Development of Label-Free Colorimetric Assay for MERS-CoV Using Gold Nanoparticles
Identifieur interne : 000125 ( Pmc/Corpus ); précédent : 000124; suivant : 000126Development of Label-Free Colorimetric Assay for MERS-CoV Using Gold Nanoparticles
Auteurs : Hanbi Kim ; Minseon Park ; Joonki Hwang ; Jin Hwa Kim ; Doo-Ryeon Chung ; Kyu-Sung Lee ; Minhee KangSource :
- ACS Sensors [ 2379-3694 ] ; 2019.
Abstract
Worldwide outbreaks of infectious diseases necessitate the development of rapid and accurate diagnostic methods. Colorimetric assays are a representative tool to simply identify the target molecules in specimens through color changes of an indicator (e.g., nanosized metallic particle, and dye molecules). The detection method is used to confirm the presence of biomarkers visually and measure absorbance of the colored compounds at a specific wavelength. In this study, we propose a colorimetric assay based on an extended form of double-stranded DNA (dsDNA) self-assembly shielded gold nanoparticles (AuNPs) under positive electrolyte (e.g., 0.1 M MgCl2) for detection of Middle East respiratory syndrome coronavirus (MERS-CoV). This platform is able to verify the existence of viral molecules through a localized surface plasmon resonance (LSPR) shift and color changes of AuNPs in the UV–vis wavelength range. We designed a pair of thiol-modified probes at either the 5′ end or 3′ end to organize complementary base pairs with upstream of the E protein gene (upE) and open reading frames (ORF) 1a on MERS-CoV. The dsDNA of the target and probes forms a disulfide-induced long self-assembled complex, which protects AuNPs from salt-induced aggregation and transition of optical properties. This colorimetric assay could discriminate down to 1 pmol/μL of 30 bp MERS-CoV and further be adapted for convenient on-site detection of other infectious diseases, especially in resource-limited settings.
Url:
DOI: 10.1021/acssensors.9b00175
PubMed: 31062580
PubMed Central: 7119221
Links to Exploration step
PMC:7119221Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Development of Label-Free Colorimetric Assay for MERS-CoV
Using Gold Nanoparticles</title>
<author><name sortKey="Kim, Hanbi" sort="Kim, Hanbi" uniqKey="Kim H" first="Hanbi" last="Kim">Hanbi Kim</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Park, Minseon" sort="Park, Minseon" uniqKey="Park M" first="Minseon" last="Park">Minseon Park</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Department of Medical Device Management and Research, SAIHST (Samsung Advanced Institute for Health Sciences & Technology),<institution>Sungkyunkwan University</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Hwang, Joonki" sort="Hwang, Joonki" uniqKey="Hwang J" first="Joonki" last="Hwang">Joonki Hwang</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kim, Jin Hwa" sort="Kim, Jin Hwa" uniqKey="Kim J" first="Jin Hwa" last="Kim">Jin Hwa Kim</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Chung, Doo Ryeon" sort="Chung, Doo Ryeon" uniqKey="Chung D" first="Doo-Ryeon" last="Chung">Doo-Ryeon Chung</name>
<affiliation><nlm:aff id="aff1a">Center for Infection Prevention and Control,<institution>Samsung Medical Center</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff4"><institution>Asia Pacific Foundation for Infectious Diseases (APFID)</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff wicri:cut=" and" id="aff5a">Division of Infectious Diseases, Department of Internal Medicine</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Lee, Kyu Sung" sort="Lee, Kyu Sung" uniqKey="Lee K" first="Kyu-Sung" last="Lee">Kyu-Sung Lee</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Department of Medical Device Management and Research, SAIHST (Samsung Advanced Institute for Health Sciences & Technology),<institution>Sungkyunkwan University</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5a">Department of Urology,<institution>Samsung Medical Center, Sungkyunkwan University School of Medicine</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kang, Minhee" sort="Kang, Minhee" uniqKey="Kang M" first="Minhee" last="Kang">Minhee Kang</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Department of Medical Device Management and Research, SAIHST (Samsung Advanced Institute for Health Sciences & Technology),<institution>Sungkyunkwan University</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">31062580</idno>
<idno type="pmc">7119221</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7119221</idno>
<idno type="RBID">PMC:7119221</idno>
<idno type="doi">10.1021/acssensors.9b00175</idno>
<date when="2019">2019</date>
<idno type="wicri:Area/Pmc/Corpus">000125</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000125</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Development of Label-Free Colorimetric Assay for MERS-CoV
Using Gold Nanoparticles</title>
<author><name sortKey="Kim, Hanbi" sort="Kim, Hanbi" uniqKey="Kim H" first="Hanbi" last="Kim">Hanbi Kim</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Park, Minseon" sort="Park, Minseon" uniqKey="Park M" first="Minseon" last="Park">Minseon Park</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Department of Medical Device Management and Research, SAIHST (Samsung Advanced Institute for Health Sciences & Technology),<institution>Sungkyunkwan University</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Hwang, Joonki" sort="Hwang, Joonki" uniqKey="Hwang J" first="Joonki" last="Hwang">Joonki Hwang</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kim, Jin Hwa" sort="Kim, Jin Hwa" uniqKey="Kim J" first="Jin Hwa" last="Kim">Jin Hwa Kim</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Chung, Doo Ryeon" sort="Chung, Doo Ryeon" uniqKey="Chung D" first="Doo-Ryeon" last="Chung">Doo-Ryeon Chung</name>
<affiliation><nlm:aff id="aff1a">Center for Infection Prevention and Control,<institution>Samsung Medical Center</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff4"><institution>Asia Pacific Foundation for Infectious Diseases (APFID)</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff wicri:cut=" and" id="aff5a">Division of Infectious Diseases, Department of Internal Medicine</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Lee, Kyu Sung" sort="Lee, Kyu Sung" uniqKey="Lee K" first="Kyu-Sung" last="Lee">Kyu-Sung Lee</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Department of Medical Device Management and Research, SAIHST (Samsung Advanced Institute for Health Sciences & Technology),<institution>Sungkyunkwan University</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5a">Department of Urology,<institution>Samsung Medical Center, Sungkyunkwan University School of Medicine</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kang, Minhee" sort="Kang, Minhee" uniqKey="Kang M" first="Minhee" last="Kang">Minhee Kang</name>
<affiliation><nlm:aff wicri:cut=" and" id="aff1a">Smart Healthcare & Device Research Center</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Department of Medical Device Management and Research, SAIHST (Samsung Advanced Institute for Health Sciences & Technology),<institution>Sungkyunkwan University</institution>
, Seoul,<country>Korea</country>
</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">ACS Sensors</title>
<idno type="eISSN">2379-3694</idno>
<imprint><date when="2019">2019</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="se9b00175_0005" id="ab-tgr1"></graphic>
</p>
<p>Worldwide
outbreaks of infectious diseases necessitate the development
of rapid and accurate diagnostic methods. Colorimetric assays are
a representative tool to simply identify the target molecules in specimens
through color changes of an indicator (e.g., nanosized metallic particle,
and dye molecules). The detection method is used to confirm the presence
of biomarkers visually and measure absorbance of the colored compounds
at a specific wavelength. In this study, we propose a colorimetric
assay based on an extended form of double-stranded DNA (dsDNA) self-assembly
shielded gold nanoparticles (AuNPs) under positive electrolyte (e.g.,
0.1 M MgCl<sub>2</sub>
) for detection of Middle East respiratory syndrome
coronavirus (MERS-CoV). This platform is able to verify the existence
of viral molecules through a localized surface plasmon resonance (LSPR)
shift and color changes of AuNPs in the UV–vis wavelength range.
We designed a pair of thiol-modified probes at either the 5′
end or 3′ end to organize complementary base pairs with upstream
of the E protein gene (upE) and open reading frames (ORF) 1a on MERS-CoV.
