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Morpholino Oligonucleotide Crosslinked Hydrogels as Portable Optical Oligonucleotide Biosensors.Geraint J. Langford,a Jaclyn Raeburn,a David C. Ferrier,b Philip J. W. Hands,b,* and Michael P. Shavera,*a School of Chemistry, David Brewster Road, University of Edinburgh, Edinburgh, EH9 3FJ, UK. b Institute for Integrated Micro and Nano Systems, School of Engineering, University of Edinburgh, Edinburgh, EH9 3JL, UK.KEYWORDS: Morpholino oligonucleotide, Oligonucleotide sensor, Biosensor, Hydrogel, microRNA.ABSTRACT: Morpholino Oligonucleotides (MOs), an uncharged DNA analogue, are functionalized with an acrylamide moiety and incorporated into polymer hydrogels as responsive crosslinks for microRNA sequence detection. The MO crosslinks can be selectively cleaved by a short target analyte single-stranded DNA (ssDNA) sequence based on microRNA, inducing a distinct swelling response measured optically. The MO crosslinks offer significant improvement over DNA based systems through improved thermal stability, no salt requirement and 1000-fold improved sensitivity over a comparative biosensor, facilitating a wider range of sensing conditions. Analysis was also achieved using a mobile phone camera, demonstrating portability.Hydrogels are crosslinked hydrophilic polymers that can swell to absorb large volumes of water. Responsive hydrogels are important smart materials able to respond to external stimuli such as pH, temperature and a wide variety of other biomarkers.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1039/b714376a","ISBN":"1744-683X","ISSN":"1744683X","abstract":"Stimuli-responsive polymer gels have received considerable attention due to their singular mechanical properties, which make them materials of choice for niche applications. Polymer gels comprising either physical or chemical cross-links can undergo controlled and reversible shape changes in response to an applied field. The stimulus or external field applied may include thermal, electrical, magnetic, pH, UV/visible light, ionic or metallic interactions or combinations thereof. The shape change can manifest itself in two-dimensional actuation, bending motion, or three-dimensional actuation, volume change. This reversible contraction and expansion of polymer gels as well as their mechanical properties are similar to that of biological muscles. This review will describe and critique some of the recent advances in the field of stimuli-responsive polymer gels including the design of new classes of polymeric gels, controlled actuation in response to external stimuli, and ability to tailor material properties for different applications.","author":[{"dropping-particle":"","family":"Ahn","given":"Suk Kyun","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kasi","given":"Rajeswari M.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kim","given":"Seong Cheol","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sharma","given":"Nitin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhou","given":"Yuxiang","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Soft Matter","id":"ITEM-1","issued":{"date-parts":[["2008"]]},"title":"Stimuli-responsive polymer gels","type":"article-journal"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1039/c6py01585a","ISBN":"1748-605X (Electronic) 1748-6041 (Linking)","ISSN":"17599962","PMID":"19261988","abstract":"Responsive polymer-based materials are capable of altering their chemical and/or physical properties upon exposure to external stimuli. These materials have been intensively studied over the years for a diverse range of applications, e.g., for on-demand drug delivery, tissue generation/repair, biosensing, smart coatings, and artificial muscles. Here, we review recent advances in the areas of sensing and biosensing, drug delivery, and actuators. Specific examples are given in each of these areas, and we highlight our group's work on poly(N-isopropylacrylamide)-based microgels and assemblies.","author":[{"dropping-particle":"","family":"Wei","given":"Menglian","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gao","given":"Yongfeng","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Li","given":"Xue","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Serpe","given":"Michael J.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Polymer Chemistry","id":"ITEM-2","issued":{"date-parts":[["2017"]]},"title":"Stimuli-responsive polymers and their applications","type":"article"},"uris":[""]},{"id":"ITEM-3","itemData":{"DOI":"10.1038/21619","ISBN":"0028-0836","ISSN":"0028-0836","PMID":"10391240","abstract":"Stimuli-responsive hydrogels that undergo abrupt changes in volume in response to external stimuli such as pH, temperature and solvent composition have potential applications in biomedicine and the creation of 'intelligent' materials systems, for example as media for drug delivery, separation processes and protein immobilization. Hydrogels have been reported that respond to pH, temperature, electric fields and saccharides. For some biomedical applications it would be very useful to have a material whose swelling response was dictated by a specific protein. Here we report such a material, which swells reversibly in a buffer solution in response to a specific antigen. The hydrogel was prepared by grafting the antigen and corresponding antibody to the polymer network, so that binding between the two introduces crosslinks in the network. Competitive binding of the free antigen triggers a change in gel volume owing to breaking of these non-covalent crosslinks. In addition, we show that the hydrogel displays shape-memory behaviour, and that stepwise changes in antigen concentration can induce pulsatile permeation of a protein through the network.","author":[{"dropping-particle":"","family":"Miyata","given":"T","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Asami","given":"N","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Uragami","given":"T","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nature","id":"ITEM-3","issue":"June","issued":{"date-parts":[["1999"]]},"page":"766-769","title":"A reversibly antigen-responsive hydrogel.","type":"article-journal","volume":"399"},"uris":[""]},{"id":"ITEM-4","itemData":{"DOI":"10.1016/j.bios.2010.04.030","ISBN":"0956-5663","ISSN":"09565663","PMID":"20471821","abstract":"A new class of optical glucose nanobiosensors with high sensitivity and selectivity at physiological pH is described. To construct these glucose nanobiosensors, the fluorescent CdS quantum dots (QDs), serving as the optical code, were incorporated into the glucose-sensitive poly(N-isopropylacrylamide-acrylamide-2-acrylamidomethyl-5-fluorophenylboronic acid) copolymer microgels, via both in situ growth method and \" breathing in\" method, respectively. The polymeric gel can adapt to surrounding glucose concentrations, and regulate the fluorescence of the embedded QDs, converting biochemical signals into optical signals. The gradual swelling of the gel would lead to the quenching of the fluorescence at the elevated glucose concentrations. The hybrid microgels displayed high selectivity to glucose over the potential primary interferents of lactate and human serum albumin in the physiologically important glucose concentration range. The stability, reversibility, and sensitivity of the organic-inorganic hybrid microgel-based biosensors were also systematically studied. These general properties of our nanobiosensors are well tunable under appropriate tailor on the hybrid microgels, in particular, simply through the change in the crosslinking degree of the microgels. The optical glucose nanobiosensors based on the organic-inorganic hybrid microgels have shown the potential for a third generation fluorescent biosensor. ?? 2010 Elsevier B.V.","author":[{"dropping-particle":"","family":"Wu","given":"Weitai","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhou","given":"Ting","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Aiello","given":"Michael","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhou","given":"Shuiqin","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biosensors and Bioelectronics","id":"ITEM-4","issue":"12","issued":{"date-parts":[["2010"]]},"page":"2603-2610","publisher":"Elsevier B.V.","title":"Construction of optical glucose nanobiosensor with high sensitivity and selectivity at physiological pH on the basis of organic-inorganic hybrid microgels","type":"article-journal","volume":"25"},"uris":[""]},{"id":"ITEM-5","itemData":{"DOI":"10.1038/nmat2614","ISBN":"1476-1122","ISSN":"1476-1122","PMID":"20094081","abstract":"Responsive polymer materials can adapt to surrounding environments, regulate transport of ions and molecules, change wettability and adhesion of different species on external stimuli, or convert chemical and biochemical signals into optical, electrical, thermal and mechanical signals, and vice versa. These materials are playing an increasingly important part in a diverse range of applications, such as drug delivery, diagnostics, tissue engineering and 'smart' optical systems, as well as biosensors, microelectromechanical systems, coatings and textiles. We review recent advances and challenges in the developments towards applications of stimuli-responsive polymeric materials that are self-assembled from nanostructured building blocks. We also provide a critical outline of emerging developments.","author":[{"dropping-particle":"","family":"Stuart","given":"Martien A. Cohen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Huck","given":"Wilhelm T. S.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Genzer","given":"Jan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Müller","given":"Marcus","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ober","given":"Christopher","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stamm","given":"Manfred","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sukhorukov","given":"Gleb B.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Szleifer","given":"Igal","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"V.","family":"Tsukruk","given":"Vladimir","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Urban","given":"Marek","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Winnik","given":"Fran?oise","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zauscher","given":"Stefan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Luzinov","given":"Igor","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Minko","given":"Sergiy","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nature materials","id":"ITEM-5","issue":"2","issued":{"date-parts":[["2010","2","22"]]},"page":"101-113","publisher":"Nature Publishing Group","title":"Emerging applications of stimuli-responsive polymer materials.","type":"article-journal","volume":"9"},"uris":[""]}],"mendeley":{"formattedCitation":"^1–5","plainTextFormattedCitation":"1–5","previouslyFormattedCitation":"^1–5"},"properties":{"noteIndex":0},"schema":""}1–5 These networks have shown promise in various applications such as controlled drug release,ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.polymer.2008.01.027","ISBN":"0032-3861","ISSN":"00323861","PMID":"19799949","abstract":"There has been considerable progress in recent years in addressing the clinical and pharmacological limitations of hydrogels for drug delivery applications but substantial challenges remain. Here we discuss recent progress in overcoming these challenges, particularly with regards to effectively delivering hydrogels inside the body without implantation, prolonging the release kinetics of drugs from hydrogels, and expanding the nature of drugs which can be delivered using hydrogel-based approaches. ? 2008 Elsevier Ltd. All rights reserved.","author":[{"dropping-particle":"","family":"Hoare","given":"Todd R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kohane","given":"Daniel S.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Polymer","id":"ITEM-1","issued":{"date-parts":[["2008"]]},"title":"Hydrogels in drug delivery: Progress and challenges","type":"article"},"uris":[""]}],"mendeley":{"formattedCitation":"⁶","plainTextFormattedCitation":"6","previouslyFormattedCitation":"⁶"},"properties":{"noteIndex":0},"schema":""}6 wound healingADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"author":[{"dropping-particle":"","family":"Banerjee","given":"Indranil","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mishra","given":"Debasish","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Das","given":"Tamal","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Biomaterials Science","id":"ITEM-1","issue":"December","issued":{"date-parts":[["2012"]]},"page":"37-41","title":"Wound pH-Responsive Sustained Release of Therapeutics from a Poly ( NIPAAm-co-AAc ) Hydrogel","type":"article-journal"},"uris":[""]}],"mendeley":{"formattedCitation":"⁷","plainTextFormattedCitation":"7","previouslyFormattedCitation":"⁷"},"properties":{"noteIndex":0},"schema":""}7 and biosensing.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/cr0684467","ISBN":"0009-2665","ISSN":"00092665","PMID":"18095717","abstract":"---","author":[{"dropping-particle":"","family":"Sassolas","given":"Audrey","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Leca-Bouvier","given":"Béatrice D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Blum","given":"Lo?c J.