid="cor1" ref-type="other">*</xref><xref rid="aff7" ref-type="aff">○</xref></contrib><aff id="aff1a"><sup>†</sup>Program in Petrochemistry, Faculty of Science,<sup>∥</sup>Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science,<sup>⊥</sup>Electrochemistry and Optical Spectroscopy Research Unit, Department of Chemistry, and<sup>#</sup>National Center of Excellence for Petroleum, Petrochemicals, and Advanced Materials,<institution>Chulalongkorn University</institution>, Pathumwan, Bangkok, 10330,<country>Thailand</country></aff><aff id="aff2"><label>‡</label>Department of Chemistry, Faculty of Science,<institution>Srinakharinwirot University</institution>, Bangkok, 10110,<country>Thailand</country></aff><aff id="aff3"><label>§</label>Nanoelectronics and MEMS Laboratory,<institution>National Electronics and Computer Technology Center</institution>, Pathumthani 12120,<country>Thailand</country></aff><aff id="aff7"><label>○</label>Departments of Chemistry and Chemical and Biological Engineering,<institution>Colorado State University</institution>, Fort Collins, Colorado 80523,<country>United States</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>E-mail: <email>chuck.henry@colostate.edu</email>.</corresp><corresp id="cor2"><label>*</label>E-mail: <email>corawon@chula.ac.th</email>.</corresp></author-notes><pub-date pub-type="epub"><day>10</day><month>04</month><year>2017</year></pub-date><pub-date pub-type="ppub"><day>16</day><month>05</month><year>2017</year></pub-date><volume>89</volume><issue>10</issue><fpage>5428</fpage><lpage>5435</lpage><history><date date-type="received"><day>20</day><month>01</month><year>2017</year></date><date date-type="accepted"><day>10</day><month>04</month><year>2017</year></date></history><permissions><copyright-statement>Copyright © 2017 American
Chemical Society</copyright-statement><copyright-year>2017</copyright-year><copyright-holder>American
Chemical Society</copyright-holder><license license-type="open-access"><license-p>This article is made available via the PMC Open Access Subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p></license></permissions><abstract><p content-type="toc-graphic"><graphic xlink:href="ac7b00255_0009" id="ab-d30e205"></graphic></p><p>The development of simple fluorescent
and colorimetric assays that
enable point-of-care DNA and RNA detection has been a topic of significant
research because of the utility of such assays in resource limited
settings. The most common motifs utilize hybridization to a complementary
detection strand coupled with a sensitive reporter molecule. Here,
a paper-based colorimetric assay for DNA detection based on pyrrolidinyl
peptide nucleic acid (acpcPNA)-induced nanoparticle aggregation is
reported as an alternative to traditional colorimetric approaches.
PNA probes are an attractive alternative to DNA and RNA probes because
they are chemically and biologically stable, easily synthesized, and
hybridize efficiently with the complementary DNA strands. The acpcPNA
probe contains a single positive charge from the lysine at C-terminus
and causes aggregation of citrate anion-stabilized silver nanoparticles
(AgNPs) in the absence of complementary DNA. In the presence of target
DNA, formation of the anionic DNA-acpcPNA duplex results in dispersion
of the AgNPs as a result of electrostatic repulsion, giving rise to
a detectable color change. Factors affecting the sensitivity and selectivity
of this assay were investigated, including ionic strength, AgNP concentration,
PNA concentration, and DNA strand mismatches. The method was used
for screening of synthetic Middle East respiratory syndrome coronavirus
(MERS-CoV), <italic>Mycobacterium tuberculosis</italic> (MTB), and human papillomavirus (HPV) DNA based on a colorimetric
paper-based analytical device developed using the aforementioned principle.