The dsDNA of the target and probes forms a disulfide-induced long
self-assembled complex, which protects AuNPs from salt-induced aggregation
and transition of optical properties. This colorimetric assay could
discriminate down to 1 pmol/μL of 30 bp MERS-CoV and further
be adapted for convenient on-site detection of other infectious diseases,
especially in resource-limited settings.</p>
</div>
</front>
<back><div1 type="bibliography"><listBibl><biblStruct></biblStruct>
<biblStruct></biblStruct>
<biblStruct><analytic><author><name sortKey="Morens, D M" uniqKey="Morens D">D. M. Morens</name>
</author>
<author><name sortKey="Fauci, A S" uniqKey="Fauci A">A. S. Fauci</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Tallury, P" uniqKey="Tallury P">P. Tallury</name>
</author>
<author><name sortKey="Malhotra, A" uniqKey="Malhotra A">A. Malhotra</name>
</author>
<author><name sortKey="Byrne, L M" uniqKey="Byrne L">L. M. Byrne</name>
</author>
<author><name sortKey="Santra, S" uniqKey="Santra S">S. Santra</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Kim, J W" uniqKey="Kim J">J. W. Kim</name>
</author>
<author><name sortKey="Kim, J H" uniqKey="Kim J">J. H. Kim</name>
</author>
<author><name sortKey="Chung, S J" uniqKey="Chung S">S. J. Chung</name>
</author>
<author><name sortKey="Chung, B H" uniqKey="Chung B">B. H. Chung</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Huang, P" uniqKey="Huang P">P. Huang</name>
</author>
<author><name sortKey="Wang, H L" uniqKey="Wang H">H. L. Wang</name>
</author>
<author><name sortKey="Cao, Z G" uniqKey="Cao Z">Z. G. Cao</name>
</author>
<author><name sortKey="Jin, H L" uniqKey="Jin H">H. L. Jin</name>
</author>
<author><name sortKey="Chi, H" uniqKey="Chi H">H. Chi</name>
</author>
<author><name sortKey="Zhao, J C" uniqKey="Zhao J">J. C. Zhao</name>
</author>
<author><name sortKey="Yu, B B" uniqKey="Yu B">B. B. Yu</name>
</author>
<author><name sortKey="Yan, F H" uniqKey="Yan F">F. H. Yan</name>
</author>
<author><name sortKey="Hu, X X" uniqKey="Hu X">X. X. Hu</name>
</author>
<author><name sortKey="Wu, F F" uniqKey="Wu F">F. F. Wu</name>
</author>
<author><name sortKey="Jiao, C C" uniqKey="Jiao C">C. C. Jiao</name>
</author>
<author><name sortKey="Hou, P F" uniqKey="Hou P">P. F. Hou</name>
</author>
<author><name sortKey="Xu, S N" uniqKey="Xu S">S. N. Xu</name>
</author>
<author><name sortKey="Zhao, Y K" uniqKey="Zhao Y">Y. K. Zhao</name>
</author>
<author><name sortKey="Feng, N" uniqKey="Feng N">N. Feng</name>
</author>
<author><name sortKey="Wang, J Z" uniqKey="Wang J">J. Z. Wang</name>
</author>
<author><name sortKey="Sun, W Y" uniqKey="Sun W">W. Y. Sun</name>
</author>
<author><name sortKey="Wang, T C" uniqKey="Wang T">T. C. Wang</name>
</author>
<author><name sortKey="Gao, Y W" uniqKey="Gao Y">Y. W. Gao</name>
</author>
<author><name sortKey="Yang, S T" uniqKey="Yang S">S. T. Yang</name>
</author>
<author><name sortKey="Xia, X Z" uniqKey="Xia X">X. Z. Xia</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Sastry, M" uniqKey="Sastry M">M. Sastry</name>
</author>
<author><name sortKey="Rao, M" uniqKey="Rao M">M. Rao</name>
</author>
<author><name sortKey="Ganesh, K N" uniqKey="Ganesh K">K. N. Ganesh</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Verveniotis, E" uniqKey="Verveniotis E">E. Verveniotis</name>
</author>
<author><name sortKey="Kromka, A" uniqKey="Kromka A">A. Kromka</name>
</author>
<author><name sortKey="Ledinsky, M" uniqKey="Ledinsky M">M. Ledinsky</name>
</author>
<author><name sortKey="Cermak, J" uniqKey="Cermak J">J. Cermak</name>
</author>
<author><name sortKey="Rezek, B" uniqKey="Rezek B">B. Rezek</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Willets, K A" uniqKey="Willets K">K. A. Willets</name>
</author>
<author><name sortKey="Van Duyne, R P" uniqKey="Van Duyne R">R. P. Van Duyne</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Abbas, A" uniqKey="Abbas A">A. Abbas</name>
</author>
<author><name sortKey="Kattumenu, R" uniqKey="Kattumenu R">R. Kattumenu</name>
</author>
<author><name sortKey="Tian, L" uniqKey="Tian L">L. Tian</name>
</author>
<author><name sortKey="Singamaneni, S" uniqKey="Singamaneni S">S. Singamaneni</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Elghanian, R" uniqKey="Elghanian R">R. Elghanian</name>
</author>
<author><name sortKey="Storhoff, J J" uniqKey="Storhoff J">J. J. Storhoff</name>
</author>
<author><name sortKey="Mucic, R C" uniqKey="Mucic R">R. C. Mucic</name>
</author>
<author><name sortKey="Letsinger, R L" uniqKey="Letsinger R">R. L. Letsinger</name>
</author>
<author><name sortKey="Mirkin, C A" uniqKey="Mirkin C">C. A. Mirkin</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Fennell Evans, H W" uniqKey="Fennell Evans H">H. W. Fennell Evans</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Valentini, P" uniqKey="Valentini P">P. Valentini</name>
</author>
<author><name sortKey="Pompa, P P" uniqKey="Pompa P">P. P. Pompa</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Li, H" uniqKey="Li H">H. Li</name>
</author>
<author><name sortKey="Rothberg, L J" uniqKey="Rothberg L">L. J. Rothberg</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Shokri, E" uniqKey="Shokri E">E. Shokri</name>
</author>
<author><name sortKey="Hosseini, M" uniqKey="Hosseini M">M. Hosseini</name>
</author>
<author><name sortKey="Davari, M D" uniqKey="Davari M">M. D. Davari</name>
</author>
<author><name sortKey="Ganjali, M R" uniqKey="Ganjali M">M. R. Ganjali</name>
</author>
<author><name sortKey="Peppelenbosch, M P" uniqKey="Peppelenbosch M">M. P. Peppelenbosch</name>
</author>
<author><name sortKey="Rezaee, F" uniqKey="Rezaee F">F. Rezaee</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Li, H" uniqKey="Li H">H. Li</name>
</author>
<author><name sortKey="Rothberg, L" uniqKey="Rothberg L">L. Rothberg</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wang, L" uniqKey="Wang L">L. Wang</name>
</author>
<author><name sortKey="Liu, X" uniqKey="Liu X">X. Liu</name>
</author>
<author><name sortKey="Hu, X" uniqKey="Hu X">X. Hu</name>
</author>
<author><name sortKey="Song, S" uniqKey="Song S">S. Song</name>
</author>
<author><name sortKey="Fan, C" uniqKey="Fan C">C. Fan</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Polavarapu, L" uniqKey="Polavarapu L">L. Polavarapu</name>
</author>
<author><name sortKey="Xu, Q H J L" uniqKey="Xu Q">Q.-H. J. L. Xu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Xia, F" uniqKey="Xia F">F. Xia</name>
</author>
<author><name sortKey="Zuo, X" uniqKey="Zuo X">X. Zuo</name>
</author>
<author><name sortKey="Yang, R" uniqKey="Yang R">R. Yang</name>
</author>
<author><name sortKey="Xiao, Y" uniqKey="Xiao Y">Y. Xiao</name>
</author>
<author><name sortKey="Kang, D" uniqKey="Kang D">D. Kang</name>
</author>
<author><name sortKey="Vallee Belisle, A" uniqKey="Vallee Belisle A">A. Vallée-Bélisle</name>
</author>
<author><name sortKey="Gong, X" uniqKey="Gong X">X. Gong</name>
</author>
<author><name sortKey="Yuen, J D" uniqKey="Yuen J">J. D. Yuen</name>
</author>
<author><name sortKey="Hsu, B B" uniqKey="Hsu B">B. B. Hsu</name>
</author>
<author><name sortKey="Heeger, A J" uniqKey="Heeger A">A. J. Heeger</name>
</author>
</analytic>
</biblStruct>
<biblStruct></biblStruct>
<biblStruct><analytic><author><name sortKey="Li, F" uniqKey="Li F">F. Li</name>
</author>
<author><name sortKey="Zhang, H" uniqKey="Zhang H">H. Zhang</name>
</author>
<author><name sortKey="Dever, B" uniqKey="Dever B">B. Dever</name>
</author>
<author><name sortKey="Li, X F" uniqKey="Li X">X. F. Li</name>
</author>
<author><name sortKey="Le, X C" uniqKey="Le X">X. C. Le</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Pensa, E" uniqKey="Pensa E">E. Pensa</name>
</author>
<author><name sortKey="Cortes, E" uniqKey="Cortes E">E. Cortes</name>
</author>
<author><name sortKey="Corthey, G" uniqKey="Corthey G">G. Corthey</name>
</author>
<author><name sortKey="Carro, P" uniqKey="Carro P">P. Carro</name>
</author>
<author><name sortKey="Vericat, C" uniqKey="Vericat C">C. Vericat</name>
</author>
<author><name sortKey="Fonticelli, M H" uniqKey="Fonticelli M">M. H. Fonticelli</name>
</author>
<author><name sortKey="Benitez, G" uniqKey="Benitez G">G. Benitez</name>
</author>
<author><name sortKey="Rubert, A A" uniqKey="Rubert A">A. A. Rubert</name>
</author>
<author><name sortKey="Salvarezza, R C" uniqKey="Salvarezza R">R. C. Salvarezza</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Frens, G" uniqKey="Frens G">G. Frens</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Sau, T K" uniqKey="Sau T">T. K. Sau</name>
</author>
<author><name sortKey="Pal, A" uniqKey="Pal A">A. Pal</name>
</author>
<author><name sortKey="Jana, N R" uniqKey="Jana N">N. R. Jana</name>
</author>
<author><name sortKey="Wang, Z L" uniqKey="Wang Z">Z. L. Wang</name>
</author>
<author><name sortKey="Pal, T" uniqKey="Pal T">T. Pal</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Kimling, J" uniqKey="Kimling J">J. Kimling</name>
</author>
<author><name sortKey="Maier, M" uniqKey="Maier M">M. Maier</name>
</author>
<author><name sortKey="Okenve, B" uniqKey="Okenve B">B. Okenve</name>
</author>
<author><name sortKey="Kotaidis, V" uniqKey="Kotaidis V">V. Kotaidis</name>
</author>
<author><name sortKey="Ballot, H" uniqKey="Ballot H">H. Ballot</name>
</author>
<author><name sortKey="Plech, A" uniqKey="Plech A">A. Plech</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Zhao, W" uniqKey="Zhao W">W. Zhao</name>
</author>
<author><name sortKey="Brook, M A" uniqKey="Brook M">M. A. Brook</name>
</author>
<author><name sortKey="Li, Y F" uniqKey="Li Y">Y. F. Li</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Dahlin, A B" uniqKey="Dahlin A">A. B. Dahlin</name>
</author>
<author><name sortKey="Chen, S" uniqKey="Chen S">S. Chen</name>
</author>
<author><name sortKey="Jonsson, M P" uniqKey="Jonsson M">M. P. Jonsson</name>
</author>
<author><name sortKey="Gunnarsson, L" uniqKey="Gunnarsson L">L. Gunnarsson</name>
</author>
<author><name sortKey="Kall, M" uniqKey="Kall M">M. Kall</name>
</author>
<author><name sortKey="Hook, F X0a J A C" uniqKey="Hook F">F.