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Chemical Reviews","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2008"]]},"page":"109-139","title":"DNA biosensors and microarrays","type":"article-journal","volume":"108"},"uris":[""]}],"mendeley":{"formattedCitation":"⁸","plainTextFormattedCitation":"8","previouslyFormattedCitation":"⁸"},"properties":{"noteIndex":0},"schema":""}8 DNA-based hydrogels using designed single-stranded DNA (ssDNA) sequences have been extensively investigated as responsive hydrogels, utilising nucleic acid Watson-Crick base pairing rules or DNA secondary structures to facilitate specific biorecognition.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.aca.2006.05.057","ISBN":"0003-2670","ISSN":"00032670","PMID":"17723692","abstract":"Selective capture and pre-concentration of target nucleic acids from relatively complicated samples may provide a method to facilitate introduction to a microfluidic-based detection system to improve detection limits. An acrylamide polymer gel modified with Acrydite??? that contained 20mer oligonucleotide probe was prepared and loaded into a capillary column. The results indicated that the amount of probe DNA that was captured into the acrylamide was about 40% of the starting monomer, and different quantities of probe could therefore be coupled into the gel. The gel was passivated by pre-treatment with non-complementary DNA oligonucleotide to block non-selective adsorption sites, and the gel was determined to be stable for multiple cycles of use. The probe could hybridize with target sequences that were introduced by electrokinetic injection from a sample solution. The target could be freed from the polymer gel by use of a combination of heating, chaotropic salt and voltage conditions. Target capture efficiency was up to 90% when using samples that did not saturate probe sites in the columns, and recovery of target from the gel could be as high as 95%. ?? 2006 Elsevier B.V. All rights reserved.","author":[{"dropping-particle":"","family":"Chan","given":"Andrew","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Krull","given":"Ulrich J.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Analytica Chimica Acta","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2006"]]},"page":"31-42","title":"Capillary electrophoresis for capture and concentrating of target nucleic acids by affinity gels modified to contain single-stranded nucleic acid probes","type":"article-journal","volume":"578"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1016/j.biomaterials.2012.09.040","ISBN":"0142-9612","ISSN":"01429612","PMID":"23083933","abstract":"Rare circulating tumor cells are a promising biomarker for the detection, diagnosis, and monitoring of cancer. However, it remains a challenge to develop biomedical devices for specific catch and nondestructive release of circulating tumor cells. The purpose of this study was to explore a unique system for cell catch and release by using aptamer-functionalized hydrogels and restriction endonucleases. The results show that the hydrogel coating was highly resistant to nonspecific cell binding with ～5-15 cells/mm2 on the hydrogel surface. In contrast, under the same condition, the aptamer-functionalized hydrogel coating could catch target cancer cells with a density over 1000 cells/mm2. When the hydrogel coating was further treated with the restriction endonucleases, the bound cells were released from the hydrogel coating because of the endonuclease-mediated sequence-specific hydrolysis of the aptamer sequences. The release efficiency reached ～99%. Importantly, ～98% of the released cells maintained viability. Taken together, this study demonstrates that it is promising to apply endonuclease-responsive aptamer-functionalized hydrogels as a coating material to develop medical devices for specific catch and nondestructive release of rare circulating tumor cells. ? 2012 Elsevier Ltd.","author":[{"dropping-particle":"","family":"Li","given":"Shihui","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chen","given":"Niancao","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhang","given":"Zhaoyang","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wang","given":"Yong","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biomaterials","id":"ITEM-2","issue":"2","issued":{"date-parts":[["2013"]]},"page":"460-469","publisher":"Elsevier Ltd","title":"Endonuclease-responsive aptamer-functionalized hydrogel coating for sequential catch and release of cancer cells","type":"article-journal","volume":"34"},"uris":[""]},{"id":"ITEM-3","itemData":{"DOI":"10.1039/c1sm05284e","ISBN":"1744-683X","ISSN":"1744-683X","abstract":"Hydrogels are crosslinked hydrophilic polymers that undergo swelling in water. The gel volume is affected by many environmental parameters including temperature, pH, ionic strength, and solvent composition. Therefore, these factors have been traditionally used for making smart hydrogels. DNA, on the other hand, is a special block copolymer. Incorporation of DNA within a hydrogel network can have several important effects. For example, DNA can serve as a reversible crosslinker modulating the mechanical and rheological properties of a hydrogel. Second, DNA can selectively bind to a variety of different molecules. Attaching these binding DNAs (aptamers) to hydrogel makes it possible to expand the range of stimuli to chemical and biological molecules. At the same time, the gel matrix can also improve DNA-based sensors and materials. For example, the hydrogel can be dried for storage and rehydrated prior to use and the immobilized DNAs are protected from nuclease cleavage. The gel backbone property can also be tuned to affect the interaction between DNA and other molecules. The rational functionalization of DNA in hydrogels has generated a diverse range of smart materials and biosensors. In the last 15 years, the field has made tremendous progress and some of the recent developments are summarized in this review. Challenges and possible future directions are also discussed.","author":[{"dropping-particle":"","family":"Liu","given":"Juewen","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Soft Matter","id":"ITEM-3","issued":{"date-parts":[["2011"]]},"page":"6757","title":"Oligonucleotide-functionalized hydrogels as stimuli responsive materials and biosensors","type":"article-journal","volume":"7"},"uris":[""]},{"id":"ITEM-4","itemData":{"DOI":"10.1002/marc.201300411","ISBN":"1022-1336; 1521-3927","ISSN":"10221336","PMID":"23857726","abstract":"The term hydrogel describes a type of soft and wet material formed by cross-linked hydrophilic polymers. The distinct feature of hydrogels is their ability to absorb a large amount of water and swell. The properties of a hydrogel are usually determined by the chemical properties of their constituent polymer(s). However, a group of hydrogels, called \"smart hydrogels,\" changes properties in response to environmental changes or external stimuli. Recently, DNA or DNA-inspired responsive hydrogels have attracted considerable attention in construction of smart hydrogels because of the intrinsic advantages of DNA. As a biological polymer, DNA is hydrophilic, biocompatible, and highly programmable by Watson-Crick base pairing. DNA can form a hydrogel by itself under certain conditions, and it can also be incorporated into synthetic polymers to form DNA-hybrid hydrogels. Functional DNAs, such as aptamers and DNAzymes, provide additional molecular recognition capabilities and versatility. In this Review, DNA-based hydrogels are discussed in terms of their stimulus response, as well as their applications.","author":[{"dropping-particle":"","family":"Xiong","given":"Xiangling","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wu","given":"Cuichen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhou","given":"Cuisong","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhu","given":"Guizhi","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chen","given":"Zhuo","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tan","given":"Weihong","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Macromolecular Rapid Communications","id":"ITEM-4","issue":"16","issued":{"date-parts":[["2013"]]},"page":"1271-1283","title":"Responsive DNA-based hydrogels and their applications","type":"article-journal","volume":"34"},"uris":[""]}],"mendeley":{"formattedCitation":"^9–12","plainTextFormattedCitation":"9–12","previouslyFormattedCitation":"^9–12"},"properties":{"noteIndex":0},"schema":""}9–12Pure DNA hydrogels have been synthesised using 3- or 4- way crosslinks, linked using T4 DNA ligase to form networked DNA, and subsequently shown to release a variety of drugs, proteins or cells during DNA degradation.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1038/nmat1741","ISBN":"1476-1122","ISSN":"14764660","PMID":"16998469","abstract":"DNA is a remarkable polymer that can be manipulated by a large number of molecular tools including enzymes. A variety of geometric objects, periodic arrays and nanoscale devices have been constructed. Previously we synthesized dendrimer-like DNA and DNA nanobarcodes from branched DNA via ligases. Here we report the construction of a hydrogel entirely from branched DNA that are three-dimensional and can be crosslinked in nature. These DNA hydrogels were biocompatible, biodegradable, inexpensive to fabricate and easily moulded into desired shapes and sizes. The distinct difference of the DNA hydrogel to other bio-inspired hydrogels (including peptide-based, alginate-based and DNA (linear)-polyacrylamide hydrogels) is that the crosslinking is realized via efficient, ligase-mediated reactions. The advantage is that the gelling processes are achieved under physiological conditions and the encapsulations are accomplished in situ-drugs including proteins and even live mammalian cells can be encapsulated in the liquid phase eliminating the drug-loading step and also avoiding denaturing conditions. Fine tuning of these hydrogels is easily accomplished by adjusting the initial concentrations and types of branched DNA monomers, thus allowing the hydrogels to be tailored for specific applications such as controlled drug delivery, tissue engineering, 3D cell culture, cell transplant therapy and other biomedical applications.","author":[{"dropping-particle":"","family":"Um","given":"Soong Ho","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lee","given":"Jong Bum","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Park","given":"Nokyoung","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kwon","given":"Sang Yeon","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Umbach","given":"Christopher C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Luo","given":"Dan","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nature Materials","id":"ITEM-1","issue":"10","issued":{"date-parts":[["2006"]]},"page":"797-801","title":"Enzyme-catalysed assembly of DNA hydrogel","type":"article-journal","volume":"5"},"uris":[""]}],"mendeley":{"formattedCitation":"¹³","plainTextFormattedCitation":"13","previouslyFormattedCitation":"¹³"},"properties":{"noteIndex":0},"schema":""}13 In sensing applications, acrydite-modified ssDNA was immobilised in polyacrylamide chains that, when mixed with partially complementary ssDNA that forms a crosslink, has been used to bring about gelation which can then be reversed in response to an analyte that displaces the DNA crosslink.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/ja801339w","ISBN":"0002-7863","ISSN":"1520-5126","PMID":"18444626","abstract":"In this communication, we report a simple, but highly adaptable, method of constructing selective target-responsive hydrogels using DNA aptamers. The simplicity of the design is accomplished by using linear polymer chains as the hydrogel backbone and a DNA aptamer as the cross-linker. in this design, competitive binding of target to the aptamer causes the decrease of cross-linking density and, hence, dissolution of the hydrogel. The adaptability of this strategy for therapeutic applications was demonstrated using two different types of targets, small molecules and proteins. Our results indicated that this molecular engineering provides a highly selective and controllable release system whereby efficient release of therapeutic agents can occur at specific environments in which the target biomarker is found.","author":[{"dropping-particle":"","family":"Yang","given":"H H","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Liu","given":"H P","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kang","given":"H Z","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tan","given":"W H","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of the American Chemical Society","id":"ITEM-1","issue":"20","issued":{"date-parts":[["2008"]]},"page":"6320-6321","title":"Engineering target-responsive hydrogels based on aptamer - Target interactions","type":"article-journal","volume":"130"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1016/0966-7822(96)00001-9","ISBN":"0966-7822","ISSN":"09667822","abstract":"Succinimido-copolymers, poly(N,N-dimethylacrylamide-co-N-acryloyloxysuccinimide)s, were coupled with 5′-terminal-amino-modified oligodeoxyribonudeotides (ODNs) to produce water-soluble copolymers partially derivatized with ODNs in their side chains. The mixing and thermal melting measurements of dilute mixed aqueous solutions of an ODN-derivatized copolymer and their complementary ODN and mixed solutions of complementary ODN-derivatized copolymers were monitored by ultraviolet spectroscopy. The results showed that hybrids were formed with their complementary ODNs at room temperature, but dissociated at high temperature. Based on the hybridization between complementary base pairs of nucleic acids and its thermal dissociation characteristics, two types of thermoresponsive hydrogels were prepared: (1) a hydrogel formed via hybridization between an oligodeoxythymidylate (oligoT)-derivatized copolymer and an oligodeoxyadenylate (oligoA), and (2) a hydrogel formed by hybridization between complementary oligoT- and oligoA-derivatized copolymers. Thus, self-association due to specific intermolecular hydrogen bonding between nucleic acid base pairs enabled the preparation of a novel thermoresponsive hydrogel. Copyright ? 