The oligonucleotide targets were detected by measuring the color change
of AgNPs, giving detection limits of 1.53 (MERS-CoV), 1.27 (MTB),
and 1.03 nM (HPV). The acpcPNA probe exhibited high selectivity for
the complementary oligonucleotides over single-base-mismatch, two-base-mismatch,
and noncomplementary DNA targets. The proposed paper-based colorimetric
DNA sensor has potential to be an alternative approach for simple,
rapid, sensitive, and selective DNA detection.</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>ac7b00255</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>ac7b00255</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value></meta-value></custom-meta></custom-meta-group></article-meta><notes id="notes-d1e21-autogenerated"><fn-group><fn fn-type="" id="d30e207"><p>This article is made available for a limited time sponsored by ACS under the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/freetoread/index.html">ACS Free to Read License</ext-link>, which permits copying and redistribution of the article for non-commercial scholarly purposes.</p></fn></fn-group></notes></front><body><p id="sec1">Infectious diseases represent
a major threat to human health in developed and developing countries
alike. DNA alterations contribute to different types of diseases;
therefore, the detection of specific DNA sequences plays a crucial
role in the development method for early stage treatment and monitoring
of genetic-related diseases. DNA diagnostics can provide sequence-specific
detection, especially for single-nucleotide polymorphisms (SNPs),<sup><xref ref-type="bibr" rid="ref1">1</xref></sup> which critical for a range of applications including
the diagnosis of human diseases and bacterial/viral infections.</p><p>Middle East respiratory syndrome (MERS), tuberculosis (TB), and
cervical cancers related to human papilloma virus (HPV) are examples
of infectious diseases caused by bacterial and viral infections that
benefit greatly from DNA detection. TB is an infectious disease caused
by mycobacteria, usually <italic>M. tuberculosis</italic> (MTB) in humans.<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> HPV has been shown
to be a major cause of cervical cancer.<sup><xref ref-type="bibr" rid="ref3">3</xref></sup> Middle East Respiratory Syndrome coronavirus (MERS-CoV) has recently
emerged as an infectious disease with a high fatality rate in humans.<sup><xref ref-type="bibr" rid="ref4">4</xref></sup> Diagnostic methods developed for these infectious
diseases include reverse transcription polymerase chain reaction (RT-PCR)
for MERS-CoV,<sup><xref ref-type="bibr" rid="ref5">5</xref></sup> sputum smear microscopy,
culture of bacilli, and molecular species diagnostics for MTB<sup><xref ref-type="bibr" rid="ref6">6</xref>−<xref ref-type="bibr" rid="ref11">11</xref></sup> and Digene Hybrid Capture assay (HC2) and Pap smear test for HPV.<sup><xref ref-type="bibr" rid="ref12">12</xref>,<xref ref-type="bibr" rid="ref13">13</xref></sup> While these techniques have been used for successful detection,
they are difficult to implement in point-of-care clinical diagnostics
particularly in developing countries lacking specialized medical facilities
and skilled personnel. Therefore, simple, rapid, low-cost, and highly
accurate on-site diagnostic platforms amenable to nucleic acid detection
remain a challenge for early detection of infectious diseases for
better patient management and infection control. Although DNA amplification
is still needed with the current method to provide high sensitivity,
we seek to further improve selectivity and assay simplicity to give
immediate and quantitative responses in resource limited settings.</p><p>Paper-based analytical devices (PADs) are a point-of-use technology
that recently received renewed interest because they are simple, inexpensive,
portable, and disposable.<sup><xref ref-type="bibr" rid="ref14">14</xref>−<xref ref-type="bibr" rid="ref16">16</xref></sup> To date, PADs have been extensively
used for applications ranging from environmental analysis to clinical
diagnostic assays.<sup><xref ref-type="bibr" rid="ref15">15</xref>,<xref ref-type="bibr" rid="ref17">17</xref>,<xref ref-type="bibr" rid="ref18">18</xref></sup> Colorimetric assays are particularly attractive when coupled with
PADs due to their ease-of-use, lack of complicated external equipment
and ability to provide semiquantitative results.<sup><xref ref-type="bibr" rid="ref19">19</xref>−<xref ref-type="bibr" rid="ref21">21</xref></sup> Moreover, quantitative
analysis of colorimetric assays can be accomplished using simple optical