J. A. c. Höök</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Lustemberg, P G" uniqKey="Lustemberg P">P. G. Lustemberg</name>
</author>
<author><name sortKey="Vericat, C" uniqKey="Vericat C">C. Vericat</name>
</author>
<author><name sortKey="Benitez, G A" uniqKey="Benitez G">G. A. Benitez</name>
</author>
<author><name sortKey="Vela, M E" uniqKey="Vela M">M. E. Vela</name>
</author>
<author><name sortKey="Tognalli, N" uniqKey="Tognalli N">N. Tognalli</name>
</author>
<author><name sortKey="Fainstein, A" uniqKey="Fainstein A">A. Fainstein</name>
</author>
<author><name sortKey="Martiarena, M L" uniqKey="Martiarena M">M. L. Martiarena</name>
</author>
<author><name sortKey="Salvarezza, R C" uniqKey="Salvarezza R">R. C. Salvarezza</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Vericat, C" uniqKey="Vericat C">C. Vericat</name>
</author>
<author><name sortKey="Vela, M E" uniqKey="Vela M">M. E. Vela</name>
</author>
<author><name sortKey="Andreasen, G" uniqKey="Andreasen G">G. Andreasen</name>
</author>
<author><name sortKey="Salvarezza, R C" uniqKey="Salvarezza R">R. C. Salvarezza</name>
</author>
<author><name sortKey="Vazquez, L" uniqKey="Vazquez L">L. Vazquez</name>
</author>
<author><name sortKey="Martin Gago, J A" uniqKey="Martin Gago J">J. A. Martin-Gago</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Koczkur, K M" uniqKey="Koczkur K">K. M. Koczkur</name>
</author>
<author><name sortKey="Hamed, E M" uniqKey="Hamed E">E. M. Hamed</name>
</author>
<author><name sortKey="Houmam, A" uniqKey="Houmam A">A. Houmam</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Castner, D G" uniqKey="Castner D">D. G. Castner</name>
</author>
<author><name sortKey="Hinds, K" uniqKey="Hinds K">K. Hinds</name>
</author>
<author><name sortKey="Grainger, D W" uniqKey="Grainger D">D. W. Grainger</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Panagiotakos, D B" uniqKey="Panagiotakos D">D. B. Panagiotakos</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Thomsen, V" uniqKey="Thomsen V">V. Thomsen</name>
</author>
<author><name sortKey="Schatzlein, D" uniqKey="Schatzlein D">D. Schatzlein</name>
</author>
<author><name sortKey="Mercuro, D" uniqKey="Mercuro D">D. Mercuro</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Min, C K" uniqKey="Min C">C.-K. Min</name>
</author>
<author><name sortKey="Cheon, S" uniqKey="Cheon S">S. Cheon</name>
</author>
<author><name sortKey="Ha, N Y" uniqKey="Ha N">N.-Y. Ha</name>
</author>
<author><name sortKey="Sohn, K M" uniqKey="Sohn K">K. M. Sohn</name>
</author>
<author><name sortKey="Kim, Y" uniqKey="Kim Y">Y. Kim</name>
</author>
<author><name sortKey="Aigerim, A" uniqKey="Aigerim A">A. Aigerim</name>
</author>
<author><name sortKey="Shin, H M" uniqKey="Shin H">H. M. Shin</name>
</author>
<author><name sortKey="Choi, J Y" uniqKey="Choi J">J.-Y. Choi</name>
</author>
<author><name sortKey="Inn, K S" uniqKey="Inn K">K.-S. Inn</name>
</author>
<author><name sortKey="Kim, J H J" uniqKey="Kim J">J.-H. J. Kim</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Kim, S K" uniqKey="Kim S">S.-K. Kim</name>
</author>
<author><name sortKey="Sung, H" uniqKey="Sung H">H. Sung</name>
</author>
<author><name sortKey="Kim, M N" uniqKey="Kim M">M.-N. Kim</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Al Abdely, H M" uniqKey="Al Abdely H">H. M. Al-Abdely</name>
</author>
<author><name sortKey="Midgley, C M" uniqKey="Midgley C">C. M. Midgley</name>
</author>
<author><name sortKey="Alkhamis, A M" uniqKey="Alkhamis A">A. M. Alkhamis</name>
</author>
<author><name sortKey="Abedi, G R" uniqKey="Abedi G">G. R. Abedi</name>
</author>
<author><name sortKey="Tamin, A" uniqKey="Tamin A">A. Tamin</name>
</author>
<author><name sortKey="Binder, A M" uniqKey="Binder A">A. M. Binder</name>
</author>
<author><name sortKey="Alanazi, K" uniqKey="Alanazi K">K. Alanazi</name>
</author>
<author><name sortKey="Lu, X" uniqKey="Lu X">X. Lu</name>
</author>
<author><name sortKey="Abdalla, O" uniqKey="Abdalla O">O. Abdalla</name>
</author>
<author><name sortKey="Sakthivel, S K" uniqKey="Sakthivel S">S. K. Sakthivel</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Kraaij Dirkzwager, M" uniqKey="Kraaij Dirkzwager M">M. Kraaij-Dirkzwager</name>
</author>
<author><name sortKey="Timen, A" uniqKey="Timen A">A. Timen</name>
</author>
<author><name sortKey="Dirksen, K" uniqKey="Dirksen K">K. Dirksen</name>
</author>
<author><name sortKey="Gelinck, L" uniqKey="Gelinck L">L. Gelinck</name>
</author>
<author><name sortKey="Leyten, E" uniqKey="Leyten E">E. Leyten</name>
</author>
<author><name sortKey="Groeneveld, P" uniqKey="Groeneveld P">P. Groeneveld</name>
</author>
<author><name sortKey="Jansen, C" uniqKey="Jansen C">C. Jansen</name>
</author>
<author><name sortKey="Jonges, M" uniqKey="Jonges M">M. Jonges</name>
</author>
<author><name sortKey="Raj, S" uniqKey="Raj S">S. Raj</name>
</author>
<author><name sortKey="Thurkow, I J E" uniqKey="Thurkow I">I. J. E. Thurkow</name>
</author>
</analytic>
</biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="research-article" xml:lang="EN"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">ACS Sens</journal-id>
<journal-id journal-id-type="iso-abbrev">ACS Sens</journal-id>
<journal-id journal-id-type="publisher-id">se</journal-id>
<journal-id journal-id-type="coden">ascefj</journal-id>
<journal-title-group><journal-title>ACS Sensors</journal-title>
</journal-title-group>
<issn pub-type="epub">2379-3694</issn>
<publisher><publisher-name>American Chemical Society</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">31062580</article-id>
<article-id pub-id-type="pmc">7119221</article-id>
<article-id pub-id-type="doi">10.1021/acssensors.9b00175</article-id>
<article-categories><subj-group><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Development of Label-Free Colorimetric Assay for MERS-CoV
Using Gold Nanoparticles</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Kim</surname>
<given-names>Hanbi</given-names>
</name>
<xref rid="aff1a" ref-type="aff">†</xref>
<xref rid="notes3" ref-type="notes">¶</xref>
</contrib>
<contrib contrib-type="author" id="ath2"><name><surname>Park</surname>
<given-names>Minseon</given-names>
</name>
<xref rid="aff1a" ref-type="aff">†</xref>
<xref rid="aff2" ref-type="aff">‡</xref>
<xref rid="notes3" ref-type="notes">¶</xref>
</contrib>
<contrib contrib-type="author" id="ath3"><name><surname>Hwang</surname>
<given-names>Joonki</given-names>
</name>
<xref rid="aff1a" ref-type="aff">†</xref>
<xref rid="notes2" ref-type="notes">●</xref>
</contrib>
<contrib contrib-type="author" id="ath4"><name><surname>Kim</surname>
<given-names>Jin Hwa</given-names>
</name>
<xref rid="aff1a" ref-type="aff">†</xref>
</contrib>
<contrib contrib-type="author" id="ath5"><name><surname>Chung</surname>
<given-names>Doo-Ryeon</given-names>
</name>
<xref rid="aff1a" ref-type="aff">§</xref>
<xref rid="aff4" ref-type="aff">∥</xref>
<xref rid="aff5a" ref-type="aff">⊥</xref>
</contrib>
<contrib contrib-type="author" corresp="yes" id="ath6"><name><surname>Lee</surname>
<given-names>Kyu-sung</given-names>
</name>
<xref rid="cor1" ref-type="other">*</xref>
<xref rid="aff1a" ref-type="aff">†</xref>
<xref rid="aff2" ref-type="aff">‡</xref>
<xref rid="aff5a" ref-type="aff">#</xref>
</contrib>
<contrib contrib-type="author" corresp="yes" id="ath7"><name><surname>Kang</surname>
<given-names>Minhee</given-names>
</name>
<xref rid="cor2" ref-type="other">*</xref>
<xref rid="aff1a" ref-type="aff">†</xref>
<xref rid="aff2" ref-type="aff">‡</xref>
</contrib>
<aff id="aff1a"><sup>†</sup>
Smart Healthcare & Device Research Center and<sup>§</sup>
Center for Infection Prevention and Control,<institution>Samsung Medical Center</institution>
, Seoul,<country>Korea</country>
</aff>
<aff id="aff2"><label>‡</label>
Department of Medical Device Management and Research, SAIHST (Samsung Advanced Institute for Health Sciences & Technology),<institution>Sungkyunkwan University</institution>
, Seoul,<country>Korea</country>
</aff>
<aff id="aff4"><label>∥</label>
<institution>Asia Pacific Foundation for Infectious Diseases (APFID)</institution>
, Seoul,<country>Korea</country>
</aff>
<aff id="aff5a"><sup>⊥</sup>
Division of Infectious Diseases, Department of Internal Medicine and<sup>#</sup>
Department of Urology,<institution>Samsung Medical Center, Sungkyunkwan University School of Medicine</institution>
, Seoul,<country>Korea</country>
</aff>
</contrib-group>
<author-notes><corresp id="cor1"><label>*</label>
E-mail: <email>minikang@skku.edu</email>
.</corresp>
<corresp id="cor2"><label>*</label>
E-mail: <email>minhee.kang@samsung.com</email>
.</corresp>
</author-notes>
<pub-date pub-type="epub"><day>07</day>
<month>05</month>
<year>2019</year>
</pub-date>
<pub-date pub-type="ppub"><day>24</day>
<month>05</month>
<year>2019</year>
</pub-date>
<volume>4</volume>
<issue>5</issue>
<fpage>1306</fpage>
<lpage>1312</lpage>
<history><date date-type="received"><day>23</day>
<month>01</month>
<year>2019</year>
</date>
<date date-type="accepted"><day>07</day>
<month>05</month>
<year>2019</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2019 American Chemical Society</copyright-statement>
<copyright-year>2019</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="se9b00175_0005" id="ab-tgr1"></graphic>
</p>
<p>Worldwide
outbreaks of infectious diseases necessitate the development
of rapid and accurate diagnostic methods. Colorimetric assays are
a representative tool to simply identify the target molecules in specimens
through color changes of an indicator (e.g., nanosized metallic particle,
and dye molecules). The detection method is used to confirm the presence
of biomarkers visually and measure absorbance of the colored compounds
at a specific wavelength. In this study, we propose a colorimetric
assay based on an extended form of double-stranded DNA (dsDNA) self-assembly
shielded gold nanoparticles (AuNPs) under positive electrolyte (e.g.,
0.1 M MgCl<sub>2</sub>
) for detection of Middle East respiratory syndrome
coronavirus (MERS-CoV). This platform is able to verify the existence
of viral molecules through a localized surface plasmon resonance (LSPR)
shift and color changes of AuNPs in the UV–vis wavelength range.
We designed a pair of thiol-modified probes at either the 5′
end or 3′ end to organize complementary base pairs with upstream
of the E protein gene (upE) and open reading frames (ORF) 1a on MERS-CoV.