1996 Elsevier Science Ltd.","author":[{"dropping-particle":"","family":"Nagahara","given":"Shunji","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Matsuda","given":"Takehisa","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Polymer Gels and Networks","id":"ITEM-2","issue":"2","issued":{"date-parts":[["1996"]]},"page":"111-127","title":"Hydrogel formation via hybridization of oligonucleotides derivatized in water-soluble vinyl polymers","type":"article-journal","volume":"4"},"uris":[""]}],"mendeley":{"formattedCitation":"^14,15","plainTextFormattedCitation":"14,15","previouslyFormattedCitation":"^14,15"},"properties":{"noteIndex":0},"schema":""}14,15 One strand of the crosslink is fully complementary to the analyte sequence, such that the analyte will displace the partially complementary strand and break the crosslink (Fig 1a and b). Similarly, partially complementary acrydite-modified ssDNA crosslinks have been copolymerised with acrylamide and a covalent crosslinker, N,N′-methylenebis(acrylamide) (MBA), to form gels to detect short DNA sequences.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/bm900218c","ISBN":"1525-7797","ISSN":"15257797","PMID":"19425572","abstract":"Development of an oligonucleotide functionalized hydrogel integrated on a high resolution interferometric readout platform capable of determining changes in optical length of the hydrogel with 2 nm resolution is described. The hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network making up a network junction point in addition to the covalent cross-links. The hybridized dioligonucleotide network junctions were made with a 10 basepair complementary region flanked by additional basepairs that could aid in destabilizing the junction points in competitive displacement hybridization by the added probe oligonucleotides. The probe oligonucleotide destabilizing the junction point thus induces swelling of the functionalized hydrogel that is sensitive to the concentration of the probe, the sequence, and matching length between the probe and sensing oligonucleotide. This design yields a molecular amplification of the change in the optical length of the gel at least 5-fold compared to a hydrogel where sensing functionality is based on hybridization with a grafted oligonucleotide that is not a part of a network junction. Concentration sensitivity applied for specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. Applications of the resulting oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequences designed with aptamer functionalities for determination of other types of molecules are discussed.","author":[{"dropping-particle":"","family":"Tierney","given":"Sven","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stokke","given":"Bj?rn Torger","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biomacromolecules","id":"ITEM-1","issue":"6","issued":{"date-parts":[["2009"]]},"page":"1619-1626","title":"Development of an oligonucleotide functionalized hydrogel integrated on a high resolution interferometric readout platform as a label-free macromolecule sensing device","type":"article-journal","volume":"10"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1039/c1sm05221g","ISBN":"1744-683X","ISSN":"1744-683X","abstract":"An oligonucleotide-polymer hybrid hydrogel displaying swelling response in a logical AND and OR fashion depending on specific stimuli by probe oligonucleotides was prepared. The hydrogel acts as a transducer of the specific recognition of DNA sequences into micro- and macroscale mechanics. Input oligonucleotide probes induce hydrogel swelling ratio at two signal levels.","author":[{"dropping-particle":"","family":"Gawe","given":"Kamila","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stokke","given":"Bj?rn Torger","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Soft Matter","id":"ITEM-2","issued":{"date-parts":[["2011"]]},"page":"4615-4618","title":"Logic swelling response of DNA–polymer hybrid hydrogel","type":"article-journal","volume":"7"},"uris":[""]},{"id":"ITEM-3","itemData":{"DOI":"10.1016/j.sbsr.2017.11.007","ISSN":"22141804","abstract":"A new method for oligonucleotide detection is presented based on oligonucleotide cross-linked polymer composites. Conductive carbon nanoparticles are incorporated into a DNA-functionalised polymer, containing partially complementary oligonucleotide cross-linkers, which is polymerised in situ upon interdigitated electrodes. In the presence of an aqueous solution of a specific analyte oligonucleotide sequence, the cross-linkers are cleaved, leading to increased swelling. As the polymer swells the relative density of the conductive particles decreases, leading to an easily measurable decrease in electrical conductivity. We demonstrate that such are capable of discriminating between analyte and control solutions, with single-base specificity, in under 3 min. The lower detection limit of these composites is of the order of 10 nM. The swelling characteristics of these composites is confirmed by optical imaging and the effects of varying temperature upon such composites are also reported.","author":[{"dropping-particle":"","family":"Ferrier","given":"David C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shaver","given":"Michael P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hands","given":"Philip J.W.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Sensing and Bio-Sensing Research","id":"ITEM-3","issue":"August 2017","issued":{"date-parts":[["2018"]]},"page":"1-6","publisher":"Elsevier","title":"Conductive composites for oligonucleotide detection","type":"article-journal","volume":"17"},"uris":[""]}],"mendeley":{"formattedCitation":"^16–18","plainTextFormattedCitation":"16–18","previouslyFormattedCitation":"^16–18"},"properties":{"noteIndex":0},"schema":""}16–18 In this case, there is no gel-sol transition due to the covalent crosslinks, rather that reduction in crosslink density allows the gel to absorb more water and swell to a greater volume (Fig 1c).DNA crosslinked hydrogels have several inherent limitations including thermal denaturation, solvent interactions and a salt requirement.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1038/nbt.3121.ChIP-nexus","ISBN":"0000000000000","ISSN":"1527-5418","PMID":"24655651","author":[{"dropping-particle":"","family":"Qiao","given":"Wanqiong","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chiang","given":"Hao-Chun","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Xie","given":"Hui","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Levicky","given":"Rastislav","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Chemical communications (Cambridge, England)","id":"ITEM-1","issue":"97","issued":{"date-parts":[["2015"]]},"page":"17245-17248","title":"Surface vs Solution Hybridization: Effects of Salt, Temperature and Probe Type","type":"article-journal","volume":"51"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁹","plainTextFormattedCitation":"19","previouslyFormattedCitation":"¹⁹"},"properties":{"noteIndex":0},"schema":""}19 These issues are exacerbated when using short ssDNA sequences (ca. 22 nucleotides) as required for microRNA (miRNA) detection.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1371/journal.pone.0005532","ISBN":"1932-6203","ISSN":"19326203","PMID":"19440243","abstract":"BACKGROUND: MicroRNAs are a family of 19- to 25-nucleotides noncoding small RNAs that primarily function as gene regulators. Aberrant microRNA expression has been described for several human malignancies, and this new class of small regulatory RNAs has both oncogenic and tumor suppressor functions. Despite this knowledge, there is little information regarding microRNAs in plasma especially because microRNAs in plasma, if exist, were thought to be digested by RNase. Recent studies, however, have revealed that microRNAs exist and escape digestion in plasma.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: We performed microRNA microaray to obtain insight into microRNA deregulation in the plasma of a leukemia patient. We have revealed that microRNA-638 (miR-638) is stably present in human plasmas, and microRNA-92a (miR-92a) dramatically decreased in the plasmas of acute leukemia patients. Especially, the ratio of miR-92a/miR-638 in plasma was very useful for distinguishing leukemia patients from healthy body.\\n\\nCONCLUSIONS/SIGNIFICANCE: The ratio of miR-92a/miR-638 in plasma has strong potential for clinical application as a novel biomarker for detection of leukemia.","author":[{"dropping-particle":"","family":"Tanaka","given":"Masami","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Oikawa","given":"Kosuke","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Takanashi","given":"Masakatsu","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kudo","given":"Motoshige","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ohyashiki","given":"Junko","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ohyashiki","given":"Kazuma","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kuroda","given":"Masahiko","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"PLoS ONE","id":"ITEM-1","issue":"5","issued":{"date-parts":[["2009"]]},"page":"1-5","title":"Down-regulation of miR-92 in human plasma is a novel marker for acute leukemia patients.","type":"article-journal","volume":"4"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1073/pnas.0804549105","ISBN":"1091-6490 (Electronic)\\n0027-8424 (Linking)","ISSN":"1091-6490","PMID":"18663219","abstract":"Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small ( approximately 22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.","author":[{"dropping-particle":"","family":"Mitchell","given":"Patrick S","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Parkin","given":"Rachael K","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kroh","given":"Evan M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Fritz","given":"Brian R","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wyman","given":"Stacia K","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pogosova-Agadjanyan","given":"Era L","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Peterson","given":"Amelia","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Noteboom","given":"Jennifer","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Briant","given":"Kathy C","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Allen","given":"April","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lin","given":"Daniel W","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Urban","given":"Nicole","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Drescher","given":"Charles W","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Knudsen","given":"Beatrice S","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stirewalt","given":"Derek L","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gentleman","given":"Robert","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Vessella","given":"Robert L","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nelson","given":"Peter S","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Martin","given":"Daniel B","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tewari","given":"Muneesh","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Proceedings of the National Academy of Sciences of the United States of America","id":"ITEM-2","issue":"30","issued":{"date-parts":[["2008"]]},"page":"10513-8","title":"Circulating microRNAs as stable blood-based markers for cancer detection.","type":"article-journal","volume":"105"},"uris":[""]}],"mendeley":{"formattedCitation":"^20,21","plainTextFormattedCitation":"20,21","previouslyFormattedCitation":"^20,21"},"properties":{"noteIndex":0},"schema":""}20,21 miRNA have garnered substantial attention as potential biomarkers able to differentiate a wide variety of pathologies.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1002/jcp.25056","ISBN":"1097-4652 (Electronic)\\r0021-9541 (Linking)","ISSN":"10974652","PMID":"26031493","abstract":"MicroRNAs (miRNAs) are a group of small non-coding RNAs that are involved in regulating a range of developmental and physiological processes; their dysregulation has been associated with development of diseases including cancer. Circulating miRNAs and exosomal miRNAs have also been proposed as being useful in diagnostics as biomarkers for diseases and different types of cancer. In this review, miRNAs are discussed as biomarkers for cancer and other diseases, including viral infections, nervous system disorders, cardiovascular disorders, and diabetes. We summarize some of the clinical evidence for the use of miRNAs as biomarkers in diagnostics and provide some general perspectives on their use in clinical situations. The analytical challenges in using miRNAs in cancer and disease diagnostics are evaluated and discussed. Validation of specific miRNA signatures as biomarkers is a critical milestone in diagnostics. This article is protected by copyright. All rights reserved.","author":[{"dropping-particle":"","family":"Wang","given":"Jin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chen","given":"Jinyun","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sen","given":"Subrata","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Cellular Physiology","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2016"]]},"page":"25-30","title":"MicroRNA as Biomarkers and Diagnostics","type":"article-journal","volume":"231"},"uris":[""]}],"mendeley":{"formattedCitation":"²²","plainTextFormattedCitation":"22","previouslyFormattedCitation":"²²"},"properties":{"noteIndex":0},"schema":""}22 In particular, circulating miRNA (either as free miRNA or contained in cell secreted exosomes) from saliva, urine, blood or other extracellular fluids have become a target for biosensor design.