technologies such as digital cameras<sup><xref ref-type="bibr" rid="ref22">22</xref>−<xref ref-type="bibr" rid="ref24">24</xref></sup> and office scanners<sup><xref ref-type="bibr" rid="ref20">20</xref>,<xref ref-type="bibr" rid="ref25">25</xref></sup> combined with image processing software to carry out color, hue,
and intensity measurements. In the field of clinical diagnostics,
the advantages of simplicity, sensitivity, and low-cost are key reasons
that make PADs coupled with colorimetric detection an effective diagnostic
tool relative to traditional methods.</p><p>Colorimetric assays based
on the aggregation of silver (AgNPs)
and gold nanoparticles (AuNPs) have attracted increasing attention
in biomedical applications. The optical properties of these nanomaterials
depend on their size and shape.<sup><xref ref-type="bibr" rid="ref26">26</xref>−<xref ref-type="bibr" rid="ref31">31</xref></sup> AgNPs are known to have a higher extinction coefficient compared
to AuNPs,<sup><xref ref-type="bibr" rid="ref32">32</xref>−<xref ref-type="bibr" rid="ref34">34</xref></sup> leading to improved optical sensitivity. Chemical
reduction of silver salts is frequently used to synthesize AgNPs;
while specific control of shape and size distribution is achieved
by varying the reducing agents and stabilizers.<sup><xref ref-type="bibr" rid="ref35">35</xref>−<xref ref-type="bibr" rid="ref37">37</xref></sup> Among stabilizing
agents, negatively charged citrate has been widely used.<sup><xref ref-type="bibr" rid="ref38">38</xref>,<xref ref-type="bibr" rid="ref39">39</xref></sup> Recently, colorimetric assays based on AgNPs aggregation for DNA
detection has been reported.<sup><xref ref-type="bibr" rid="ref34">34</xref></sup> Colorimetric
DNA detection using AgNPs usually involves modifying the particles
with a DNA probe and mixing them with the DNA target containing the
complementary sequence. When the hybridization of probe and target
DNA occurs, the AgNPs aggregate and change color.<sup><xref ref-type="bibr" rid="ref33">33</xref>,<xref ref-type="bibr" rid="ref34">34</xref></sup> The assay principal has been further adopted using charge-neutral
peptide nucleic acids (PNA)<sup><xref ref-type="bibr" rid="ref40">40</xref>,<xref ref-type="bibr" rid="ref41">41</xref></sup> as the hybridization
agent. PNA causes aggregation of metal nanoparticles in solution without
immobilization, thus, simplifying the assay.<sup><xref ref-type="bibr" rid="ref42">42</xref>,<xref ref-type="bibr" rid="ref43">43</xref></sup> Finally, PNA-based nanoparticle aggregation assays also provide
a high hybridization efficiency of PNA-DNA duplexes leading to a rapid
color change.<sup><xref ref-type="bibr" rid="ref43">43</xref></sup></p><p>Recently, Vilaivan’s
group proposed a new conformationally
constrained pyrrolidinyl PNA system which possesses an α,β-peptide
backbone derived from <sc>d</sc>-proline/2-aminocyclopentanecarboxylic
acid (known as acpcPNA).<sup><xref ref-type="bibr" rid="ref44">44</xref>,<xref ref-type="bibr" rid="ref45">45</xref></sup> Compared to Nielsen’s
PNA,<sup><xref ref-type="bibr" rid="ref40">40</xref></sup> acpcPNA exhibits a stronger affinity
and higher sequence specificity binding to DNA. acpcPNA exhibits the
characteristic selectivity of antiparallel binding to the target DNA
and low tendency to self-hybridize. Moreover, the nucleobases and
backbone of acpcPNA can be modified to increase molecular functionality.
These combined properties make acpcPNA an attractive candidate as
a probe for biological applications.<sup><xref ref-type="bibr" rid="ref46">46</xref>−<xref ref-type="bibr" rid="ref48">48</xref></sup></p><p>Here, the multiplex
colorimetric PAD for DNA detection based on
the aggregation of AgNPs induced by acpcPNA is reported. acpcPNA bearing
a positively charged lysine modification at C-terminus was designed
as the probe. The cationic PNA probe can interact with the negatively
charged AgNPs leading to nanoparticle aggregation and a significant
color change. This proposed sensor was used for simultaneous detection
of MERS-CoV, MTB, and HPV. The developed paper-based DNA sensor has
potential as an alternative diagnostic device for simple, rapid, sensitive,
and selective DNA/RNA detection.</p><sec id="sec2"><title>Experimental Section</title><sec id="sec2.1"><title>Chemicals
and Materials</title><p>Analytical grade reagents,
including AgNO<sub>3</sub>, NaBH<sub>4</sub>, and sodium citrate from
Sigma-Aldrich, KH<sub>2</sub>PO<sub>4</sub> and KCl from Fisher Scientific,
Na<sub>2</sub>HPO<sub>4</sub> from Mallinckrodt, and NaCl from Macron,