The dsDNA of the target and probes forms a disulfide-induced long
self-assembled complex, which protects AuNPs from salt-induced aggregation
and transition of optical properties. This colorimetric assay could
discriminate down to 1 pmol/μL of 30 bp MERS-CoV and further
be adapted for convenient on-site detection of other infectious diseases,
especially in resource-limited settings.</p>
</abstract>
<kwd-group><kwd>colorimetric assay</kwd>
<kwd>Middle East respiratory syndrome
coronavirus (MERS-CoV)</kwd>
<kwd>molecular diagnosis</kwd>
<kwd>gold
nanoparticle</kwd>
<kwd>label-free detection</kwd>
</kwd-group>
<custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name>
<meta-value>se9b00175</meta-value>
</custom-meta>
<custom-meta><meta-name>document-id-new-14</meta-name>
<meta-value>se9b00175</meta-value>
</custom-meta>
<custom-meta><meta-name>ccc-price</meta-name>
<meta-value></meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
</front>
<body><p id="sec1">The point-of-care
testing (POCT)
market of infectious disease represents promising and significant
growth in the global <italic>in vitro</italic>
diagnostics (IVD) industry.<sup><xref ref-type="bibr" rid="ref1">1</xref>
</sup>
There are several factors that are stimulating
the demand for infectious disease POCT, including the increasing spread
of human immunodeficiency virus (HIV), tuberculosis (TB), and malaria
in developing countries, and the threat of emerging and re-emerging
infectious diseases such as the Middle East respiratory syndrome (MERS),
severe acute respiratory syndrome (SARS), ZIKA, a variety of influenza
strains, and the West Nile virus.<sup><xref ref-type="bibr" rid="ref2">2</xref>
</sup>
Infectious
diseases pose a significant risk to human health and has led to more
than half of the deaths worldwide.<sup><xref ref-type="bibr" rid="ref3">3</xref>
</sup>
Additionally,
widespread infectious diseases have caused a continuous increase in
fatality rates in developing countries. The best way for containment
of the epidemic is an early diagnosis, which is difficult using ordinary
methods because of costly and large equipment, the necessity of experts,
and slow data output.<sup><xref ref-type="bibr" rid="ref4">4</xref>
</sup>
Therefore, rapid
POCT methods are crucial for overcoming these limitations by miniaturizing
and reducing the device cost and providing simple, fast, easy-to-use
diagnostic tests without specialized training.</p>
<p>Colorimetric
detection originating from gold nanoparticles (AuNPs)
have been intensively studied because of their particular optical
properties, i.e., localized surface plasmon resonance (LSPR), which
represent a color with maximal absorbance wavelength. AuNPs have been
utilized in colorimetric assays for the detection of diverse biological
molecules (e.g., proteins<sup><xref ref-type="bibr" rid="ref5">5</xref>
</sup>
and nucleic
acids<sup><xref ref-type="bibr" rid="ref6">6</xref>
</sup>
) in that the change in particle
color is generated by sensitive reactivity of nanosized particles
to external condition. In addition, the disperse state is adjustably
modified from artificial electrostatic force control by ion, pH, biomacromolecules,
and so forth.<sup><xref ref-type="bibr" rid="ref7">7</xref>
,<xref ref-type="bibr" rid="ref8">8</xref>
</sup>
For example, a positive electrolyte
(salt) causes metallic nanoparticle aggregation, causing a significant
color change as a red-shift in the LSPR spectrum.<sup><xref ref-type="bibr" rid="ref9">9</xref>
,<xref ref-type="bibr" rid="ref10">10</xref>
</sup>
Based
on that, a modified AuNP cross-linking method was demonstrated in
Mirkin’s group for DNA identification.<sup><xref ref-type="bibr" rid="ref11">11</xref>
</sup>
In the research, the surfaces of AuNPs were conjugated with two
thiolated single-stranded DNA (ssDNA) via strong Au–S interactions.
The hybridization of target DNAs with the ssDNAs on the surface of
the AuNPs induces the formation of double-stranded DNA (dsDNA)-AuNP
networks, resulting in AuNPs aggregation and dramatic color changes.
The target DNA acts as a cross-linker between two ssDNA-functionalized
AuNPs. On the other hand, colorimetric DNA sensing methods using functionalized
AuNPs have been demonstrated using non-cross-linking DNA hybridization
with the benefit of the powerful salting-out effect of dsDNA compared
to ssDNA.<sup><xref ref-type="bibr" rid="ref12">12</xref>
</sup>
Since the ssDNA density on
the surface of AuNPs and the ionic strength of the solution affect
the stability of functionalized AuNPs, control of the aggregated state
by modification is quite difficult in practical applications. Rothberg
et al. suggested a label-free detection method based on the fact that
ssDNA in the solution could bind to citrate-capped AuNPs and electrostatically
stabilize them at high ionic strength. In such a system, AuNPs are
stabilized in the presence of ssDNA, but aggregated with dsDNA in
a highly concentrated electrolyte solution. This strategy does not
need the direct binding of ssDNA on the surface of nanosized metallic
particles, as the DNA amplification step prior to detection is inevitable
due to the low sensitivity of this method.<sup><xref ref-type="bibr" rid="ref13">13</xref>
</sup>
Since both ssDNA and dsDNA allow AuNPs to stabilize at low salt
concentration,<sup><xref ref-type="bibr" rid="ref14">14</xref>
−<xref ref-type="bibr" rid="ref17">17</xref>
</sup>
cationic agents are mainly used to identify the target with probes.<sup><xref ref-type="bibr" rid="ref18">18</xref>
,<xref ref-type="bibr" rid="ref19">19</xref>
</sup>
In this regard, Farhad Rezaee et al. demonstrated a modified non-cross-linking
AuNP aggregation using disulfide self-assembly of terminal modified
DNA. Long and flexible sulfur-rich, self-assembled products were well
combined on the surface of AuNPs and effectively inhibit the particles
from salt-induced aggregation.<sup><xref ref-type="bibr" rid="ref15">15</xref>
</sup>
</p>
<p>Inspired by this research, we developed a colorimetric assay that
relies on bare AuNPs that employs a disulfide-induced self-assembly.
The bare AuNP-based colorimetric method consists of two thiol modified
probes at the 3′ or 5′ ends for targeting partial genomic
regions (30 bp) of MERS-CoV along with upstream E protein gene (upE)
and encoding open reading frames (ORF) 1a. This assay could be highly
reliable for MERS-CoV diagnosis as we have followed WHO updated recommendations
for infectious disease laboratory testing, which targets the two regions
on MERS-CoV considered for potential preclinical screening and high
sensitivity<sup><xref ref-type="bibr" rid="ref20">20</xref>
</sup>
</p>
<p>The developed assay
platform was able to detect the target DNA
through optical properties of the gold nanoparticles such as color
changes with the naked eye and spectral shifts on UV–vis wavelength.
The specific thiolated probes form the complementary dsDNA with the
target and make a disulfide-induced long self-assembled complex due
to continuous disulfide bond formation. The extended self-assembled
complex can protect bare AuNPs for stability against salt-induced
aggregation since sulfur-group at ends of dsDNA are mediated covalent
bond with gold surface. The interaction is known to generate stable
conjugation within disulfide-induced self-assembled complex and AuNPs
and intermolecular force between Au–S attains about 40 to 50
kcal mol<sup>–1</sup>
.<sup><xref ref-type="bibr" rid="ref21">21</xref>
,<xref ref-type="bibr" rid="ref22">22</xref>
</sup>
Otherwise, probes build
a disulfide induced interconnection with each other in the absence
of targeting DNA. The probe DNA terminal coupling is unable to cover
the surface of AuNPs exposed to aggregation, leading to significant
color changes as well as a broader red-shift of the LSPR peak (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
). With this method,
we can visually observe the results with the naked eye, not using
costly equipment. This colorimetric assay is an important step to
use of infectious disease POCT across the developing world. Moreover,
this concept of a disulfide bond based colorimetric assay could be
applied for diagnosis of other infectious diseases.</p>
<fig id="fig1" position="float"><label>Figure 1</label>
<caption><p>Colorimetric detection
of DNA based on disulfide induced self-assembly.
(a) Salt-induced aggregation of AuNPs in the absence of targets. (b)
Procedures for preventing AuNPs from salt-induced aggregation by disulfide
induced self-assembly in the presence of targets.</p>
</caption>
<graphic xlink:href="se9b00175_0001" id="gr1" position="float"></graphic>
</fig>
<sec id="sec2"><title>Results and Discussion</title>
<sec id="sec2.1"><title>Preparation of the Ideal Gold Nanoparticle
upon a Spectral Centroid</title>
<p>We developed various sized gold
nanoparticles for optimization
as a colorimetric indicator. The citrate reduction method, i.e., Turkevich-Frens,
is generally applied to synthesize stabilized gold nanoparticles under
electrostatic repulsion.<sup><xref ref-type="bibr" rid="ref23">23</xref>
</sup>
Additionally,
the ratio of the reducing agent and HAuCl<sub>4</sub>
allow for the
preparation of a diverse size of AuNPs. We changed the volume of trisodium
citrate acid from 300 to 1600 μL to acquire different-sized
AuNPs. The morphology of the prepared AuNPs and DLS records are reported
in <xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
a. According
to the TEM images and DLS measurements, the average diameter of AuNPs
decreased as the amount of trisodium citrate dehydrate increased.
For instance, the addition of reducing agents (300, 500, 1200, 1600
μL) allow us to synthesize 72-, 41-, 26-, and 19-nm-sized gold
nanospheres, respectively. This was the result of changed citrate
and gold ratio to control the reduction and stabilization of the nanoparticle
surface.<sup><xref ref-type="bibr" rid="ref24">24</xref>
,<xref ref-type="bibr" rid="ref25">25</xref>
</sup>
We optimized an average of 19 nm AuNP through
reduction with 1600 μL of trisodium citrate and called that
formed particle “bare gold”, which means “unmodified
and inartificial conjugation with any molecules after synthesized
process” and “citrate ion capped gold nanoparticle derived
from reduction and stabilization effects of sodium citrate”
for this experiment. The structural and spectroscopic properties are
presented as (iv) in <xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
.</p>
<fig id="fig2" position="float"><label>Figure 2</label>
<caption><p>Morphological and optical properties of gold nanoparticles based
on the synthesized conditions and electric charge in solution. (a)
Size distribution of AuNPs measured from TEM images and DLS of gold
nanoparticles (AuNPs). AuNPs, i.e., average size (iv) 19 nm, was used
in this work. (b) UV–vis spectra of as-prepared AuNPs and (c)
AuNPs after salt addition.</p>
</caption>
<graphic xlink:href="se9b00175_0002" id="gr2" position="float"></graphic>
</fig>
<p>To evaluate the effects of salt on as-prepared AuNPs, the
absorbance
spectra of the bare AuNPs and those with MgCl<sub>2</sub>
were measured
using a conventional UV–visible spectrometer (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
b,c). Since the LSPR spectral
band of nanosized metallic particle is derived from dielectric factors,
particle size and shape, the absorption peak shift indicated a changed
environment surrounding AuNPs such as by conjugation of biomolecules.
