TY - JOUR
T1 - Dip-pen microarraying of molecular beacon probes on microgel thin-film substrates
AU - Dai, Xiaoguang
AU - Libera, Matthew
N1 - Publisher Copyright:
© the Partner Organisations 2014.
PY - 2014
Y1 - 2014
N2 - The integration of microarray-based nucleic acid detection technologies and microfluidics is attractive, because the combination of small sample volumes, relatively short diffusion distances, and solid-phase detection enhances the development of multiplexed assays with improved sensitivity and minimal sample size. However, traditional microarray spotting methods typically create probe spot sizes of ∼50-100 μm diameter, comparable to the dimensions of many microfluidic channels. In addition, detection of hybridization events typically requires a post-hybridization labeling step. We address both issues by exploring the use of dip-pen nanolithography (DPN) to pattern linear oligonucleotides and self-reporting molecular beacon (MB) probes on streptavidin-functionalized poly(ethylene glycol) microgel thin-film substrates. In contrast to many systems involving DPN deposition, the fluorescence of the labeled probes enables their amount and spatial distribution to be characterized by optical microscopy. Their deposition rate decreases with increasing DPN dwell time, consistent with a Langmuir adsorption model, but the linear relationship between spot diameter and time1/2 indicates that spot size is diffusion controlled. We then use DPN to pattern MB probes for the mecA and spa genes in Staphylococcus aureus as a 2-column array with 1 μm spot sizes and 5 μm spot spacings, and we use this array to differentiate targets characteristic of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus. This duplexed self-reporting gel-tethered MB microarray not only shows high specificity but also a high signal-to-background ratio.
AB - The integration of microarray-based nucleic acid detection technologies and microfluidics is attractive, because the combination of small sample volumes, relatively short diffusion distances, and solid-phase detection enhances the development of multiplexed assays with improved sensitivity and minimal sample size. However, traditional microarray spotting methods typically create probe spot sizes of ∼50-100 μm diameter, comparable to the dimensions of many microfluidic channels. In addition, detection of hybridization events typically requires a post-hybridization labeling step. We address both issues by exploring the use of dip-pen nanolithography (DPN) to pattern linear oligonucleotides and self-reporting molecular beacon (MB) probes on streptavidin-functionalized poly(ethylene glycol) microgel thin-film substrates. In contrast to many systems involving DPN deposition, the fluorescence of the labeled probes enables their amount and spatial distribution to be characterized by optical microscopy. Their deposition rate decreases with increasing DPN dwell time, consistent with a Langmuir adsorption model, but the linear relationship between spot diameter and time1/2 indicates that spot size is diffusion controlled. We then use DPN to pattern MB probes for the mecA and spa genes in Staphylococcus aureus as a 2-column array with 1 μm spot sizes and 5 μm spot spacings, and we use this array to differentiate targets characteristic of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus. This duplexed self-reporting gel-tethered MB microarray not only shows high specificity but also a high signal-to-background ratio.
UR - http://www.scopus.com/inward/record.url?scp=84908330051&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84908330051&partnerID=8YFLogxK
U2 - 10.1039/c4an01220h
DO - 10.1039/c4an01220h
M3 - Article
AN - SCOPUS:84908330051
SN - 0003-2654
VL - 139
SP - 5568
EP - 5575
JO - Analyst
JF - Analyst
IS - 21
ER -