were used without further purification. A total of 18 M Ω·cm<sup>–1</sup> resistance water was obtained from a Millipore Milli-Q
water system. Synthetic DNA oligonucleotides were obtained from Biosearch
Technologies. The sequences of DNA oligonucleotides are shown in <xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref></xref>.</p><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><title>List of Oligonucleotide Used in This
Study</title></caption><table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col><col align="left"></col></colgroup><thead><tr><th style="border:none;" align="center">oligonucleotide</th><th style="border:none;" align="center">sequence
(5′-3′)</th></tr></thead><tbody><tr><td style="border:none;" align="left">MERS-CoV</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">complementary DNA</td><td style="border:none;" align="left">5′-CGATTATGTGAAGAG-3′</td></tr><tr><td style="border:none;" align="left">two-base-mismatch</td><td style="border:none;" align="left">5′-CGATTAT<underline>C</underline>TGA<underline>G</underline>GAG-3′</td></tr><tr><td style="border:none;" align="left">noncomplementary DNA</td><td style="border:none;" align="left">5′-TTCGCACAGTGGTCA-3′</td></tr><tr><td style="border:none;" align="left">MTB</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">complementary DNA</td><td style="border:none;" align="left">5′-ATAACGTGTTTCTTG-3′</td></tr><tr><td style="border:none;" align="left">single-base-mismatch</td><td style="border:none;" align="left">5′-ATAACGT<underline>C</underline>TTTCTTG-3′</td></tr><tr><td style="border:none;" align="left">noncomplementary
DNA 1</td><td style="border:none;" align="left">5′-TGGCTAGCCGCTCCT-3′</td></tr><tr><td style="border:none;" align="left">noncomplementary DNA 2</td><td style="border:none;" align="left">5′-CACTTGCCTACACCA-3′</td></tr><tr><td style="border:none;" align="left">HPV</td><td style="border:none;" align="left"> </td></tr><tr><td style="border:none;" align="left">complementary DNA (HPV type 16)</td><td style="border:none;" align="left">5′-GCTGGAGGTGTATG-3′</td></tr><tr><td style="border:none;" align="left">noncomplementary DNA 1 (HPV type 18)</td><td style="border:none;" align="left">5′-GGATGCTGCACCGG-3′</td></tr><tr><td style="border:none;" align="left">noncomplementary
DNA 2 (HPV type 31)</td><td style="border:none;" align="left">5′-CCAAAAGCCCAAGG-3′</td></tr><tr><td style="border:none;" align="left">noncomplementary DNA 3 (HPV type 33)</td><td style="border:none;" align="left">5′-CACATCCACCCGCA-3′</td></tr></tbody></table></table-wrap></sec><sec id="sec2.2"><title>Synthesis of AgNPs</title><p>The AgNPs were
synthesized using
the citrate-stabilization method.<sup><xref ref-type="bibr" rid="ref49">49</xref></sup> Briefly,
4 mL of 12.6 mM sodium citrate and 50 mL of 0.3 mM AgNO<sub>3</sub> were mixed together. Then, 1 mL of 37 mM NaBH<sub>4</sub> was added
to the mixture under vigorous stirring and the solution turned yellow.
The formation of AgNPs and their size distribution were verified by
dynamic light scattering measurement, and the average size of AgNPs
was found to be 19 nm (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b00255/suppl_file/ac7b00255_si_001.pdf">Figure S1</ext-link>).</p></sec><sec id="sec2.3"><title>Synthesis
of acpcPNA Probes</title><p>The acpcPNA probes were
designed to detect the synthetic oligonucleotide targets with sequences
corresponding to MERS-CoV, MTB, and HPV type 16. The sequences of
acpcPNA probes are as follows:</p><p>MERS-CoV: CTCTTCACATAATCG-LysNH<sub>2</sub></p><p>MTB: CAAGAAACACGTTAT-LysNH<sub>2</sub></p><p>HPV type
16: CATACACCTCCAGC-LysNH<sub>2</sub></p><p>*(written in the N →
C direction)</p><p>The acpcPNA probe was synthesized by solid-phase
peptide synthesis
using Fmoc chemistry, as previously described.<sup><xref ref-type="bibr" rid="ref44">44</xref></sup> At the C-terminus, lysinamide was included as a positively
charged group that could induce nanoparticle aggregation. All PNA
were purified by reverse-phase HPLC (C18 column, 0.1% (v/v) trifluoroacetic
acid (TFA) in H<sub>2</sub>O–MeOH gradient). The identity of
the acpcPNA was verified by MALDI-TOF MS analysis (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b00255/suppl_file/ac7b00255_si_001.pdf">Figure S2</ext-link>), and the purity was confirmed to be >90% by
reverse-phase
HPLC.</p></sec><sec id="sec2.4"><title>Design and Operation of Paper-Based Multiplex DNA Sensor</title><p>A wax-printing technique was used to create PADs.<sup><xref ref-type="bibr" rid="ref50">50</xref></sup> The sensor was designed using Adobe Illustrator. The wax