Thus, the addition of MgCl<sub>2</sub>
induces a significant change
in the absorption spectra. For example, the LSPR band has a decrease
in intensity and increase in bandwidth, as well as new bands at longer
wavelength. These LSPR band shifts represent the aggregation of AuNPs
caused by the loss of interparticle repulsive force as salt disrupts
the charge interaction surround AuNPs and promotes the interparticle
van der Waals attractive forces.<sup><xref ref-type="bibr" rid="ref26">26</xref>
</sup>
As shown
in the salt induced results, the solutions of gold nanoparticles,
excluding 19 nm AuNPs, became gradually transparent due to severe
aggregation of metal particles and also a broader, flattened spectra
appeared in the UV–vis measurement as <xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
c. Therefore, we selected the 19-nm-diameter
AuNPs that detected the LSPR changes on the surface of the nanosized
metallic particles in this experiment.</p>
<p>Furthermore, a spectral
centroid was derived by calculating the
centroid wavelength in each experiment spectrum to determine a credible
and accurate point of the LSPR band. The spectral centroid represents
several parameters of the LSPR band including the peak wavelength,
bandwidth, and intensity that depend on the size, shape, concentration,
and interparticle interactions of the AuNPs.<sup><xref ref-type="bibr" rid="ref15">15</xref>
,<xref ref-type="bibr" rid="ref27">27</xref>
</sup>
The shift of the centroid can be a comprehensive indicator for the
overall transition of the spectral distribution toward shorter or
longer wavelengths. For example, 19-nm-diameter AuNPs possess a spectral
centroid at the orange-red wavelength 516 nm, whereas larger 72-nm-diameter
AuNPs exhibit a purple color with a spectral centroid at 576 nm.</p>
</sec>
<sec id="sec2.2"><title>Optimization of Disulfide-Induced Long Self-Assembly and the
Salting Agent</title>
<p>In this report, bare 19-nm-diameter AuNPs were
used, and we selected a target and control upstream of the E protein
gene (upE) and open reading frames (ORF) 1a on MERS-CoV, and the tobacco
mosaic virus (TMV), respectively. Furthermore, we synthesized forward
and reverse thiol modified probes specifically binding the target
DNA and forming complementary base pairs. Each of the thiolated probes
were modified at the 5′ site (Right: 5′R) and 3′
site (Left: 3′L). Once two probes simultaneously recognize
a specific half of the target, the disulfide bonds at the 3′
and 5′ terminals readily form a sulfur-rich self-assembled
complex. Specifically, target DNA samples were mixed with thiolated
probes (5′ R, 3′ L) in distilled water (D.W.) and then
incubated at 90 °C for a few minutes to establish disulfide-induced
long self-assembled products. 90 μL of the AuNPs solution was
then add to the above-mentioned mixture (60 μL). The solution
was incubated at room temperature for 30 min and various amounts of
MgCl<sub>2</sub>
solutions were added. Each test was evaluated in
at least triplicate.</p>
<p>We analyzed the color of the resulting
solutions with the naked eye and UV–vis spectrometer. Disulfide
self-assembled products were visualized along with the gel electrophoresis,
where the presence of the target (positive reaction) displayed bands
with a characteristic ladder due to the formation of long assemblies
of dsDNA (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
a).
Polymorphous bands appeared with the formation of disulfide self-assembly
between the target and probes on original full-length native poly-acrylamide
gel electrophoresis (PAGE) analysis image (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acssensors.9b00175/suppl_file/se9b00175_si_001.pdf">Figure S1</ext-link>
). Additionally, we confirm the sizes of DNA fragments with
4% agarose gel which is commonly used to separate and visualize the
amplified DNA outcomes depended on number of base pairs (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acssensors.9b00175/suppl_file/se9b00175_si_001.pdf">Figure S2</ext-link>
).</p>
<fig id="fig3" position="float"><label>Figure 3</label>
<caption><p>Visible results analysis depending on
(i) negative (TMV) and (ii)
positive control (ORF1a). (a) Native polyacrylamide gel electrophoresis
(PAGE) analysis for confirmation of disulfide-induced self-assembly
with target DNA and probes (lane 1: ORF1a, lane 2: upE), and nonextended
result of negative control and equal probes with positive control
(lane 3: TMV). (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acssensors.9b00175/suppl_file/se9b00175_si_001.pdf">Figure S1</ext-link>
shows the original
full-length native polyacrylamide gel electrophoresis (PAGE) analysis
image.) (b) X-ray photoelectron spectroscopy (XPS) analysis depends
on S<sub>2p 3/2</sub>
binding energy for confirming the presence
of sulfur group based layers on gold surface (162.2 eV: S̅,
SH̅, or SH<sub>2</sub>
species, 163.7 eV: thiol containing short
layers, 163.37 and 164.55 eV: S multilayer). (c) Absorption spectra
of AuNPs exposed to various concentrations of salt (0.05, 0.1, 0.16,
0.3, and 0.5 M of MgCl<sub>2</sub>
). (d) Spectral centroid shifts
depending on the salt concentration.</p>
</caption>
<graphic xlink:href="se9b00175_0003" id="gr3" position="float"></graphic>
</fig>
<p>Furthermore, we confirmed the adsorbed components on the
surface
of gold nanoparticle via X-ray photoelectron spectroscopy (XPS) (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
b). In the case where
thiolated probe met the incompatible DNA, the S<sub>2p</sub>
signals
mainly indicated the absorbed thiol component at S<sub>2p3/2</sub>
photoelectron binding energy (BE) of 162.2 eV since a sulfur monolayer
was formed on the surface of AuNPs from the probe solution (S̅,
SH̅, or SH<sub>2</sub>
species) (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
b(i)).<sup><xref ref-type="bibr" rid="ref28">28</xref>
,<xref ref-type="bibr" rid="ref29">29</xref>
</sup>
Meanwhile, XPS spectrum
can be fitted at 163.37 and 164.55 eV of S<sub>2p3/2</sub>
binding
energy when target DNA and probes compose the disulfide-induced self-assembled
multilayers on AuNPs (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
b(ii)). The observed components at 163–164 eV of BE
are assigned to some S in multilayer which highly defect to the gold
surface or domain boundaries.<sup><xref ref-type="bibr" rid="ref29">29</xref>
,<xref ref-type="bibr" rid="ref30">30</xref>
</sup>
Although BE of 163.7
appeared in <xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
b(i), the area in the XPS spectrum was lower than thiol species (1:0.64),
and in PAGE, the size of DNA fragments only appeared close to 20 bp
ladder that indicates thiol containing short layers formed, not the
disulfide based multilayer, and unbound thiol assigned that BE of
S<sub>2p 3/2</sub>
when target DNA is absent.<sup><xref ref-type="bibr" rid="ref31">31</xref>
</sup>
Also, the increased area at higher binding energy indicates
higher coverage with absorbed polysulfides.<sup><xref ref-type="bibr" rid="ref22">22</xref>
</sup>
Based on results mentioned earlier from PAGE and XPS analyses, disulfide-induced
self-assembled complex composed the multilayers on AuNPs, but thiolated
ssDNA probes form monolayer which is hard to cover the surface of
gold nanoparticle from salt-induced aggregation. DLS measurements
also supported the size increasing of DNA covered AuNPs which depended
on extended layers (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acssensors.9b00175/suppl_file/se9b00175_si_001.pdf">Figure S3</ext-link>
). Dynamic
light scattering (DLS) was used to monitor the distribution of the
hydrodynamic size of the gold nanoparticles after immobilized ssDNA
or dsDNA complex layer on the gold surface, respectively. As shown
in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acssensors.9b00175/suppl_file/se9b00175_si_001.pdf">Figure S3</ext-link>
, the average hydrodynamic
size of the gold nanoparticle protected by disulfide-induced self-assembly
from the positive reaction was increased by approximately 8.11 nm
compared to immobilized ssDNA on gold nanoparticle from the negative
reaction.</p>
<p>The stability of the assay was investigated through
the addition
of various concentrations of MgCl<sub>2</sub>
into the bare AuNPs
solution in the (i) absence or (ii) existence of the target (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
c). Mixed products
with target DNA and probe solutions shielded the particles, which
tolerated the salting more than gold nanoparticles conjugated with
thiol from disulfide-interconnected probes, since the disulfide-induced
self-assembled complex was used to protect bare AuNPs against salt
addition. The spectral peak of the AuNPs colloid was not quite different
with bare AuNPs when the target DNA is in the solution with 0.05 to
0.5 M MgCl<sub>2</sub>
, while AuNPs linked to disulfide-interconnected
probes aggregated with any concentration of salt. However, the absorbance
declined slightly, corresponding to an increased salt concentration
whether extended disulfide assemblies were formed or not. Therefore,
the spectral centroid shifts depending on salt concentration were
plotted and are shown in <xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
d. At all concentrations (0.05 to 0.5 M), the delta
centroid of the negative control showed more shift than positive controls
due to intergold nanoparticle aggregation, while the centroid shift
of the positive control was the smallest at 0.1 M of MgCl<sub>2.</sub>
As previously mentioned, the absorbance declined conversely according
to the increase in salt concentration. Hence, 0.1 M of MgCl<sub>2</sub>
was chosen as the optimized concentration.</p>
</sec>
<sec id="sec2.3"><title>Verification of the Colorimetric
Assay</title>
<p>To consider
the disulfide self-assembled reaction dependence on target DNAs and
their concentrations, we measured the absorption change of the AuNP
solution before and after adding 0.1 M MgCl<sub>2</sub>
in the presence
or absence of each target using a UV–vis spectrometer. When
the target was absent, the wavelength was shifted more toward the
red side of the spectrum compared to when the target was present.
The wavelength peak shifts were 11, 19, and 51 nm after complementary
binding reactions between probes and (i) ORF1a, (ii) upE, and (iii)
TMV, respectively ((i) 522 to 533 nm, (ii) 520 to 541 nm, and (iii)
521 to 572 nm) (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref>
</xref>
a,b). Due to the broad spectrum of the negative control, delta centroid
was estimated as 2.5 times greater than a positive reaction (avg.
29 nm vs avg 12 nm). These changes indicate that positive samples
(ORF1a and upE) that are a mixture of thiol-modified probes and their
target DNA that hindered the aggregation of AuNPs through composition
of the long self-assembled structures. Negative samples (TMV) were
unable to form long self-assembled structures due to mismatched targets.
Furthermore, we calculated a <italic>p</italic>
-value to prove the
reproducibility of this colorimetric platform (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref>
</xref>
b). The <italic>p</italic>
-value is the probability
that might yield the same results observed in the biological study,
if the null hypothesis is true. All data groups were found to pass
the null-hypothesis assuming a normal distribution was followed.<sup><xref ref-type="bibr" rid="ref32">32</xref>
</sup>
Therefore, the <italic>p</italic>
-value of the
delta centroid implies that this assay is considered statistically
significant (<italic>p</italic>
-value <0.01).</p>
<fig id="fig4" position="float"><label>Figure 4</label>
<caption><p>Spectral analysis of
the colorimetric assay for detection of partial
MERS-CoV including (i) ORF1a and (ii) upE and negative control of
(iii) TMV. (a) UV–vis spectra of the AuNPs solution before
and after adding salt in the presence or absence of disulfide-induced
self-assembled targets. (b) Average delta centroid of positive controls
and a negative control at 0.1 M MgCl2 ((i) avg. 10.7 nm, (ii) avg.