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.3390/s140509117","ISBN":"1424-8220","ISSN":"14248220","PMID":"24858962","abstract":"MicroRNAs or miRNAs are a form of small non-coding RNAs (ncRNAs) of 19-22 nucleotides in length in their mature form. miRNAs are transcribed in the nucleus of all cells from large precursors, many of which have several kilobases in length. Originally identified as intracellular modulators of protein synthesis via posttranscriptional gene silencing, more recently it has been found that miRNAs can travel in extracellular human fluids inside specialized vesicles known as exosomes. We will be referring to this miRNAs as circulating microRNAs. More interestingly, the miRNA content inside exosomes changes during pathological events. In the present review we analyze the literature about circulating miRNAs and their possible use as biomarkers. Furthermore, we explore their future in point-of-care (POC) diagnostics and provide an example of a portable POC apparatus useful in the detection of circulating miRNAs.","author":[{"dropping-particle":"","family":"Lubbe","given":"G.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Die Sonde","id":"ITEM-1","issue":"1","issued":{"date-parts":[["1969"]]},"page":"10","title":"Point-of-care Diagnostic Tools to Detect Circulating MicroRNAS as Biomarkers of Disease","type":"article-journal","volume":"11"},"uris":[""]}],"mendeley":{"formattedCitation":"²³","plainTextFormattedCitation":"23","previouslyFormattedCitation":"²³"},"properties":{"noteIndex":0},"schema":""}23 Despite this, difficulties in detection remain, in particular due to the low concentrations of miRNA (~1 pM - ~10 fM in serum).ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1007/s00216-013-7459-z","ISSN":"16182642","PMID":"24247551","abstract":"MicroRNAs (miRNAs), new stars of human genetics, are naturally occurring, 19-25 base pair, noncoding RNAs that regulate gene expression posttranscriptionally and have been demonstrated to be excellent biomarkers for cancer diagnosis/prognosis. Because of their short length, sequence similarity, and very low concentration, their detection in real samples is challenging. Among other methods for miRNA detection, electrochemical nucleic acid biosensors exhibit relevant advantages in terms of high sensitivity, ease of use, short assay time, nontoxic experimental steps, and adaptability to point-of-care testing. This article gives a brief overview of recent advances in the rapidly developing area of electrochemical biosensors for miRNA detection. The fundamentals of the different strategies developed to achieve novel signal amplification and sensitive electrochemical detection are discussed, and some examples of relevant approaches are highlighted, along with future prospects and challenges.","author":[{"dropping-particle":"","family":"Campuzano","given":"Susana","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pedrero","given":"María","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pingarrón","given":"José M.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Analytical and Bioanalytical Chemistry","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2014"]]},"page":"27-33","title":"Electrochemical genosensors for the detection of cancer-related miRNAs","type":"article-journal","volume":"406"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1002/med","ISBN":"0125-2208","ISSN":"01252208","PMID":"23140041","abstract":"Acomprehensive focus on 4-hydroxynonenal (HNE) as candidate molecule in a variety of pathophysiological conditions occurring inhumans is here provided. Despite an active,nowwell characterized, metabolism in most cells and tissues, HNE can be easily detected and quantified by means of several methods, although with different sensitivity. Measurements ofHNEand/or stable metabolites in biological fluids are already applied as lipid peroxidation/oxidative stress markers in a huge number of human disease processes, often sustained by inflammatory reactions.Aprimary involvement of this aldehydic product of membrane lipid oxidation in inflammation-related events, as well as in regulation of cell proliferation and growth, in necrotic or apoptotic cell death, appears supported by its marked ability to modulate several major pathways of cell signaling and, consequently, gene expression. The actual knowledge of HNE reactivity, metabolism, signaling and modulatory effect in the various human organs should provide a solid background to the investigation of the aldehyde’s contribution to the pathogenesis of human major chronic diseases and would likely promote advanced and oriented applications not only in diagnosis and prevention but also in molecular treatment of human diseases.?2007 Wiley Periodicals, Inc. Med Res Rev, 28, No. 4, 569–631, 2008 Key","author":[{"dropping-particle":"","family":"Zen","given":"Ke","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhang","given":"Chen-Yu","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Medicinal Research Reviews","id":"ITEM-2","issue":"2","issued":{"date-parts":[["2012"]]},"page":"326-348","title":"Circulating MicroRNAs: A Novel Class of Biomarkers to Diagnose and Monitor Human Cancers","type":"article-journal","volume":"32"},"uris":[""]}],"mendeley":{"formattedCitation":"^24,25","plainTextFormattedCitation":"24,25","previouslyFormattedCitation":"^24,25"},"properties":{"noteIndex":0},"schema":""}24,25 Established miRNA detection methods such as RT-qPCR and microarrays (Limit of detection (LoD) fM - aM and nM - pM respectively)ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1039/c3an01677c","ISBN":"0003-2654","ISSN":"0003-2654","abstract":"The current state of biosensor-based techniques for amplification-free microRNA (miRNA) detection is critically reviewed. Comparison with non-sensor and amplification-based molecular techniques (MTs), such as polymerase-based methods, is made in terms of transduction mechanism, associated protocol, and sensitivity. Challenges associated with miRNA hybridization thermodynamics which affect assay selectivity and amplification bias are briefly discussed. Electrochemical, electromechanical, and optical classes of miRNA biosensors are reviewed in terms of transduction mechanism, limit of detection (LOD), time-to-results (TTR), multiplexing potential, and measurement robustness. Current trends suggest that biosensor-based techniques (BTs) for miRNA assay will complement MTs due to the advantages of amplification-free detection, LOD being femtomolar (fM)-attomolar (aM), short TTR, multiplexing capability, and minimal sample preparation requirement. Areas of future importance in miRNA BT development are presented which include focus on achieving high measurement confidence and multiplexing capabilities.","author":[{"dropping-particle":"","family":"Johnson","given":"Blake N.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mutharasan","given":"Raj","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Analyst","id":"ITEM-1","issue":"7","issued":{"date-parts":[["2014"]]},"page":"1576-1589","title":"Biosensor-based microRNA detection: techniques, design, performance, and challenges","type":"article-journal","volume":"139"},"uris":[""]}],"mendeley":{"formattedCitation":"²⁶","plainTextFormattedCitation":"26","previouslyFormattedCitation":"²⁶"},"properties":{"noteIndex":0},"schema":""}26offer high sensitivity and multiplexing, yet typically require centralised labs, expensive reagents and relatively long times to results (1 day or more). Techniques reliant on fluorescence (LoD nM - fM)ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/cr0684467","ISBN":"0009-2665","ISSN":"00092665","PMID":"18095717","abstract":"---","author":[{"dropping-particle":"","family":"Sassolas","given":"Audrey","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Leca-Bouvier","given":"Béatrice D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Blum","given":"Lo?c J.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Chemical Reviews","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2008"]]},"page":"109-139","title":"DNA Biosensors and Microarrays","type":"article-journal","volume":"108"},"uris":[""]}],"mendeley":{"formattedCitation":"²⁷","plainTextFormattedCitation":"27","previouslyFormattedCitation":"²⁷"},"properties":{"noteIndex":0},"schema":""}27 have issues such as bleaching or auto-fluorescence, while microelectromechanical systems (MEMS) or nanoelectromechanical systems (NEMS) (LoD aM - ?M)ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.bios.2015.01.031","ISSN":"09565663","author":[{"dropping-particle":"","family":"Ferrier","given":"David C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shaver","given":"Michael P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hands","given":"Philip J.W.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biosensors and Bioelectronics","id":"ITEM-1","issued":{"date-parts":[["2015"]]},"page":"798-810","publisher":"Elsevier","title":"Micro- and nano-structure based oligonucleotide sensors","type":"article-journal","volume":"68"},"uris":[""]}],"mendeley":{"formattedCitation":"²⁸","plainTextFormattedCitation":"28","previouslyFormattedCitation":"²⁸"},"properties":{"noteIndex":0},"schema":""}28 typically have a relatively complex fabrication process.Recent work showed the benefits of using synthetic DNA analogues in hydrogels, utilising a mixture of peptide nucleic acid (PNA) in a “hybrid” crosslink with ssDNA to form crosslinked salt-free hydrogels,ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1161/CIRCRESAHA.116.303790.The","ISBN":"0324141122","ISSN":"1527-5418","PMID":"24655651","author":[{"dropping-particle":"","family":"Chu","given":"Te-Wei","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Feng","given":"Jiayue","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Yang","given":"Jiyuan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kope?ek","given":"J.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Controlled Release","id":"ITEM-1","issued":{"date-parts":[["2015"]]},"page":"608-616","title":"Hybrid Polymeric Hydrogels via Peptide Nucleic Acid (PNA)/DNA Complexation Te-Wei","type":"article-journal","volume":"220, Part"},"uris":[""]}],"mendeley":{"formattedCitation":"²⁹","plainTextFormattedCitation":"29","previouslyFormattedCitation":"²⁹"},"properties":{"noteIndex":0},"schema":""}29 although some challenges remain. We investigate herein Morpholino Oligonucleotide (MO) crosslinked hydrogels. MOs are synthetic DNA analogues with an uncharged backbone consisting of morpholine rings connected by phosphorodiamidate groups (Figure 1d).ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"author":[{"dropping-particle":"","family":"Summerton","given":"J","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Drug Discovery, Development and Delivery","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2016"]]},"page":"1-7","title":"History and Properties of Morpholino Antisense Oligos","type":"article-journal","volume":"3"},"uris":[""]}],"mendeley":{"formattedCitation":"³⁰","plainTextFormattedCitation":"30","previouslyFormattedCitation":"³⁰"},"properties":{"noteIndex":0},"schema":""}30 This uncharged backbone results in stronger DNA binding and reduced salt dependence whilst maintaining high water solubility and avoiding enzymatic degradation.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1007/BF02484560","ISBN":"978-0-306-48230-4","ISSN":"09295666","author":[{"dropping-particle":"","family":"Summerton","given":"James E.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Letters in Peptide Science","id":"ITEM-1","issue":"3-4","issued":{"date-parts":[["2003"]]},"page":"215-236","title":"Morpholinos and PNAs compared","type":"article-journal","volume":"10"},"uris":[""]}],"mendeley":{"formattedCitation":"³¹","plainTextFormattedCitation":"31","previouslyFormattedCitation":"³¹"},"properties":{"noteIndex":0},"schema":""}31 The increased rigidity of the backbone also reduces self-hybridisation. The benefits of MOs have been assessed in electrochemical studiesADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1149/1.3571981","ISBN":"9781607682189","ISSN":"19385862 19386737","abstract":"Surface hybridization, in which nucleic acids from solution bind to complementary \"probe\" strands immobilized on a solid support, is widely used to analyze composition of nucleic acid mixtures. Most often, detection is accomplished with fluorescent techniques whose sensitivity can be extended down to individual molecules. Applications, however, benefit as much if not more from convenience, accuracy, and affordability of the diagnostic test. By eliminating the need for fluorescent labeling and more complex sample workup, label-free electrochemical assays have significant advantages provided transduction remains sufficiently sensitive for applications. To this end, we have been exploring morpholinos, which are uncharged DNA analogues, as the immobilized probe species in surface hybridization assays based on measurement of interfacial capacitance. Through comparison of experimental trends with those predicted from basic physical models, the origins of diagnostic contrast in capacitive sensing are reviewed for assays based on morpholino as well as on DNA probes. ?The Electrochemical Society.","author":[{"dropping-particle":"","family":"O'Connor","given":"R.