colors were selected to be complementary to the colorimetric reactions
to enhance visualization. For paper-based device fabrication, the
wax design was printed onto Whatman grade 1 filter paper (VWR) using
a wax printer (Xerox Phaser 8860). The wax pattern was subsequently
melted at 175 °C for 50 s to generate the hydrophobic barriers
and hydrophilic channels. The sensor was based on Origami concept
consisting of two layers.<sup><xref ref-type="bibr" rid="ref51">51</xref>,<xref ref-type="bibr" rid="ref52">52</xref></sup> As shown in <xref rid="sch1" ref-type="scheme">Scheme <xref rid="sch1" ref-type="scheme">1</xref></xref>A, the base layer
contains four wax-defined channels extending outward from the sample
reservoir (6 mm i.d.) and the top layer contains four detection and
control zones (4 mm i.d.). <xref rid="sch1" ref-type="scheme">Scheme <xref rid="sch1" ref-type="scheme">1</xref></xref>B illustrates operation of the multiplex sensor. First,
the sample reservoir of the top layer was punched to provide a solution
connection directly from the top to the bottom layer, and then the
device was assembled by folding the top layer over the base layer
to create the three-dimension origami paper-based device. A polydimethylsiloxane
(PDMS) lid was used for holding the two layers together. The lid consisting
of one 6 mm diameter hole over the sample reservoir and eight 4 mm
diameter holes over the colorimetric detection and control zones was
aligned over the device to provide consistent pressure across the
surface of the device. Next, the acpcPNA probe and AgNPs solution
were added onto the detection and control zones. Finally, the sample
solution was added onto the sample reservoir and flow through the
channels to wet the colorimetric detection zones.</p><fig id="sch1" position="float"><label>Scheme 1</label><caption><title>(A) Design and (B)
Operation of Multiplex Paper-Based Colorimetric
Device</title></caption><graphic xlink:href="ac7b00255_0006" id="gr1" position="float"></graphic></fig></sec><sec id="sec2.5"><title>Colorimetric Detection
of MERS-CoV, MTB, and HPV DNA Target</title><p>According to the concept
of PNA-induced AgNPs aggregation,<sup><xref ref-type="bibr" rid="ref42">42</xref>,<xref ref-type="bibr" rid="ref43">43</xref></sup> acpcPNA was
designed as a specific probe for quantitative detection
of synthetic MERS-CoV, MTB, and HPV DNA targets. For colorimetric
detection, the detection zone was prepared by adding 10 μL of
AgNPs in 0.1 M phosphate buffer saline (PBS) pH 7.4 in a ratio of
5:1 (AgNPs: PBS), followed by 1 μL of specific acpcPNA probe.
Control zones were prepared using the same conditions as the colorimetric
detection zones. Next, 25 μL of DNA target was added to the
open sample reservoir. Upon sample addition, solution moved outward
through the channels to wet the colorimetric detection zone of the
top layer. Finally, the AgNPs aggregation occurred and the color intensity
was measured.</p></sec><sec id="sec2.6"><title>Image Processing</title><p>The detection images
were recorded
using a scanner (XEROX DocuMate 3220) and saved in JPEG format at
600 dpi. ImageJ software (National Institutes of Health) was used
to analyze the mean intensity of the color for each colorimetric reaction
zone by applying a color threshold window for removing the blue background.
Images were then inverted, and the mean intensity was measured.<sup><xref ref-type="bibr" rid="ref20">20</xref>,<xref ref-type="bibr" rid="ref53">53</xref></sup></p></sec></sec><sec id="sec3"><title>Results and Discussion</title><sec id="sec3.1"><title>acpcPNA-Induced AgNPs Aggregation</title><p>The process of acpcPNA-induced
AgNPs aggregation is shown in <xref rid="sch2" ref-type="scheme">Scheme <xref rid="sch2" ref-type="scheme">2</xref></xref>. The anionic AgNPs are initially well dispersed due
to electrostatic repulsion. On addition of the cationic acpcPNA, the
electrostatic repulsion is shielded, resulting in nanoparticle aggregation.
When complementary DNA (DNA<sub>com</sub>) is present, the specific
PNA–DNA interaction outcompetes the less specific PNA–AgNPs
interaction, resulting in a negatively charged PNA–DNA<sub>com</sub> duplex and deaggregation of the anionic nanoparticles.
Upon addition of noncomplementary DNA (DNA<sub>nc</sub>), the acpcPNA
should remain bound to the AgNPs and no color change occurs. To prove
the concept, we designed and synthesized acpcPNA probes to detect
synthetic oligonucleotide targets with sequences corresponding to
MERS-CoV, MTB, and HPV type 16. The photographs of the results are
shown in <xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>.