13.4 nm, (iii) avg. 29.1 nm)). (c) LOD graph of the positive control
according to target concentration.</p>
</caption>
<graphic xlink:href="se9b00175_0004" id="gr4" position="float"></graphic>
</fig>
<p>The limit-of-detection (LOD) was determined in order to apply
to
the medical laboratory field. The LOD of this colorimetric platform
was 1 pmol/μL, which was calculated by the equation Mean<sub>blank</sub>
+ 3σ<sub>blank</sub>
defined from the IUPAC standard
(<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref>
</xref>
c).<sup><xref ref-type="bibr" rid="ref33">33</xref>
</sup>
1 pmol/μL is about 6 × 10<sup>11</sup>
copies/μL, which requires only 26 PCR cycles to diagnose MERS
patients who release 1.5 × 10<sup>3</sup>
to 6.7 × 10<sup>3</sup>
copies/μL in the sputum for 10 days and have recovered
without specific treatment for MERS-CoV. This assay can reduce the
number of PCR cycles for diagnosis of MERS even with a small amount
of viral molecules, compared to conventional PCR reactions that require
more than about 34 cycles to detect MERS-CoV.<sup><xref ref-type="bibr" rid="ref34">34</xref>
−<xref ref-type="bibr" rid="ref37">37</xref>
</sup>
</p>
</sec>
</sec>
<sec id="sec3"><title>Conclusions</title>
<p>A
simple and fast colorimetric assay for detecting infectious disease
that can be seen by the naked eye without costly equipment was developed.
We proposed a colorimetric assay using disulfide bonds formed by hybridizing
with thiolated probes and a target; this method inhibited the aggregation
of AuNPs by salt and limits the color change for diagnosis of MERS.
This assay can confirm the presence of MERS-CoV within 10 min without
electrophoresis or other procedures. The assay could discriminate
MERS-CoV with a potential detection limit of 1 pmol/μL, which
means it could distinguish a lower amount of target with a lower level
of amplification, or even without amplification. A follow-up study
will be conducted to apply this method to clinical samples with longer
targeting regions. We anticipate that this method will provide rapid
and accurate diagnostic results in epidemic areas, especially in resource-limited
settings and will evolve by combining with a mobile platform and artificial
intelligence in the near future.</p>
</sec>
</body>
<back><notes id="notes1" 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/acssensors.9b00175">10.1021/acssensors.9b00175</ext-link>
.<list id="silist" list-type="simple"><list-item><p>PAGE analysis;
Agarose gel electrophoresis analysis;
Hydrodynamic size distribution measured by DLS; Experimental methods
(<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acssensors.9b00175/suppl_file/se9b00175_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="se9b00175_si_001.pdf"><caption><p>se9b00175_si_001.pdf</p>
</caption>
</media>
</supplementary-material>
</sec>
<notes notes-type="" id="notes2"><title>Author Present Address</title>
<p><sup>●</sup>
J. Hwang: Life Science Laboratory, SG Medical, Seoul, Korea</p>
</notes>
<notes notes-type="" id="notes3"><title>Author Contributions</title>
<p><sup>¶</sup>
H. Kim and M. Park contributed equally to this work.
The manuscript
was written by contributions from all authors who have given approval
to this final version of the manuscript.</p>
</notes>
<notes notes-type="COI-statement" id="notes4"><p>The authors
declare no competing financial interest.</p>
</notes>
<ack><title>Acknowledgments</title>
<p>This research was supported by a grant of the Korea
Health Technology R&D Project through the Korea Health Industry
Development Institute (KHIDI), funded by the Ministry of Health &
Welfare, Republic of Korea (HG18C0062, HI14C3229) and the Bio &
Medical Technology Development Program of the National Research Foundation
(NRF) funded by the Ministry of Science (2016M3A9B6919189).</p>
</ack>
<ref-list><title>References</title>
<ref id="ref1"><mixed-citation publication-type="journal" id="cit1"><article-title>State
of Knowledge and Data Gaps of Middle East Respiratory Syndrome Coronavirus
(MERS-CoV) in Humans</article-title>
. <source>PLoS Curr.</source>
<year>2013</year>
, <fpage>1</fpage>
<pub-id pub-id-type="doi">10.1371/currents.outbreaks.0bf719e352e7478f8ad85fa30127ddb8</pub-id>
.</mixed-citation>
</ref>
<ref id="ref2"><mixed-citation publication-type="report" id="cit2"><source>MediPoint: Point of Care Diagnostics - Global Analysis
and Market
Forecasts</source>
<year>2016</year>
; pp <fpage>1</fpage>
–<lpage>187</lpage>
.</mixed-citation>
</ref>
<ref id="ref3"><mixed-citation publication-type="journal" id="cit3"><name><surname>Morens</surname>
<given-names>D. M.</given-names>
</name>
; <name><surname>Fauci</surname>
<given-names>A. S.</given-names>
</name>
<article-title>Emerging infectious
diseases: threats to human health
and global stability</article-title>
. <source>PLoS Pathog.</source>
<year>2013</year>
, <volume>9</volume>
(<issue>7</issue>
), <fpage>e1003467</fpage>
<pub-id pub-id-type="doi">10.1371/journal.ppat.1003467</pub-id>
.<pub-id pub-id-type="pmid">23853589</pub-id>
</mixed-citation>
</ref>
<ref id="ref4"><mixed-citation publication-type="journal" id="cit4"><name><surname>Tallury</surname>
<given-names>P.</given-names>
</name>
; <name><surname>Malhotra</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Byrne</surname>
<given-names>L. M.</given-names>
</name>
; <name><surname>Santra</surname>
<given-names>S.</given-names>
</name>
<article-title>Nanobioimaging and
sensing of infectious diseases</article-title>
. <source>Adv. Drug Delivery
Rev.</source>
<year>2010</year>
, <volume>62</volume>
(<issue>4–5</issue>
), <fpage>424</fpage>
–<lpage>437</lpage>
. <pub-id pub-id-type="doi">10.1016/j.addr.2009.11.014</pub-id>
.</mixed-citation>
</ref>
<ref id="ref5"><mixed-citation publication-type="journal" id="cit5"><name><surname>Kim</surname>
<given-names>J. W.</given-names>
</name>
; <name><surname>Kim</surname>
<given-names>J. H.</given-names>
</name>
; <name><surname>Chung</surname>
<given-names>S. J.</given-names>
</name>
; <name><surname>Chung</surname>
<given-names>B. H.</given-names>
</name>
<article-title>An operationally
simple colorimetric assay of hyaluronidase activity using cationic
gold nanoparticles</article-title>
. <source>Analyst</source>
<year>2009</year>
, <volume>134</volume>
(<issue>7</issue>
), <fpage>1291</fpage>
–<lpage>1293</lpage>
. <pub-id pub-id-type="doi">10.1039/b822917a</pub-id>
.<pub-id pub-id-type="pmid">19562190</pub-id>
</mixed-citation>
</ref>
<ref id="ref6"><mixed-citation publication-type="journal" id="cit6"><name><surname>Huang</surname>
<given-names>P.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>H. L.</given-names>
</name>
; <name><surname>Cao</surname>
<given-names>Z. G.</given-names>
</name>
; <name><surname>Jin</surname>
<given-names>H. L.</given-names>
</name>
; <name><surname>Chi</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Zhao</surname>
<given-names>J. C.</given-names>
</name>
; <name><surname>Yu</surname>
<given-names>B. B.</given-names>
</name>
; <name><surname>Yan</surname>
<given-names>F. H.</given-names>
</name>
; <name><surname>Hu</surname>
<given-names>X. X.</given-names>
</name>
; <name><surname>Wu</surname>
<given-names>F. F.</given-names>
</name>
; <name><surname>Jiao</surname>
<given-names>C. C.</given-names>
</name>
; <name><surname>Hou</surname>
<given-names>P. F.</given-names>
</name>
; <name><surname>Xu</surname>
<given-names>S. N.</given-names>
</name>
; <name><surname>Zhao</surname>
<given-names>Y. K.</given-names>
</name>
; <name><surname>Feng</surname>
<given-names>N.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>J. Z.</given-names>
</name>
; <name><surname>Sun</surname>
<given-names>W. Y.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>T. C.</given-names>
</name>
; <name><surname>Gao</surname>
<given-names>Y. W.</given-names>
</name>
; <name><surname>Yang</surname>
<given-names>S. T.</given-names>
</name>
; <name><surname>Xia</surname>
<given-names>X. Z.</given-names>
</name>
<article-title>A Rapid
and Specific Assay for the Detection of MERS-CoV</article-title>
. <source>Front. Microbiol.</source>
<year>2018</year>
, <fpage>1</fpage>
<pub-id pub-id-type="doi">10.3389/fmicb.2018.01101</pub-id>
.<pub-id pub-id-type="pmid">29403456</pub-id>
</mixed-citation>
</ref>
<ref id="ref7"><mixed-citation publication-type="journal" id="cit7"><name><surname>Sastry</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Rao</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Ganesh</surname>
<given-names>K. N.</given-names>
</name>
<article-title>Electrostatic assembly of nanoparticles and biomacromolecules</article-title>
. <source>Acc. Chem. Res.</source>
<year>2002</year>
, <volume>35</volume>
(<issue>10</issue>
), <fpage>847</fpage>
–<lpage>855</lpage>
. <pub-id pub-id-type="doi">10.1021/ar010094x</pub-id>
.<pub-id pub-id-type="pmid">12379137</pub-id>
</mixed-citation>
</ref>
<ref id="ref8"><mixed-citation publication-type="journal" id="cit8"><person-group person-group-type="allauthors"><name><surname>Verveniotis</surname>
<given-names>E.</given-names>
</name>
; <name><surname>Kromka</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Ledinsky</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Cermak</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Rezek</surname>
<given-names>B.</given-names>
</name>
</person-group>
, <article-title>Guided assembly
of nanoparticles on electrostatically charged nanocrystalline diamond
thin films.</article-title>
<source>Nanoscale Res. Lett.</source>
<year>2011</year>
, <pub-id pub-id-type="doi">10.1186/1556-276X-6-144</pub-id>
.</mixed-citation>
</ref>
<ref id="ref9"><mixed-citation publication-type="journal" id="cit9"><name><surname>Willets</surname>
<given-names>K. A.</given-names>
</name>
; <name><surname>Van Duyne</surname>
<given-names>R. P.</given-names>
</name>
<article-title>Localized
surface plasmon resonance spectroscopy and
sensing</article-title>
. <source>Annu. Rev. Phys. Chem.</source>
<year>2007</year>
, <volume>58</volume>
, <fpage>267</fpage>
–<lpage>97</lpage>
. <pub-id pub-id-type="doi">10.1146/annurev.physchem.58.032806.104607</pub-id>
.<pub-id pub-id-type="pmid">17067281</pub-id>
</mixed-citation>
</ref>
<ref id="ref10"><mixed-citation publication-type="journal" id="cit10"><name><surname>Abbas</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Kattumenu</surname>
<given-names>R.</given-names>
</name>
; <name><surname>Tian</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Singamaneni</surname>
<given-names>S.</given-names>