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tercero","given":"N.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Qiao","given":"W.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Levicky","given":"R.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"ECS Transactions","id":"ITEM-1","issue":"7","issued":{"date-parts":[["2011"]]},"page":"99-110","title":"Electrochemical studies of morpholino-DNA surface hybridization","type":"article-journal","volume":"35"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1021/ja202631b","ISBN":"0002-7863","ISSN":"00027863","PMID":"21699181","abstract":"Morpholinos (MOs) are DNA analogues whose uncharged nature can bring fundamental advantages to surface hybridization technologies such as DNA microarrays, by using MOs as the immobilized, or \"probe\", species. Advancement of MO-based diagnostics, however, is challenged by limited understanding of the surface organization of MO molecules and of how this organization impacts hybridization kinetics and thermodynamics. The present study focuses on hybridization kinetics between monolayers of MO probes and DNA targets as a function of the instantaneous extent of hybridization (i.e., duplex coverage), total probe coverage, and ionic strength. Intriguingly, these experiments reveal distinct kinetic stages, none of which are consistent with Langmuir kinetics. The initial stage, in which duplex coverage remains relatively sparse, indicates confluence of two effects: blockage of target access to unhybridized probes by previously formed duplexes and deactivation of the solid support due to consumption of probe molecules. This interpretation is consistent with a surface organization in which unhybridized MO probes localize near the solid support, underneath a layer of MO-DNA duplexes. As duplex coverage builds, provided saturation is not reached first, the initial stage can transition to an unusual regime characterized by near independence of hybridization rate on duplex coverage, followed by a prolonged approach to equilibrium. The possible origins of these more complex latter behaviors are discussed. Comparison with published data for DNA and peptide nucleic acid (PNA) probes is carried out to look for universal trends in kinetics. This comparison reveals qualitative similarities when comparable surface organization of probes is expected. In addition, MO monolayers are found capable of a broad range of reactivities that span reported values for PNA and DNA probes.","author":[{"dropping-particle":"","family":"Liu","given":"Yatao","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Irving","given":"Damion","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Qiao","given":"Wanqiong","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ge","given":"Dongbiao","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Levicky","given":"Rastislav","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of the American Chemical Society","id":"ITEM-2","issue":"30","issued":{"date-parts":[["2011"]]},"page":"11588-11596","title":"Kinetic mechanisms in morpholino-DNA surface hybridization","type":"article-journal","volume":"133"},"uris":[""]}],"mendeley":{"formattedCitation":"^32,33","plainTextFormattedCitation":"32,33","previouslyFormattedCitation":"^32,33"},"properties":{"noteIndex":0},"schema":""}32,33 and microarraysADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/nl061786n.Core-Shell","ISBN":"0009-2665","ISSN":"00092665","PMID":"19772347","author":[{"dropping-particle":"","family":"Borja-Cacho","given":"Daniel","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Matthews","given":"Jeffrey","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nano","id":"ITEM-1","issue":"9","issued":{"date-parts":[["2008"]]},"page":"2166-2171","title":"NIH Public Access","type":"article-journal","volume":"6"},"uris":[""]}],"mendeley":{"formattedCitation":"³⁴","plainTextFormattedCitation":"34","previouslyFormattedCitation":"³⁴"},"properties":{"noteIndex":0},"schema":""}34 but have hitherto been unused in responsive hydrogels or in other fields using oligonucleotide interactions such as DNA nanotechnology. We present the first MO crosslinked hydrogels (MOCHs) as a means of label-free ssDNA detection and assess the benefits of MO crosslinks with regards to future automation and processing. MOCHs included both physical (hydrogen bonded MOs) and covalent crosslinks (MBA) (Figure 1e). The MO crosslinks consisted of two strands, the “sensor” strand had full complementarity for the chosen “analyte” ssDNA sequence, while the “blocker” strand was partially complementary to the sensor strand, such that the blocker strand will be displaced by the analyte ssDNA sequence. Displacement of the blocker strand breaks the physical crosslinks of the gel facilitating greater swelling while binding of the ssDNA will also change the ionic charge of the gel macrostructure. In this work we selected the miRNA sequence miR-92a (Table 1, A1), which has potential as a biomarker for leukaemia when compared to miR-638 concentrations.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1371/journal.pone.0005532","ISBN":"1932-6203","ISSN":"19326203","PMID":"19440243","abstract":"BACKGROUND: MicroRNAs are a family of 19- to 25-nucleotides noncoding small RNAs that primarily function as gene regulators. Aberrant microRNA expression has been described for several human malignancies, and this new class of small regulatory RNAs has both oncogenic and tumor suppressor functions. Despite this knowledge, there is little information regarding microRNAs in plasma especially because microRNAs in plasma, if exist, were thought to be digested by RNase. Recent studies, however, have revealed that microRNAs exist and escape digestion in plasma.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: We performed microRNA microaray to obtain insight into microRNA deregulation in the plasma of a leukemia patient. We have revealed that microRNA-638 (miR-638) is stably present in human plasmas, and microRNA-92a (miR-92a) dramatically decreased in the plasmas of acute leukemia patients. Especially, the ratio of miR-92a/miR-638 in plasma was very useful for distinguishing leukemia patients from healthy body.\\n\\nCONCLUSIONS/SIGNIFICANCE: The ratio of miR-92a/miR-638 in plasma has strong potential for clinical application as a novel biomarker for detection of leukemia.","author":[{"dropping-particle":"","family":"Tanaka","given":"Masami","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Oikawa","given":"Kosuke","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Takanashi","given":"Masakatsu","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kudo","given":"Motoshige","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ohyashiki","given":"Junko","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ohyashiki","given":"Kazuma","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kuroda","given":"Masahiko","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"PLoS ONE","id":"ITEM-1","issue":"5","issued":{"date-parts":[["2009"]]},"page":"1-5","title":"Down-regulation of miR-92 in human plasma is a novel marker for acute leukemia patients.","type":"article-journal","volume":"4"},"uris":[""]}],"mendeley":{"formattedCitation":"²⁰","plainTextFormattedCitation":"20","previouslyFormattedCitation":"²⁰"},"properties":{"noteIndex":0},"schema":""}20 This sensor design can be easily adapted to target any miRNA sequence and the blocker toehold length can be altered to account for weaker sequences with fewer GC base pairs. Figure 1 (a) Sequences of the morpholino oligonucleotide (MO) crosslink, tethered in polymer at the 5’ end. (b) Displacement of the MO blocker strand by the analyte strand to break the crosslink. (c) Morpholino Oligonucleotide Crosslinked Hydrogel (MOCH) system showing displacement of blocker strand (yellow) from sensor strand (blue) by analyte sequence (red), facilitating greater swelling. (d) Structure of MO with 5’ acrylamide for copolymerization with acrylamide where “R” is any of nucleobases ACGT. Full structure in Figure S1. (e) UV-initiated radical polymerization of acrylamide (10 wt%), functionalized MOs (0.4 mol %) and MBA (0.6 mol %) to form MOCH through radical initiation (0.125 mol %) where mol % is relative to acrylamide. Experimental SectionMaterials. All materials were purchased from Sigma Aldrich and used as received, except for the Morpholino Oligonucleotides and ssDNA oligonucleotide sequences (Table 1) which were purchased from GeneTools and IDT Technologies, respectively.Table 1. Morpholino Oligonucleotide sequences. S1:5′-ACA GGC CGG GAC AAG TGC AAT A-3′B1:5′-TAT TGC ACT TGT-3′ A1:5′-TAT TGC ACT TGT CCC GGC CTG T-3′AMM1:5’-TAT TGC CCT TGT CCC GGC CTG T-3’AMM5:5’-TAG TGC ACT TGT GCG GCC CTG G-3’R1:5′-ACG TCT AGA CGT AAC GAA GGT C-3′ Sensor (S1) and blocker (B1) units have acrylamide moiety at 5’ end for integration in polyacrylamide hydrogels. The analyte sequence (A1) is taken from miRNA 92a-1.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1371/journal.pone.0005532","ISBN":"1932-6203","ISSN":"19326203","PMID":"19440243","abstract":"BACKGROUND: MicroRNAs are a family of 19- to 25-nucleotides noncoding small RNAs that primarily function as gene regulators. Aberrant microRNA expression has been described for several human malignancies, and this new class of small regulatory RNAs has both oncogenic and tumor suppressor functions. Despite this knowledge, there is little information regarding microRNAs in plasma especially because microRNAs in plasma, if exist, were thought to be digested by RNase. Recent studies, however, have revealed that microRNAs exist and escape digestion in plasma.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: We performed microRNA microaray to obtain insight into microRNA deregulation in the plasma of a leukemia patient. We have revealed that microRNA-638 (miR-638) is stably present in human plasmas, and microRNA-92a (miR-92a) dramatically decreased in the plasmas of acute leukemia patients. Especially, the ratio of miR-92a/miR-638 in plasma was very useful for distinguishing leukemia patients from healthy body.\\n\\nCONCLUSIONS/SIGNIFICANCE: The ratio of miR-92a/miR-638 in plasma has strong potential for clinical application as a novel biomarker for detection of leukemia.","author":[{"dropping-particle":"","family":"Tanaka","given":"Masami","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Oikawa","given":"Kosuke","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Takanashi","given":"Masakatsu","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kudo","given":"Motoshige","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ohyashiki","given":"Junko","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ohyashiki","given":"Kazuma","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kuroda","given":"Masahiko","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"PLoS ONE","id":"ITEM-1","issue":"5","issued":{"date-parts":[["2009"]]},"page":"1-5","title":"Down-regulation of miR-92 in human plasma is a novel marker for acute leukemia patients.","type":"article-journal","volume":"4"},"uris":[""]}],"mendeley":{"formattedCitation":"²⁰","plainTextFormattedCitation":"20","previouslyFormattedCitation":"²⁰"},"properties":{"noteIndex":0},"schema":""}20 Complementarity with S1 are highlighted in red and underlined. S1 and B1 are 12 base pair complementary. A1 is fully complementary to S1, AMM1 and AMM5 have 1 and 5 mismatches with regard to S1 respectively. R1 is a randomly generated sequence and has only 6 non-contiguous base pair complementary to S1.Morpholino Oligonucleotide Preparation. Equimolar S1 and B1 MOs were dissolved in distilled water (at a concentration of 1 mM) and aliquoted in appropriate quantities. Mixtures were then heated to 95 °C for 2 min, cooled to room temperature and subsequently freeze-dried as smaller aliquots for use in gel preparation.Sample Preparation. Pre-gel solutions were prepared from stock monomer solutions of acrylamide (AAm), N,N′-methylene bisacrylamide (MBA) and NaCl in pH 7.4 phosphate buffer with 1-hydroxycyclohexyl phenyl ketone (HPK) in ethylene glycol as a radical photoinitiator. Mixing of these stocks with 0-150 mM NaCl gave final concentrations of 10 wt% AAm with 0.6 mol % MBA and 0.13 mol % HPK with regard to AAm. Pre-gel stocks contained carbon nanopowder (<50 nm particle size, 10 mg/mL) as an additive to increase contrast between gel and swelling solution. The combined stock solution was then pipetted into a 1.5 mL Eppendorf centrifuge tube containing MOs to a final concentration of 0.4 mol % (5.6 mM in the gel assuming 100% conversion).Gelation. Hydrogel samples were prepared in 1 or 2 ?L quantities by pipetting the pre-gel (and MO) stock onto a 7.7 x 22.8 mm silicon oxide chip with a silanized layer for polymer attachment to the chip surface. Silicon oxide chips were cleaned using 10 wt% NaOH for 4 hours, rinsed with deionised water, soaked in 0.01 M HCl for 10 min before pipetting 2 ?L of 3-(trimethoxysilyl)propyl methacrylate onto the gelation area for 16 hours. Chips were then rinsed with acetone and deionised water and dried before use. Once deposited, the pre-gelator droplet on each chip was irradiated with a Dymax Bluewave 75 UV curing light source (280 – 450 nm, 19+ W/cm2) for 60 s to initiate polymerisation and gelation. Gels were washed in 1 mM phosphate buffer solution (pH 7.4) with the same NaCl concentration as the pre-gelator solution (0-150 mM) at 4 °C for 1 hour, the wafer was then patted dry and stored at 4 °C for 16-24 hours until constant mass and moved into the test environment (20 1 °C, 40 % humidity) before swelling.Optical Swelling Measurements. Swelling properties of the hydrogels were characterised by taking images of the gels using a Sony XCD-X710 Firewire Camera with a MEDALight LP-300 lightbox as backlight using IC Capture image acquisition software. For swelling kinetics studies gels were imaged in solution. For end volume only gels were dried carefully to remove excess liquid on the outside of the gels before imaging. Images were processed and analysed using custom written MatLab code which calculated the gel volume using the contrast between the background and the gel and calibrated using the width of the chip.