The yellow AgNPs turned red when the acpcPNA was added. When the solution
contained of the acpcPNA and DNA<sub>nc</sub>, the color also changed
to red due to aggregation of the AgNPs. On the other hand, the color
changed from red (aggregated) to yellow (nonaggregated) in the presence
of DNA<sub>com</sub>, with the intensity dependent on the DNA concentration.
Next, the sequence of adding the PNA probe and DNA target was investigated.
As shown in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b00255/suppl_file/ac7b00255_si_001.pdf">Figure S3</ext-link>, when equimolar DNA<sub>com</sub> was added either before or after the addition of acpcPNA
probe into the AgNPs, the same color intensities were obtained indicating
that the sequence of adding acpcPNA and DNA<sub>com</sub> did not
impact the final signal.</p><fig id="sch2" position="float"><label>Scheme 2</label><caption><title>Process of acpcPNA-Induced AgNP Aggregation
in the Presence of DNA<sub>com</sub> and DNA<sub>nc</sub></title></caption><graphic xlink:href="ac7b00255_0007" id="gr2" position="float"></graphic></fig><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Photograph of visual color changes obtained
from detection of MERS-CoV,
MTB, and HPV in the presence of DNA<sub>com</sub>.</p></caption><graphic xlink:href="ac7b00255_0001" id="gr3" position="float"></graphic></fig></sec><sec id="sec3.2"><title>Critical Coagulation Concentration (CCC)</title><p>The influence
of electrolyte solution on the aggregation behavior of citrate-stabilized
AgNPs was investigated based on the CCC.<sup><xref ref-type="bibr" rid="ref54">54</xref></sup>The CCC represents the electrolyte concentration required to cause
aggregation of the nanoparticles in the absence of acpcPNA. In <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b00255/suppl_file/ac7b00255_si_001.pdf">Figure S4</ext-link>, the color intensity of citrate-stabilized
AgNPs in the absence of acpcPNA probe is shown as a function of NaCl
concentration. The intensity and, therefore, the degree of aggregation,
increased with the concentration of NaCl, indicating that increasing
ionic strength led to enhanced aggregation.<sup><xref ref-type="bibr" rid="ref55">55</xref></sup> We believe that the ionic strength can decrease the electrostatic
repulsion of citrate-stabilized AgNPs as a result of shielding, accelerating
the AgNPs aggregation. The CCC was obtained when the degree of aggregation
reached a maximum and became independent of NaCl concentration. In
this experiment, the CCC of citrate-stabilized AgNPs was found to
be 30 mM. Above this concentration, PNA-induced aggregation was not
observed.</p></sec><sec id="sec3.3"><title>Optimization of Assay Parameters</title><p>For a colorimetric
assay based on acpcPNA-induced AgNPs aggregation, assay parameters
including 0.1 M PBS (pH 7.4) ratio and acpcPNA concentration were
optimized using a simple paper-based design. The degree of AgNPs aggregation
was determined by measuring the color intensity of the resulting solution
in the presence of acpcPNA without target DNA. First, the impact of
the PBS concentration on AgNPs aggregation was measured. The differential
color intensity (Δ intensity, Δ<italic>I</italic>) obtained
before and after addition of acpcPNA as a function of AgNPs to PBS
ratio is shown in <xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>A. Δ<italic>I</italic> increased until the ratio of
AgNPs/PBS reached 5:1 and then decreased until it plateaued at 5:2.
Thus, the ratio of 5:1 AgNPs/PBS was selected as the optimal condition
because it gave the largest Δ<italic>I</italic>. Another important
aspect for the DNA assay is probe concentration. The influence of
acpcPNA probe concentration on absolute intensity was studied. As
shown in <xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>B,
the acpcPNA concentration was varied within a range of 0–2.5
μM, and the highest aggregation was obtained at the concentration
of 1.0 μM. At this concentration, the aggregation became independent
of acpcPNA concentration, which was desirable for simplifying the
assay. Higher concentrations of AgNPs were not tested in order to
minimize reagent consumption. As a result, the optimal conditions
consisting of AgNPs/PBS ratio of 5:1 and acpcPNA concentration of
1.0 μM were selected for further experiments.</p><fig id="fig2" position="float"><label>Figure 2</label><caption><p>Influence of (A) AgNPs/PBS
ratio and (B) acpcPNA probe concentration
on color intensity for MERS-CoV, MTB, and HPV detection. The error
bars represent one standard deviation (SD) obtained from three independent
measurements (<italic>n</italic> = 3).</p></caption><graphic xlink:href="ac7b00255_0002" id="gr4" position="float"></graphic></fig></sec><sec id="sec3.4"><title>Selectivity of MERS-CoV, MTB, and HPV Detection</title><p>To
investigate the selectivity of this system, the color intensity obtained
from the DNA<sub>com</sub> of MERS-CoV, MTB, and HPV was compared
to that of single-base mismatch (DNA<sub>m1</sub>), two-base mismatch
(DNA<sub>m2</sub>), and DNA<sub>nc</sub> sequences. The color intensity
decreased significantly in the presence of DNA<sub>com</sub>; whereas,
the intensity did not change for the mismatched and noncomplementary
targets (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>).