</name>
<article-title>Molecular
linker-mediated self-assembly of gold nanoparticles: understanding
and controlling the dynamics</article-title>
. <source>Langmuir</source>
<year>2013</year>
, <volume>29</volume>
(<issue>1</issue>
), <fpage>56</fpage>
–<lpage>64</lpage>
. <pub-id pub-id-type="doi">10.1021/la303368q</pub-id>
.<pub-id pub-id-type="pmid">23205505</pub-id>
</mixed-citation>
</ref>
<ref id="ref11"><mixed-citation publication-type="journal" id="cit11"><name><surname>Elghanian</surname>
<given-names>R.</given-names>
</name>
; <name><surname>Storhoff</surname>
<given-names>J. J.</given-names>
</name>
; <name><surname>Mucic</surname>
<given-names>R. C.</given-names>
</name>
; <name><surname>Letsinger</surname>
<given-names>R. L.</given-names>
</name>
; <name><surname>Mirkin</surname>
<given-names>C. A.</given-names>
</name>
<article-title>Selective colorimetric
detection of polynucleotides
based on the distance-dependent optical properties of gold nanoparticles</article-title>
. <source>Science</source>
<year>1997</year>
, <volume>277</volume>
(<issue>5329</issue>
), <fpage>1078</fpage>
–<lpage>1081</lpage>
. <pub-id pub-id-type="doi">10.1126/science.277.5329.1078</pub-id>
.<pub-id pub-id-type="pmid">9262471</pub-id>
</mixed-citation>
</ref>
<ref id="ref12"><mixed-citation publication-type="book" id="cit12"><person-group person-group-type="allauthors"><name><surname>Fennell Evans</surname>
<given-names>H. W.</given-names>
</name>
</person-group>
<source>The colloidal
domain: Where physics, chemistry, biology, and technology meet</source>
, <edition>2</edition>
nd ed.; <publisher-name>Wiley-VCH</publisher-name>
: <year>1999</year>
.</mixed-citation>
</ref>
<ref id="ref13"><mixed-citation publication-type="journal" id="cit13"><name><surname>Valentini</surname>
<given-names>P.</given-names>
</name>
; <name><surname>Pompa</surname>
<given-names>P. P.</given-names>
</name>
<article-title>Gold nanoparticles
for naked-eye DNA detection: smart
designs for sensitive assays</article-title>
. <source>RSC Adv.</source>
<year>2013</year>
, <volume>3</volume>
(<issue>42</issue>
), <fpage>19181</fpage>
–<lpage>19190</lpage>
. <pub-id pub-id-type="doi">10.1039/c3ra43729a</pub-id>
.</mixed-citation>
</ref>
<ref id="ref14"><mixed-citation publication-type="journal" id="cit14"><name><surname>Li</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Rothberg</surname>
<given-names>L. J.</given-names>
</name>
<article-title>Label-free colorimetric
detection of specific sequences
in genomic DNA amplified by the polymerase chain reaction</article-title>
. <source>J. Am. Chem. Soc.</source>
<year>2004</year>
, <volume>126</volume>
(<issue>35</issue>
), <fpage>10958</fpage>
–<lpage>61</lpage>
. <pub-id pub-id-type="doi">10.1021/ja048749n</pub-id>
.<pub-id pub-id-type="pmid">15339181</pub-id>
</mixed-citation>
</ref>
<ref id="ref15"><mixed-citation publication-type="journal" id="cit15"><name><surname>Shokri</surname>
<given-names>E.</given-names>
</name>
; <name><surname>Hosseini</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Davari</surname>
<given-names>M. D.</given-names>
</name>
; <name><surname>Ganjali</surname>
<given-names>M. R.</given-names>
</name>
; <name><surname>Peppelenbosch</surname>
<given-names>M. P.</given-names>
</name>
; <name><surname>Rezaee</surname>
<given-names>F.</given-names>
</name>
<article-title>Disulfide-induced self-assembled
targets: A novel strategy
for the label free colorimetric detection of DNAs/RNAs via unmodified
gold nanoparticles</article-title>
. <source>Sci. Rep.</source>
<year>2017</year>
, <volume>7</volume>
, <fpage>1</fpage>
<pub-id pub-id-type="doi">10.1038/srep45837</pub-id>
.<pub-id pub-id-type="pmid">28127051</pub-id>
</mixed-citation>
</ref>
<ref id="ref16"><mixed-citation publication-type="journal" id="cit16"><name><surname>Li</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Rothberg</surname>
<given-names>L.</given-names>
</name>
<article-title>Colorimetric detection of DNA sequences based on electrostatic
interactions with unmodified gold nanoparticles</article-title>
. <source>Proc. Natl. Acad. Sci. U. S. A.</source>
<year>2004</year>
, <volume>101</volume>
(<issue>39</issue>
), <fpage>14036</fpage>
–<lpage>14039</lpage>
. <pub-id pub-id-type="doi">10.1073/pnas.0406115101</pub-id>
.<pub-id pub-id-type="pmid">15381774</pub-id>
</mixed-citation>
</ref>
<ref id="ref17"><mixed-citation publication-type="journal" id="cit17"><name><surname>Wang</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Hu</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Song</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Fan</surname>
<given-names>C.</given-names>
</name>
<article-title>Unmodified gold nanoparticles
as a colorimetric probe for potassium DNA aptamers</article-title>
. <source>Chem. Commun.</source>
<year>2006</year>
, (<issue>36</issue>
), <fpage>3780</fpage>
–<lpage>3782</lpage>
. <pub-id pub-id-type="doi">10.1039/b607448k</pub-id>
.</mixed-citation>
</ref>
<ref id="ref18"><mixed-citation publication-type="journal" id="cit18"><name><surname>Polavarapu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Xu</surname>
<given-names>Q.-H. J. L.</given-names>
</name>
<source>Langmuir</source>
<year>2008</year>
, <volume>24</volume>
(<issue>19</issue>
), <fpage>10608</fpage>
–<lpage>10611</lpage>
. <pub-id pub-id-type="doi">10.1021/la802319c</pub-id>
.<pub-id pub-id-type="pmid">18729527</pub-id>
</mixed-citation>
</ref>
<ref id="ref19"><mixed-citation publication-type="journal" id="cit19"><name><surname>Xia</surname>
<given-names>F.</given-names>
</name>
; <name><surname>Zuo</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Yang</surname>
<given-names>R.</given-names>
</name>
; <name><surname>Xiao</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Kang</surname>
<given-names>D.</given-names>
</name>
; <name><surname>Vallée-Bélisle</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Gong</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Yuen</surname>
<given-names>J. D.</given-names>
</name>
; <name><surname>Hsu</surname>
<given-names>B. B.</given-names>
</name>
; <name><surname>Heeger</surname>
<given-names>A. J.</given-names>
</name>
<source>Proc. Natl. Acad.
Sci. U. S. A.</source>
<year>2010</year>
, <volume>107</volume>
(<issue>24</issue>
), <fpage>10837</fpage>
–<lpage>10841</lpage>
. <pub-id pub-id-type="doi">10.1073/pnas.1005632107</pub-id>
.<pub-id pub-id-type="pmid">20534499</pub-id>
</mixed-citation>
</ref>
<ref id="ref20"><mixed-citation publication-type="weblink" id="cit20"><person-group person-group-type="allauthors"><collab>W.
W. H. Organization</collab>
</person-group>
, <article-title>Laboratory
testing for Middle East
Respiratory Syndrome Coronavirus Interim guidance</article-title>
; <uri xlink:href="https://www.who.int/csr/disease/coronavirus_infections/mers-laboratory-testing/en/">https://www.who.int/csr/disease/coronavirus_infections/mers-laboratory-testing/en/</uri>
.</mixed-citation>
</ref>
<ref id="ref21"><mixed-citation publication-type="journal" id="cit21"><name><surname>Li</surname>
<given-names>F.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Dever</surname>
<given-names>B.</given-names>
</name>
; <name><surname>Li</surname>
<given-names>X. F.</given-names>
</name>
; <name><surname>Le</surname>
<given-names>X. C.</given-names>
</name>
<article-title>Thermal
stability of DNA functionalized gold nanoparticles</article-title>
. <source>Bioconjugate Chem.</source>
<year>2013</year>
, <volume>24</volume>
(<issue>11</issue>
), <fpage>1790</fpage>
–<lpage>1797</lpage>
. <pub-id pub-id-type="doi">10.1021/bc300687z</pub-id>
.</mixed-citation>
</ref>
<ref id="ref22"><mixed-citation publication-type="journal" id="cit22"><name><surname>Pensa</surname>
<given-names>E.</given-names>
</name>
; <name><surname>Cortes</surname>
<given-names>E.</given-names>
</name>
; <name><surname>Corthey</surname>
<given-names>G.</given-names>
</name>
; <name><surname>Carro</surname>
<given-names>P.</given-names>
</name>
; <name><surname>Vericat</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Fonticelli</surname>
<given-names>M. H.</given-names>
</name>
; <name><surname>Benitez</surname>
<given-names>G.</given-names>
</name>
; <name><surname>Rubert</surname>
<given-names>A. A.</given-names>
</name>
; <name><surname>Salvarezza</surname>
<given-names>R. C.</given-names>
</name>
<article-title>The chemistry
of the sulfur-gold interface: in search of a unified model</article-title>
. <source>Acc. Chem. Res.</source>
<year>2012</year>
, <volume>45</volume>
(<issue>8</issue>
), <fpage>1183</fpage>
–<lpage>1192</lpage>
. <pub-id pub-id-type="doi">10.1021/ar200260p</pub-id>
.<pub-id pub-id-type="pmid">22444437</pub-id>
</mixed-citation>
</ref>
<ref id="ref23"><mixed-citation publication-type="journal" id="cit23"><name><surname>Frens</surname>
<given-names>G.</given-names>
</name>
<article-title>Controlled
Nucleation for the Regulation of the Particle Size in Monodisperse
Gold Suspensions</article-title>
. <source>Nature, Phys. Sci.</source>
<year>1973</year>
, <volume>241</volume>
, <fpage>20</fpage>
–<lpage>22</lpage>
. <pub-id pub-id-type="doi">10.1038/physci241020a0</pub-id>
.</mixed-citation>
</ref>
<ref id="ref24"><mixed-citation publication-type="journal" id="cit24"><name><surname>Sau</surname>
<given-names>T. K.</given-names>
</name>
; <name><surname>Pal</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Jana</surname>
<given-names>N. R.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>Z. L.</given-names>
</name>
; <name><surname>Pal</surname>
<given-names>T.</given-names>
</name>
<article-title>Size controlled synthesis
of gold nanoparticles using photochemically prepared seed particles</article-title>
. <source>J. Nanopart. Res.</source>
<year>2001</year>
, <volume>3</volume>
(<issue>4</issue>
), <fpage>257</fpage>
–<lpage>261</lpage>
. <pub-id pub-id-type="doi">10.1023/A:1017567225071</pub-id>
.</mixed-citation>
</ref>
<ref id="ref25"><mixed-citation publication-type="journal" id="cit25"><name><surname>Kimling</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Maier</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Okenve</surname>
<given-names>B.</given-names>
</name>
; <name><surname>Kotaidis</surname>
<given-names>V.</given-names>
</name>
; <name><surname>Ballot</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Plech</surname>
<given-names>A.</given-names>
</name>
<article-title>Turkevich method for
gold nanoparticle synthesis revisited</article-title>
. <source>J. Phys.