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.sbsr.2017.11.007","ISSN":"22141804","abstract":"A new method for oligonucleotide detection is presented based on oligonucleotide cross-linked polymer composites. Conductive carbon nanoparticles are incorporated into a DNA-functionalised polymer, containing partially complementary oligonucleotide cross-linkers, which is polymerised in situ upon interdigitated electrodes. In the presence of an aqueous solution of a specific analyte oligonucleotide sequence, the cross-linkers are cleaved, leading to increased swelling. As the polymer swells the relative density of the conductive particles decreases, leading to an easily measurable decrease in electrical conductivity. We demonstrate that such are capable of discriminating between analyte and control solutions, with single-base specificity, in under 3 min. The lower detection limit of these composites is of the order of 10 nM. The swelling characteristics of these composites is confirmed by optical imaging and the effects of varying temperature upon such composites are also reported.","author":[{"dropping-particle":"","family":"Ferrier","given":"David C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shaver","given":"Michael P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hands","given":"Philip J.W.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Sensing and Bio-Sensing Research","id":"ITEM-1","issue":"August 2017","issued":{"date-parts":[["2018"]]},"page":"1-6","publisher":"Elsevier","title":"Conductive composites for oligonucleotide detection","type":"article-journal","volume":"17"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁸","plainTextFormattedCitation":"18","previouslyFormattedCitation":"¹⁸"},"properties":{"noteIndex":0},"schema":""}18Polymer Swelling. Prepared hydrogels (2 or 1 ?L) were submerged in 5 mL of 1 mM phosphate buffer solution with 150 mM NaCl containing either the ssDNA ‘analyte’ or ‘random’ sequence at various concentrations of ssDNA (1 ?M – 1 pM or buffer only) at 20 °C 1 °C. Samples were imaged every 10 seconds as described above. Samples were measured in triplicate.Salt Study. Sample solutions and gels were prepared as described above without NaCl and washed in 1 mM phosphate buffer without NaCl. Samples were then placed in a 5 mL solution containing the ‘analyte’ or ‘random’ ssDNA sequence (1 ?M) with varying NaCl concentrations (0-300 mM) at 20 1 °C. Swelling was monitored in triplicate optically as above.Temperature Study. Gels (1 ?L) were prepared without NaCl as above. 3 samples were placed in a 5 mL solution of 1 mM phosphate buffer pre-heated to temperatures varying from 20-65 °C 2 °C and kept at the set temperature for 1 hour. Gels were then removed from solution, patted dry and imaged. Mobile Measurements. Gels (1 ?L) were prepared without NaCl as above. Samples were placed in 5 mL solution of 1 mM phosphate buffer with either A1 or R1 ssDNA (10 pM) in triplicate for 30 minutes. Gels were then removed from solution, the wafer dried, and the gel imaged using a OnePlus 5t camera (20 MP) with a SODIAL(R) 30X Zoom LED Magnifier Clip-On Cell Phone Mobile Phone Microscope Micro Lens attachment. Images were analysed using Digimizer (MedCalc Software bvba) to measure gel areas.Results and DiscussionSensitivity to Target (A1) vs a Random (R1) Control Sequence. Morpholino oligonucleotide crosslinked hydrogels with 0.4 mol % MO and 0.6 mol % MBA relative to AAm (10 wt %) were investigated as a comparison to previous ssDNA crosslinked hydrogel work. Swelling was calculated as the % volume change (%) (where % = (Vm – Vi)/Vi × 100 and Vm and Vi represent measured volume and initial deposited volume respectively). Established ssDNA crosslinked systems exhibit selective analyte sequence recognition with high fidelity;ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/bm900218c","ISBN":"1525-7797","ISSN":"15257797","PMID":"19425572","abstract":"Development of an oligonucleotide functionalized hydrogel integrated on a high resolution interferometric readout platform capable of determining changes in optical length of the hydrogel with 2 nm resolution is described. The hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network making up a network junction point in addition to the covalent cross-links. The hybridized dioligonucleotide network junctions were made with a 10 basepair complementary region flanked by additional basepairs that could aid in destabilizing the junction points in competitive displacement hybridization by the added probe oligonucleotides. The probe oligonucleotide destabilizing the junction point thus induces swelling of the functionalized hydrogel that is sensitive to the concentration of the probe, the sequence, and matching length between the probe and sensing oligonucleotide. This design yields a molecular amplification of the change in the optical length of the gel at least 5-fold compared to a hydrogel where sensing functionality is based on hybridization with a grafted oligonucleotide that is not a part of a network junction. Concentration sensitivity applied for specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. Applications of the resulting oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequences designed with aptamer functionalities for determination of other types of molecules are discussed.","author":[{"dropping-particle":"","family":"Tierney","given":"Sven","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stokke","given":"Bj?rn Torger","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biomacromolecules","id":"ITEM-1","issue":"6","issued":{"date-parts":[["2009"]]},"page":"1619-1626","title":"Development of an oligonucleotide functionalized hydrogel integrated on a high resolution interferometric readout platform as a label-free macromolecule sensing device","type":"article-journal","volume":"10"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1016/j.sbsr.2017.11.007","ISSN":"22141804","abstract":"A new method for oligonucleotide detection is presented based on oligonucleotide cross-linked polymer composites. Conductive carbon nanoparticles are incorporated into a DNA-functionalised polymer, containing partially complementary oligonucleotide cross-linkers, which is polymerised in situ upon interdigitated electrodes. In the presence of an aqueous solution of a specific analyte oligonucleotide sequence, the cross-linkers are cleaved, leading to increased swelling. As the polymer swells the relative density of the conductive particles decreases, leading to an easily measurable decrease in electrical conductivity. We demonstrate that such are capable of discriminating between analyte and control solutions, with single-base specificity, in under 3 min. The lower detection limit of these composites is of the order of 10 nM. The swelling characteristics of these composites is confirmed by optical imaging and the effects of varying temperature upon such composites are also reported.","author":[{"dropping-particle":"","family":"Ferrier","given":"David C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shaver","given":"Michael P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hands","given":"Philip J.W.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Sensing and Bio-Sensing Research","id":"ITEM-2","issue":"August 2017","issued":{"date-parts":[["2018"]]},"page":"1-6","publisher":"Elsevier","title":"Conductive composites for oligonucleotide detection","type":"article-journal","volume":"17"},"uris":[""]}],"mendeley":{"formattedCitation":"^16,18","plainTextFormattedCitation":"16,18","previouslyFormattedCitation":"^16,18"},"properties":{"noteIndex":0},"schema":""}16,18 it is essential that this is maintained when using MO crosslinks. MOCH swelling was measured in the presence of the A1 sequence (fully complementary to the sensor strand) or in the presence of a randomly generated R1 sequence (6 non-contiguous nucleobase complementary to the sensor strand). Swelling kinetics were measured optically at a range of A1 and R1 concentrations (1 ?M – 10 pM) to probe the selectivity and detection limits of this system. MOCHs remained sensitive and selective at 100 pM (Fig 2a), a 100-fold improvement on the comparative ssDNA crosslinked system which had a limit-of-detection of 10 nM.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.sbsr.2017.11.007","ISSN":"22141804","abstract":"A new method for oligonucleotide detection is presented based on oligonucleotide cross-linked polymer composites. Conductive carbon nanoparticles are incorporated into a DNA-functionalised polymer, containing partially complementary oligonucleotide cross-linkers, which is polymerised in situ upon interdigitated electrodes. In the presence of an aqueous solution of a specific analyte oligonucleotide sequence, the cross-linkers are cleaved, leading to increased swelling. As the polymer swells the relative density of the conductive particles decreases, leading to an easily measurable decrease in electrical conductivity. We demonstrate that such are capable of discriminating between analyte and control solutions, with single-base specificity, in under 3 min. The lower detection limit of these composites is of the order of 10 nM. The swelling characteristics of these composites is confirmed by optical imaging and the effects of varying temperature upon such composites are also reported.","author":[{"dropping-particle":"","family":"Ferrier","given":"David C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shaver","given":"Michael P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hands","given":"Philip J.W.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Sensing and Bio-Sensing Research","id":"ITEM-1","issue":"August 2017","issued":{"date-parts":[["2018"]]},"page":"1-6","publisher":"Elsevier","title":"Conductive composites for oligonucleotide detection","type":"article-journal","volume":"17"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁸","plainTextFormattedCitation":"18","previouslyFormattedCitation":"¹⁸"},"properties":{"noteIndex":0},"schema":""}18 MOs have a stronger affinity than ssDNA for binding RNA or ssDNA (As shown in Table S1)ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/ja809933h","ISBN":"1520-5126 (Electronic)\\n0002-7863 (Linking)","ISSN":"00027863","PMID":"19708646","abstract":"Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these molecular mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chemical technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successful-ly applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies.","author":[{"dropping-particle":"","family":"Ouyang","given":"Xiaohu","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shestopalov","given":"Ilya A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sinha","given":"Surajit","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zheng","given":"Genhua","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pitt","given":"Cameron L.W.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Li","given":"Wen Hong","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Olson","given":"Andrew J.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chen","given":"James K.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of the American Chemical Society","id":"ITEM-1","issue":"37","issued":{"date-parts":[["2009"]]},"page":"13255-13269","title":"Versatile synthesis and rational design of caged morpholinos","type":"article-journal","volume":"131"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1093/nar/gkm234","ISBN":"1362-4962 (Electronic)\\r0305-1048 (Linking)","ISSN":"03051048","PMID":"17452344","abstract":"We developed OligoCalc as a web-accessible, client-based computational engine for reporting DNA and RNA single-stranded and double-stranded properties, including molecular weight, solution concentration, melting temperature, estimated absorbance coefficients, inter-molecular self-complementarity estimation and intra-molecular hairpin loop formation. OligoCalc has a familiar 'calculator' look and feel, making it readily understandable and usable. OligoCalc incorporates three common methods for calculating oligonucleotide-melting temperatures, including a nearest-neighbor thermodynamic model for melting temperature. Since it first came online in 1997, there have been more than 900,000 accesses of OligoCalc from nearly 200,000 distinct hosts, excluding search engines. OligoCalc is available at , with links to the full source code, usage patterns and statistics at that link as well.","author":[{"dropping-particle":"","family":"Kibbe","given":"Warren A.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nucleic Acids Research","id":"ITEM-2","issue":"SUPPL.2","issued":{"date-parts":[["2007"]]},"page":"43-46","title":"OligoCalc: An online oligonucleotide properties calculator","type":"article-journal","volume":"35"},"uris":[""]}],"mendeley":{"formattedCitation":"^35,36","plainTextFormattedCitation":"35,36","previouslyFormattedCitation":"^35,36"},"properties":{"noteIndex":0},"schema":""}35,36 and so may be expected to form a fully displaced system at a lower concentration of A1. Similarly, the effective tethering of the ssDNA strands from solution confers a charged moiety to the hydrogel which can contribute to overall swelling.