We believe the affinity of PNA–DNA hybridization was reduced
due to the contribution of one- and two-base mismatches, leaving free
PNA to aggregate the nanoparticles. PNA–DNA<sub>com</sub> complex
can retard the ability of PNA to induce AgNPs aggregation as discussed
above and result in different color intensities. These results suggest
that the fully complementary DNA selectively hybridized the acpcPNA
probe and yielded measurable signals. In addition, bovine serum albumin
(BSA), which is commonly used in cell culture protocols, was used
to investigate the protein interference of the proposed system. The
DNA target was prepared in the presence of 3% BSA solution. It was
observed that the color intensities of the DNA targets for MERS-CoV,
MTB, and HPV in 3% BSA solution were statistically identical to the
ones without BSA (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b00255/suppl_file/ac7b00255_si_001.pdf">Figure S5</ext-link>). Hence, common
proteins should not negatively affect the analysis of this system.</p><fig id="fig3" position="float"><label>Figure 3</label><caption><p>Color
intensity of (A) MERS-CoV, (B) MTB, and (C) HPV detection
after hybridization of DNA<sub>m1</sub>, DNA<sub>m2</sub>, and DNA<sub>nc</sub>. The error bars represent one standard deviation (SD) obtained
from three independent measurements (<italic>n</italic> = 3).</p></caption><graphic xlink:href="ac7b00255_0003" id="gr5" position="float"></graphic></fig></sec><sec id="sec3.5"><title>Analytical Performance</title><p>To assess the sensitivity of
the proposed method for DNA quantification, the intensity as a function
of the target DNA concentration was determined. The color intensity
decreases with the target DNA concentration. The calibration curves
for each species are shown in <xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>A, B, and C for MERS-CoV, MTB, and HPV, respectively.
The linear range for each DNA target using a logarithmic DNA concentration
and color intensity (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>, inset) was also obtained. The analytical performances for
all three DNA targets are summarized in <xref rid="tbl2" ref-type="other">Table <xref rid="tbl2" ref-type="other">2</xref></xref>. It can be seen that a sufficiently low
detection limit could be obtained for MERS-CoV, MTB, and HPV detection
without the need for multiple PCR cycles. Moreover, this multiplex
system can provide sensitive and selective detection for simultaneous
analysis of multiple DNA targets in a single device, which simplifies
the analysis compared to traditional diagnostics.<sup><xref ref-type="bibr" rid="ref9">9</xref>,<xref ref-type="bibr" rid="ref56">56</xref>−<xref ref-type="bibr" rid="ref59">59</xref></sup></p><fig id="fig4" position="float"><label>Figure 4</label><caption><p>Change
of probe color intensity vs DNA target concentration (Δ<italic>I</italic>) and calibration graph between Δ<italic>I</italic> and log DNA target concentration (inset) for (A) MERS-CoV, (B) MTB,
and (C) HPV detection. The error bars represent standard deviation
(SD) obtained from three independent measurement (<italic>n</italic> = 3).</p></caption><graphic xlink:href="ac7b00255_0004" id="gr6" position="float"></graphic></fig><table-wrap id="tbl2" position="float"><label>Table 2</label><caption><title>Summarized Analytical
Performance
of the Multiplexed 3DPAD for Colorimetric DNA Assay</title></caption><table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col><col align="left"></col><col align="justify"></col><col align="left"></col></colgroup><thead><tr><th style="border:none;" align="center">DNA target</th><th style="border:none;" align="center">linearity (nM)</th><th style="border:none;" align="center">LOD (nM)</th><th style="border:none;" align="center">%RSD (<italic>n</italic> = 3)</th></tr></thead><tbody><tr><td style="border:none;" align="left">MERS-CoV</td><td style="border:none;" align="left">20–1000</td><td style="border:none;" align="justify">1.53</td><td style="border:none;" align="left">0.17–0.50</td></tr><tr><td style="border:none;" align="left">MTB</td><td style="border:none;" align="left">50–2500</td><td style="border:none;" align="justify">1.27</td><td style="border:none;" align="left">0.12–0.67</td></tr><tr><td style="border:none;" align="left">HPV</td><td style="border:none;" align="left">20–2500</td><td style="border:none;" align="justify">1.03</td><td style="border:none;" align="left">0.43–0.93</td></tr></tbody></table></table-wrap></sec><sec id="sec3.6"><title>Device Design</title><p>Next, a multiplex device (<xref rid="sch1" ref-type="scheme">Scheme <xref rid="sch1" ref-type="scheme">1</xref></xref>) was designed for simultaneous
detection of MERS-CoV, MTB, and HPV. The top layer contained four
detection zones and four control zones. Each zone contained AgNPs
with a single acpcPNA probe to provide selectivity for DNA. The base
layer contained four wax-defined channels extending outward from a
sample inlet. After the device was folded and stacked together, the
channels of the base layer were connected to four detection zones
of the top layer. Upon sample addition, the solution moved outward
through the channels of the base layer to wet the colorimetric detection
zones of the top layer and lead to color change. <xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref> illustrates the ability of
the proposed sensor for detection of 100 nM MERS-CoV, MTB, and HPV.