Chem. B</source>
<year>2006</year>
, <volume>110</volume>
(<issue>32</issue>
), <fpage>15700</fpage>
–<lpage>15707</lpage>
. <pub-id pub-id-type="doi">10.1021/jp061667w</pub-id>
.<pub-id pub-id-type="pmid">16898714</pub-id>
</mixed-citation>
</ref>
<ref id="ref26"><mixed-citation publication-type="journal" id="cit26"><name><surname>Zhao</surname>
<given-names>W.</given-names>
</name>
; <name><surname>Brook</surname>
<given-names>M. A.</given-names>
</name>
; <name><surname>Li</surname>
<given-names>Y. F.</given-names>
</name>
<article-title>Design of Gold Nanoparticle-Based
Colorimetric Biosensing Assays</article-title>
. <source>ChemBioChem</source>
<year>2008</year>
, <volume>9</volume>
(<issue>15</issue>
), <fpage>2363</fpage>
–<lpage>2371</lpage>
. <pub-id pub-id-type="doi">10.1002/cbic.200800282</pub-id>
.<pub-id pub-id-type="pmid">18821551</pub-id>
</mixed-citation>
</ref>
<ref id="ref27"><mixed-citation publication-type="journal" id="cit27"><name><surname>Dahlin</surname>
<given-names>A. B.</given-names>
</name>
; <name><surname>Chen</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Jonsson</surname>
<given-names>M. P.</given-names>
</name>
; <name><surname>Gunnarsson</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Kall</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Höök</surname>
<given-names>F.
J. A. c.</given-names>
</name>
<source>Anal. Chem.</source>
<year>2009</year>
, <volume>81</volume>
(<issue>16</issue>
), <fpage>6572</fpage>
–<lpage>6580</lpage>
. <pub-id pub-id-type="doi">10.1021/ac901175k</pub-id>
.<pub-id pub-id-type="pmid">19621881</pub-id>
</mixed-citation>
</ref>
<ref id="ref28"><mixed-citation publication-type="journal" id="cit28"><name><surname>Lustemberg</surname>
<given-names>P. G.</given-names>
</name>
; <name><surname>Vericat</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Benitez</surname>
<given-names>G. A.</given-names>
</name>
; <name><surname>Vela</surname>
<given-names>M. E.</given-names>
</name>
; <name><surname>Tognalli</surname>
<given-names>N.</given-names>
</name>
; <name><surname>Fainstein</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Martiarena</surname>
<given-names>M. L.</given-names>
</name>
; <name><surname>Salvarezza</surname>
<given-names>R. C.</given-names>
</name>
<article-title>Spontaneously
formed sulfur adlayers on gold in electrolyte solutions: Adsorbed
sulfur or gold sulfide?</article-title>
. <source>J. Phys. Chem. C</source>
<year>2008</year>
, <volume>112</volume>
(<issue>30</issue>
), <fpage>11394</fpage>
–<lpage>11402</lpage>
. <pub-id pub-id-type="doi">10.1021/jp8029055</pub-id>
.</mixed-citation>
</ref>
<ref id="ref29"><mixed-citation publication-type="journal" id="cit29"><name><surname>Vericat</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Vela</surname>
<given-names>M. E.</given-names>
</name>
; <name><surname>Andreasen</surname>
<given-names>G.</given-names>
</name>
; <name><surname>Salvarezza</surname>
<given-names>R. C.</given-names>
</name>
; <name><surname>Vazquez</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Martin-Gago</surname>
<given-names>J. A.</given-names>
</name>
<article-title>Sulfur-substrate
interactions in
spontaneously formed sulfur adlayers on Au(111)</article-title>
. <source>Langmuir</source>
<year>2001</year>
, <volume>17</volume>
(<issue>16</issue>
), <fpage>4919</fpage>
–<lpage>4924</lpage>
. <pub-id pub-id-type="doi">10.1021/la0018179</pub-id>
.</mixed-citation>
</ref>
<ref id="ref30"><mixed-citation publication-type="journal" id="cit30"><name><surname>Koczkur</surname>
<given-names>K. M.</given-names>
</name>
; <name><surname>Hamed</surname>
<given-names>E. M.</given-names>
</name>
; <name><surname>Houmam</surname>
<given-names>A.</given-names>
</name>
<article-title>Sulfur multilayer formation on Au(111):
new insights from the study of hexamethyldisilathiane</article-title>
. <source>Langmuir</source>
<year>2011</year>
, <volume>27</volume>
(<issue>20</issue>
), <fpage>12270</fpage>
–<lpage>4</lpage>
. <pub-id pub-id-type="doi">10.1021/la202591j</pub-id>
.<pub-id pub-id-type="pmid">21916512</pub-id>
</mixed-citation>
</ref>
<ref id="ref31"><mixed-citation publication-type="journal" id="cit31"><name><surname>Castner</surname>
<given-names>D. G.</given-names>
</name>
; <name><surname>Hinds</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Grainger</surname>
<given-names>D. W.</given-names>
</name>
<article-title>X-ray photoelectron
spectroscopy
sulfur 2p study of organic thiol and disulfide binding interactions
with gold surfaces</article-title>
. <source>Langmuir</source>
<year>1996</year>
, <volume>12</volume>
(<issue>21</issue>
), <fpage>5083</fpage>
–<lpage>5086</lpage>
. <pub-id pub-id-type="doi">10.1021/la960465w</pub-id>
.</mixed-citation>
</ref>
<ref id="ref32"><mixed-citation publication-type="journal" id="cit32"><name><surname>Panagiotakos</surname>
<given-names>D. B.</given-names>
</name>
<article-title>Value of
p-value in biomedical research</article-title>
. <source>Open Cardiovasc.
Med. J.</source>
<year>2008</year>
, <volume>2</volume>
, <fpage>97</fpage>
–<lpage>99</lpage>
. <pub-id pub-id-type="doi">10.2174/1874192400802010097</pub-id>
.<pub-id pub-id-type="pmid">19430522</pub-id>
</mixed-citation>
</ref>
<ref id="ref33"><mixed-citation publication-type="journal" id="cit33"><name><surname>Thomsen</surname>
<given-names>V.</given-names>
</name>
; <name><surname>Schatzlein</surname>
<given-names>D.</given-names>
</name>
; <name><surname>Mercuro</surname>
<given-names>D.</given-names>
</name>
<article-title>Limits of detection in spectroscopy</article-title>
. <source>Spectroscopy.</source>
<year>2003</year>
, <volume>18</volume>
(<issue>12</issue>
), <fpage>112</fpage>
–<lpage>114</lpage>
.</mixed-citation>
</ref>
<ref id="ref34"><mixed-citation publication-type="journal" id="cit34"><name><surname>Min</surname>
<given-names>C.-K.</given-names>
</name>
; <name><surname>Cheon</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Ha</surname>
<given-names>N.-Y.</given-names>
</name>
; <name><surname>Sohn</surname>
<given-names>K. M.</given-names>
</name>
; <name><surname>Kim</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Aigerim</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Shin</surname>
<given-names>H. M.</given-names>
</name>
; <name><surname>Choi</surname>
<given-names>J.-Y.</given-names>
</name>
; <name><surname>Inn</surname>
<given-names>K.-S.</given-names>
</name>
; <name><surname>Kim</surname>
<given-names>J.-H. J.</given-names>
</name>
<article-title>Comparative and kinetic analysis of viral shedding
and immunological responses in MERS patients representing a broad
spectrum of disease severity</article-title>
. <source>Sci. Rep.</source>
<year>2016</year>
, <volume>6</volume>
, <fpage>25359</fpage>
<pub-id pub-id-type="doi">10.1038/srep25359</pub-id>
.<pub-id pub-id-type="pmid">27146253</pub-id>
</mixed-citation>
</ref>
<ref id="ref35"><mixed-citation publication-type="journal" id="cit35"><name><surname>Kim</surname>
<given-names>S.-K.</given-names>
</name>
; <name><surname>Sung</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Kim</surname>
<given-names>M.-N.</given-names>
</name>
<article-title>Kinetic
Studies and Infection Control
of Respiratory Viruses</article-title>
. <source>Korean J Healthc Assoc
Infect Control Prev.</source>
<year>2018</year>
, <volume>23</volume>
(<issue>1</issue>
), <fpage>1</fpage>
–<lpage>7</lpage>
. <pub-id pub-id-type="doi">10.14192/kjhaicp.2018.23.1.1</pub-id>
.</mixed-citation>
</ref>
<ref id="ref36"><mixed-citation publication-type="book" id="cit36"><person-group person-group-type="allauthors"><name><surname>Al-Abdely</surname>
<given-names>H. M.</given-names>
</name>
; <name><surname>Midgley</surname>
<given-names>C. M.</given-names>
</name>
; <name><surname>Alkhamis</surname>
<given-names>A. M.</given-names>
</name>
; <name><surname>Abedi</surname>
<given-names>G. R.</given-names>
</name>
; <name><surname>Tamin</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Binder</surname>
<given-names>A. M.</given-names>
</name>
; <name><surname>Alanazi</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Lu</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Abdalla</surname>
<given-names>O.</given-names>
</name>
; <name><surname>Sakthivel</surname>
<given-names>S. K.</given-names>
</name>
</person-group>
In <source>Infectious MERS-CoV Isolated From
a Mildly Ill Patient, Saudi Arabia.</source>
; <publisher-name>Open
Forum Infectious Diseases, Oxford University Press</publisher-name>
: <publisher-loc>US</publisher-loc>
, <year>2018</year>
; p <elocation-id>ofy111</elocation-id>
.</mixed-citation>
</ref>
<ref id="ref37"><mixed-citation publication-type="report" id="cit37"><person-group person-group-type="allauthors"><name><surname>Kraaij-Dirkzwager</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Timen</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Dirksen</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Gelinck</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Leyten</surname>
<given-names>E.</given-names>
</name>
; <name><surname>Groeneveld</surname>
<given-names>P.</given-names>
</name>
; <name><surname>Jansen</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Jonges</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Raj</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Thurkow</surname>
<given-names>I. J. E.</given-names>
</name>
</person-group>
, <article-title>Middle East respiratory syndrome
coronavirus (MERS-CoV) infections in two returning travellers in the
Netherlands</article-title>
, May <year>2014</year>
, <pub-id pub-id-type="doi">10.2807/1560-7917.ES2014.19.21.20817</pub-id>
.</mixed-citation>
</ref>
</ref-list>
</back>
</pmc>
</record>
Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000125 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000125 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien |wiki= Sante |area= MersV1 |flux= Pmc |étape= Corpus |type= RBID |clé= PMC:7119221 |texte= Development of Label-Free Colorimetric Assay for MERS-CoV Using Gold Nanoparticles }}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:31062580" \ | HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \ | NlmPubMed2Wicri -a MersV1
![]() | This area was generated with Dilib version V0.6.33. | ![]() |