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1039/c1sm05221g","ISBN":"1744-683X","ISSN":"1744-683X","abstract":"An oligonucleotide-polymer hybrid hydrogel displaying swelling response in a logical AND and OR fashion depending on specific stimuli by probe oligonucleotides was prepared. The hydrogel acts as a transducer of the specific recognition of DNA sequences into micro- and macroscale mechanics. Input oligonucleotide probes induce hydrogel swelling ratio at two signal levels.","author":[{"dropping-particle":"","family":"Gawe","given":"Kamila","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stokke","given":"Bj?rn Torger","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Soft Matter","id":"ITEM-1","issued":{"date-parts":[["2011"]]},"page":"4615-4618","title":"Logic swelling response of DNA–polymer hybrid hydrogel","type":"article-journal","volume":"7"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁷","plainTextFormattedCitation":"17","previouslyFormattedCitation":"¹⁷"},"properties":{"noteIndex":0},"schema":""}17 As seen in Figure 2b, the maximal response to A1 occurs at or above 100 pM, whereas the response to R1 increases with concentration. This may be from competitive displacement of the blocker shifting the equilibrium towards more broken crosslinks, or from association of R1 strands, and the associated charges, with the unhybridized segment of S1. Displacement of the blocking strand resulting in crosslink breakdown is the biggest factor causing increased swelling capacity and rate in response to the A1 sequence.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1039/c0sm00915f","ISSN":"1744-683X","abstract":"Hydrogel-based label-free nucleic acids sensor materials using high resolution interferometric readout of hydrogel swelling changes was prepared and characterized with respect to molecular design parameters. The DNA-sensitive hydrogel comprised sensing (S) and blocking (B) oligonucleotide pairs copolymerized with the network and formed reversible crosslinks in addition to stable covalent ones. Oligonucleotide probes (P) complementary to S with longer complementary regions comparing to B results in competitive replacement of S-B strands. The associated destabilization of DNA crosslinks results in changes of the hydrogel swelling. S-B dioligonucleotidic crosslinkers were designed with 8{,} 12 and 16 basepairs in complementary regions and were destabilized by probes with matching sequences with 2{,} 6 and 10 excess basepairs in the so-called {","author":[{"dropping-particle":"","family":"Gao","given":"Ming","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gawel","given":"Kamila","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stokke","given":"Bj?rn Torger","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Soft Matter","id":"ITEM-1","issue":"5","issued":{"date-parts":[["2011"]]},"page":"1741","title":"Toehold of dsDNA exchange affects the hydrogel swelling kinetics of a polymer–dsDNA hybrid hydrogel","type":"article-journal","volume":"7"},"uris":[""]}],"mendeley":{"formattedCitation":"³⁷","plainTextFormattedCitation":"37","previouslyFormattedCitation":"³⁷"},"properties":{"noteIndex":0},"schema":""}37 Increasing the strength of the crosslink through elongating the “toehold” overlap, the number of continuous complementary nucleobases, between blocker and sensor strands results in a slower response profile in ssDNA crosslinked hydrogels, as displacement of the blocker is then less thermodynamically favored. As MO crosslinks are stronger than ssDNA crosslinks a similar trend is seen and the response rate is slower than our previous ssDNA crosslinked system. Diffusion of the ssDNA in solution into the hydrogel is another influential factor on the rate of swelling. This is dependent on the concentration gradient between solution and hydrogel,ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/bm900218c","ISBN":"1525-7797","ISSN":"15257797","PMID":"19425572","abstract":"Development of an oligonucleotide functionalized hydrogel integrated on a high resolution interferometric readout platform capable of determining changes in optical length of the hydrogel with 2 nm resolution is described. The hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network making up a network junction point in addition to the covalent cross-links. The hybridized dioligonucleotide network junctions were made with a 10 basepair complementary region flanked by additional basepairs that could aid in destabilizing the junction points in competitive displacement hybridization by the added probe oligonucleotides. The probe oligonucleotide destabilizing the junction point thus induces swelling of the functionalized hydrogel that is sensitive to the concentration of the probe, the sequence, and matching length between the probe and sensing oligonucleotide. This design yields a molecular amplification of the change in the optical length of the gel at least 5-fold compared to a hydrogel where sensing functionality is based on hybridization with a grafted oligonucleotide that is not a part of a network junction. Concentration sensitivity applied for specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. Applications of the resulting oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequences designed with aptamer functionalities for determination of other types of molecules are discussed.","author":[{"dropping-particle":"","family":"Tierney","given":"Sven","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stokke","given":"Bj?rn Torger","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biomacromolecules","id":"ITEM-1","issue":"6","issued":{"date-parts":[["2009"]]},"page":"1619-1626","title":"Development of an oligonucleotide functionalized hydrogel integrated on a high resolution interferometric readout platform as a label-free macromolecule sensing device","type":"article-journal","volume":"10"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁶","plainTextFormattedCitation":"16","previouslyFormattedCitation":"¹⁶"},"properties":{"noteIndex":0},"schema":""}16 the porosity and charge of the hydrogel,ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1007/3-540-56791-7_1","ISBN":"9783540567912","ISSN":"0065-3195","abstract":"This review covers the recent advances in studies of the volume phase transition and critical phenomena of polymer gels mostly carried out in our group from 1973 to the present. We aimed here to discuss intensively (i) the basic understanding of the transition from the viewpoints of structure, dynamics, kinetics, and equilibrium thermodynamics, (ii) technological applications of the volume transition, and (iii) the relation between the phase transition and biological interactions.","author":[{"dropping-particle":"","family":"Shibayama","given":"Mitsuhiro","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tanaka","given":"Toyoichi","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Responsive Gels: Volume ransitions I Advances in Polymer Science1","id":"ITEM-1","issue":"1993","issued":{"date-parts":[["1979"]]},"page":"1-62","title":"Volume Phase Transition and Related Phenomena of Polymer Gels","type":"article-journal","volume":"109"},"uris":[""]}],"mendeley":{"formattedCitation":"³⁸","plainTextFormattedCitation":"38","previouslyFormattedCitation":"³⁸"},"properties":{"noteIndex":0},"schema":""}38 as well as retardation of diffusion through the gel, as ssDNA interacts with the MO crosslink even if not resulting in blocker strand displacement. ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.dci.2009.07.003.Characterization","ISBN":"8587847562","ISSN":"1946-6242","PMID":"20371490","author":[{"dropping-particle":"","family":"Allen","given":"Peter B.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ellington","given":"Andrew D.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Molecules","id":"ITEM-1","issue":"11","issued":{"date-parts":[["2013"]]},"page":"13390-13402","title":"Spatial Control of DNA Reaction Networks by DNA Sequence","type":"article-journal","volume":"17"},"uris":[""]},{"id":"ITEM-2","itemData":{"ISBN":"7095135231","author":[{"dropping-particle":"","family":"Livshits","given":"Mikhail a","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mirzabekov","given":"Andrei D","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biophysical Journal","id":"ITEM-2","issue":"November","issued":{"date-parts":[["1996"]]},"page":"2795-2801","title":"Theoretical Analysis of the Kinetics of DNA Hybridization with","type":"article-journal","volume":"71"},"uris":[""]}],"mendeley":{"formattedCitation":"^39,40","plainTextFormattedCitation":"39,40","previouslyFormattedCitation":"^39,40"},"properties":{"noteIndex":0},"schema":""}39,40Figure 2b shows some concentration dependent responsivity, whereby oversaturation with a high R1 solution generates a response indistinguishable from a lower concentration of A1. We believe this is due to the competitive displacement of the MO crosslinks by a sequence with lower affinity but higher concentration. The hydrophilicity of the hydrogel is lower than in ssDNA crosslinked hydrogels as the charged ssDNA species has been replaced with the uncharged MOs. When interacting with MOs in the gel, the ssDNA in solution can become tethered within the hydrogel due to interaction with the MO strands. Once the blocker strand has been displaced, it is likely that it could also hybridize with a ssDNA strand from solution. Each ssDNA strand that becomes tethered would add 22 phosphate groups, if the blocker strand also hybridizes with a solution ssDNA strand that would double to 44 phosphate groups per MO crosslink. This would equate to a charged density of 8.8 or 17.6 mol %. This localized tethering of charge ssDNA sequences within the MOCHs is believed to cause the concentration dependent increase rate of swelling exhibited at concentrations of 100 pM and above, whereas at concentrations below this the response to the R1 sequence is indistinguishable from swelling in buffer alone.Sequence Specificity. ssDNA crosslinked systems have been shown with high sequence fidelity, able to differentiate between a full sequence match and a sequence with one or more mismatches.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/bm900218c","ISBN":"1525-7797","ISSN":"15257797","PMID":"19425572","abstract":"Development of an oligonucleotide functionalized hydrogel integrated on a high resolution interferometric readout platform capable of determining changes in optical length of the hydrogel with 2 nm resolution is described. The hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network making up a network junction point in addition to the covalent cross-links. The hybridized dioligonucleotide network junctions were made with a 10 basepair complementary region flanked by additional basepairs that could aid in destabilizing the junction points in competitive displacement hybridization by the added probe oligonucleotides. The probe oligonucleotide destabilizing the junction point thus induces swelling of the functionalized hydrogel that is sensitive to the concentration of the probe, the sequence, and matching length between the probe and sensing oligonucleotide. This design yields a molecular amplification of the change in the optical length of the gel at least 5-fold compared to a hydrogel where sensing functionality is based on hybridization with a grafted oligonucleotide that is not a part of a network junction. Concentration sensitivity applied for specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. Applications of the resulting oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequences designed with aptamer functionalities for determination of other types of molecules are discussed.","author":[{"dropping-particle":"","family":"Tierney","given":"Sven","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stokke","given":"Bj?rn Torger","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biomacromolecules","id":"ITEM-1","issue":"6","issued":{"date-parts":[["2009"]]},"page":"1619-1626","title":"Development of an oligonucleotide functionalized hydrogel integrated on a high resolution interferometric readout platform as a label-free macromolecule sensing device","type":"article-journal","volume":"10"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁶","plainTextFormattedCitation":"16","previouslyFormattedCitation":"¹⁶"},"properties":{"noteIndex":0},"schema":""}16 To investigate sequence fidelity, MOCHs were swollen with sequences AMM1 and AMM5 (identical to A1 but with 1 and 5 mismatches respectively). Figure 2c details the swelling of MOCHs in 100 pM solutions of AMM1 and AMM5 compared to swelling in buffer only, A1 or R1. The greatest response is to A1, with decreasing swelling in AMM1, AMM5, R1 and finally buffer. As before, this is likely to the thermodynamic favorability of displacement, whereby S1 has the strongest hybridization with so B1 is most rapidly displaced by A1, then AMM1, AMM5, R1 and is not displaced in buffer only. The competitive displacement is quantifiable and can be accounted for during analysis.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1529/biophysj.106.097121","ISSN":"00063495","PMID":"17056736","abstract":"Using real-time dual-color fluorescence detection, we have experimentally tracked individual target species during competitive DNA surface hybridization in a two-component sample. Our experimental results demonstrate displacement of the lower affinity species by the higher affinity species and corroborate recent theoretical models describing competitive DNA surface hybridization. Competition at probe sites complementary to one of the two DNA species was monitored in separate experiments for two different target pairs. Each pair differs in sequence by a single nucleotide polymorphism, and one pair includes a folding target. We propose a mechanistic interpretation of the differences between hybridization curves of targets in multi-component and single-component experiments. ? 