Only the colorimetric detection zones that contained the selective
probes changed color compared to their control zones. This result
indicated that the multiplex paper-based colorimetric sensor is promising
for simultaneous determination of MERS-CoV, MTB, and HPV.</p><fig id="fig5" position="float"><label>Figure 5</label><caption><p>Selectivity
of 100 nM MERS-CoV, MTB, and HPV detection using multiplex
colorimetric PAD (1, C<sub>1</sub>= AgNPs + MERS-CoV acpcPNA probe;
2, C<sub>2</sub> = AgNPs + MTB acpcPNA probe; 3, C<sub>3</sub> = AgNPs
+ HPV acpcPNA probe).</p></caption><graphic xlink:href="ac7b00255_0005" id="gr7" position="float"></graphic></fig></sec></sec><sec id="sec4"><title>Conclusions</title><p>A multiplex colorimetric PAD was developed
for simultaneous detection
of DNA associated with viral and bacterial infectious diseases, including
Middle East respiratory syndrome coronavirus (MERS-CoV), <italic>Mycobacterium tuberculosis</italic> (MTB), and human papillomavirus
(HPV). AgNPs were used as a colorimetric reagent for DNA detection
based on acpcPNA-induced nanoparticle aggregation. This colorimetric
DNA sensor exhibited high selectivity against single-base mismatch,
two-base mismatch and noncomplementary target DNA. Under the optimized
condition, the limit of detection for MERS-CoV, MTB, and HPV were
found to be 1.53, 1.27, and 1.03 nM, respectively. As a result, this
developed multiplex colorimetric PAD could be a low-cost and disposable
alternative tool for rapid screening and detecting in infectious diagnostics.</p></sec></body><back><notes id="NOTES-d96e905-autogenerated" notes-type="si"><title>Supporting Information Available</title><p>The Supporting Information
is available
free of charge on the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org">ACS Publications
website</ext-link> at DOI: <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/abs/10.1021/acs.analchem.7b00255">10.1021/acs.analchem.7b00255</ext-link>.<list id="silist" list-type="simple"><list-item><p>Supporting Figures
S1–S5 (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b00255/suppl_file/ac7b00255_si_001.pdf">PDF</ext-link>).</p></list-item></list></p></notes><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="sifile1"><media xlink:href="ac7b00255_si_001.pdf"><caption><p>ac7b00255_si_001.pdf</p></caption></media></supplementary-material></sec><notes notes-type="COI-statement" id="NOTES-d96e921-autogenerated"><p>The authors
declare no
competing financial interest.</p></notes><ack><title>Acknowledgments</title><p>P.T. gratefully appreciates the financial
supports from Thailand
Graduate Institute of Science and Technology (TGIST 01-55-014) and
The Thailand Research Fund (RTA5780005). C.S.H. acknowledges financial
support from Colorado State University and the United States Department
of Agriculture through the National Wildlife Research Center (1574000859CA).
T.V. acknowledges technical assistance of Ms. Chotima Vilaivan (Organic
Synthesis Research Unit, Chulalongkorn University) and the financial
support from Thailand Research Fund (DPG5780002, to T.V.) for the
PNA synthesis. The authors thank Dr. Yuanyuan Yang for assistance
with manuscript editing. The authors also acknowledge important discussions
with Dr. Christopher Ackerson surrounding the critical coagulation
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