2007 by the Biophysical Society.","author":[{"dropping-particle":"","family":"Bishop","given":"J.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wilson","given":"C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chagovetz","given":"A. M.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Blair","given":"S.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biophysical Journal","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2007"]]},"page":"10-12","title":"Competitive displacement of DNA during surface hybridization","type":"article-journal","volume":"92"},"uris":[""]}],"mendeley":{"formattedCitation":"⁴¹","plainTextFormattedCitation":"41","previouslyFormattedCitation":"⁴¹"},"properties":{"noteIndex":0},"schema":""}41Figure 2 (a) Comparison of swelling kinetics showing lower LoD of 2 ?L MOCHs in A1 or R1 at 10-100 pM or buffer only. (b) Comparison of swollen volume at 60 minutes in A1 (solid red) or R1 (dotted blue) at 10 pM – 1 ?M or buffer. Full swelling kinetics of 1 nM - 1 ?M in S2. (c) Swollen volume at 60 minutes to test mismatch response, at 100 pM or A1, R1, AMM1, AMM5 and buffer. Synthesis and swelling all with 150 mM NaCl. Error bars in figures a-c show standard error of the mean (SEM).Sensitivity to Salt. DNA hybridization is well known to require salt to shield the charges of the phosphate groups in the backbone and prevent backbone repulsion between strands.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/jp106754k","ISBN":"1520-5207 (Electronic)\\r1520-5207 (Linking)","ISSN":"15205207","PMID":"21062084","abstract":"DNA hybridization is of tremendous importance in biology, bionanotechnology, and biophysics. Molecular beacons are engineered DNA hairpins with a fluorophore and a quencher labeled on each of the two ends. A target DNA can open the hairpin to give an increased fluorescence signal. To date, the majority of molecular beacon detections have been performed only in aqueous buffers. We describe herein DNA detection in nine different organic solvents, methanol, ethanol, isopropanol, acetonitrile, formamide, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethylene glycol, and glycerol, varying each up to 75% (v/v). In comparison with detection in water, the detection in organic solvents showed several important features. First, the molecular beacon hybridizes to its target DNA in the presence of all nine solvents up to a certain percentage. Second, the rate of this hybridization was significantly faster in most organic solvents compared with water. For example, in 56% ethanol, the beacon showed a 70-fold rate enhancement. Third, the ability of the molecular beacon to discriminate single-base mismatch is still maintained. Lastly, the DNA melting temperature in the organic solvents showed a solvent concentration-dependent decrease. This study suggests that molecular beacons can be used for applications where organic solvents must be involved or organic solvents can be intentionally added to improve the molecular beacon performance.","author":[{"dropping-particle":"","family":"Dave","given":"Neeshma","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Liu","given":"Juewen","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Physical Chemistry B","id":"ITEM-1","issue":"47","issued":{"date-parts":[["2010"]]},"page":"15694-15699","title":"Fast molecular beacon hybridization in organic solvents with improved target specificity","type":"article-journal","volume":"114"},"uris":[""]}],"mendeley":{"formattedCitation":"⁴²","plainTextFormattedCitation":"42","previouslyFormattedCitation":"⁴²"},"properties":{"noteIndex":0},"schema":""}42 MO-DNA hybridization has been shown to be possible with minimal salt present as the phosphate groups are replaced with uncharged phosphorodiamidate groups.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1007/BF02484560","ISBN":"978-0-306-48230-4","ISSN":"09295666","author":[{"dropping-particle":"","family":"Summerton","given":"James E.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Letters in Peptide Science","id":"ITEM-1","issue":"3-4","issued":{"date-parts":[["2003"]]},"page":"215-236","title":"Morpholinos and PNAs compared","type":"article-journal","volume":"10"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1021/ac302915a","ISBN":"1520-6882 (Electronic)\\r0003-2700 (Linking)","ISSN":"00032700","PMID":"23215633","abstract":"Exploring local pH in micro/nanoscale is fundamentally important for understanding microprocesses including the corrosion of metal and the metabolism of cell. Regular fluorescence pH probes and potentiometric electrodes show either low signal intensity or lack of spatial resolution when being applied in a micro/nanoenvironment. Here, we developed a nanoscale reversible pH probe based on the plasmonic coupling effect of i-motif modulated gold nanoparticle (AuNP) assembly. The pH probe shows a reversible and highly sensitive response to pH variation between 4.5 and 7.5. Introduction of morpholino oligomers (MO), a neutral analog of DNA, into the assembly endows the pH probe with high stability even under low salt concentration. The intense optical signal of a AuNP enables local pH to be read out not only in the micro/nanofluidic channel but also on a single i-motif-MO-AuNP assembly. Recording of the strong plasmonic resonance scattering spectrum of AuNP provides a promising method for extracting chemical information in nanospace of biological systems.","author":[{"dropping-particle":"","family":"Zhao","given":"Yun","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Cao","given":"Lei","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ouyang","given":"Jun","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wang","given":"Min","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wang","given":"Kang","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Xia","given":"Xing Hua","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Analytical Chemistry","id":"ITEM-2","issue":"2","issued":{"date-parts":[["2013"]]},"page":"1053-1057","title":"Reversible plasmonic probe sensitive for pH in micro/nanospaces based on i-motif-modulated morpholino-gold nanoparticle assembly","type":"article-journal","volume":"85"},"uris":[""]}],"mendeley":{"formattedCitation":"^31,43","plainTextFormattedCitation":"31,43","previouslyFormattedCitation":"^31,43"},"properties":{"noteIndex":0},"schema":""}31,43 MO-MO interactions were therefore expected to be possible without salt. Figure 3a shows the effect of altering the NaCl concentration (0-300 mM) in solution on MOCHs (2 ?L) synthesized with no salt. As expected, gels synthesized without NaCl retain their sensing capability, indicating that the MO crosslinks remain intact. At each salt concentration, there is a clear increase in swelling in A1 over R1. With no NaCl there is the greatest response to both A1 and R1. This is expected to be both due to increased hydrophilicity and motility of ssDNA and all charges from strands that become tethered through interactions with S1 or R1 being unshielded. Above 50 mM both A1 and R1 responses are lowered. MOCHs offer a solution to salt sensitivity challenges with the anionic DNA crosslinked gels. Removing salt during synthesis enables the use of electrostatic additives that can otherwise aggregate and precipitate in the presence of counter ions. Furthermore, if processing or storage requires the material to be dried, salt crystallization may lead to non-uniform damage and inconsistent responses.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1179/sic.2006.51.1.41","ISBN":"0039-3630","ISSN":"0039-3630","abstract":"Laboratory weathering tests are important in the field of restoration as they provide a means of estimating, in a relatively short time, the longer-term performance of conservation materials when applied in practice. Accelerated tests to simulate the damage caused to porous materials by soluble salts such as sodium sulphate are well known and highly effective. However, when using sodium chloride the existing test methods are not particularly successful. Research on case studies has shown that the environmental conditions play an important role in determining the occurrence of the decay. Therefore understanding the effect of the environmental conditions on the damage constitutes a first step in the development of an effective weathering test for sodium chloride. The research described in this paper studies the effect of environmental conditions on the decay due to sodium chloride with the ultimate aim of defining an effective laboratory test. The experiments have been performed on two plasters with very dffierent physical, mechanical and mineralogical properties. Different techniques of analysis have been used: mercury intrusion porosimetry, optical polarized microscopy, environmental scanning electron microscopy, chemical analyses and X-ray diffraction. The results obtained highlighted which of the test conditions under evaluation were the most effective. Using this information, a new procedure for an effective accelerated salt weathering test using sodium chloride is proposed.","author":[{"dropping-particle":"","family":"Lubelli","given":"B.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hees","given":"R. P. J.","non-dropping-particle":"van","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Groot","given":"C. J. W. P.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Studies in Conservation","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2006"]]},"page":"41-56","title":"The effect of environmental conditions on sodium chloride damage: A step in the development of an effective weathering test","type":"article-journal","volume":"51"},"uris":[""]}],"mendeley":{"formattedCitation":"⁴⁴","plainTextFormattedCitation":"44","previouslyFormattedCitation":"⁴⁴"},"properties":{"noteIndex":0},"schema":""}44 Although salt is plentiful in human serum, removal is possible using simple methods, such as magnetic beads that can be used as part of an extraction protocol, and would prevent any self-complementarity induced hairpin or self-dimerization that may occur with other miRNA sequences.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"ISBN":"1312013125","author":[{"dropping-particle":"","family":"Hawkins","given":"Trevor","non-dropping-particle":"","parse-names":false,"suffix":""}],"id":"ITEM-1","issue":"54","issued":{"date-parts":[["1985"]]},"page":"2989-2997","title":"DNA Purification and Isolation using Magnetic Particles. U.S. Patent 5,705,628","type":"patent","volume":"19"},"uris":[""]}],"mendeley":{"formattedCitation":"⁴⁵","plainTextFormattedCitation":"45","previouslyFormattedCitation":"⁴⁵"},"properties":{"noteIndex":0},"schema":""}45 Figure 3 MOCH stability studies (a) Influence of salt (mM) on response to 1 ?M A1 and R1. Full data with SEM shown in S3. (b) Thermal effects on MOCHs and crosslink integrity, error bars show SEM. Sensitivity to Temperature. Figure 3b shows the thermal stability of MO crosslinks. Thermal dehybridization is an important consideration when designing oligonucleotide based sensors, particularly when targeting short sequences like miRNA. When the analyte sequence is only 22 nucleotides long, the blocker sequence must be long enough to be stable at room temperature and during synthesis, but not so strong that displacement is excessively slow. The use of more stable, high-GC, crosslinks is not always possible as the crosslink sequence will be dependent on the target sequence. Due to the lack of phosphate-backbone repulsion, MO-ssDNA interactions have a higher Tm than ssDNA-ssDNA, and similarly MO-MO interactions are expected to be higher again. Figure 3b shows gel stability at or below 45 °C and apparent crosslink dehybridization at 55 °C and above. Although we predicted a Tm of 66 °C (Table S1),ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/ja809933h","ISBN":"1520-5126 (Electronic)\\n0002-7863 (Linking)","ISSN":"00027863","PMID":"19708646","abstract":"Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these molecular mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chemical technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successful-ly applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies.","author":[{"dropping-particle":"","family":"Ouyang","given":"Xiaohu","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shestopalov","given":"Ilya A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sinha","given":"Surajit","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zheng","given":"Genhua","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pitt","given":"Cameron L.W.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Li","given":"Wen Hong","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Olson","given":"Andrew J.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chen","given":"James K.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of the American Chemical Society","id":"ITEM-1","issue":"37","issued":{"date-parts":[["2009"]]},"page":"13255-13269","title":"Versatile synthesis and rational design of caged morpholinos","type":"article-journal","volume":"131"},"uris":[""]}],"mendeley":{"formattedCitation":"³⁵","plainTextFormattedCitation":"35","previouslyFormattedCitation":"³⁵"},"properties":{"noteIndex":0},"schema":""}35 this will be affected by the incorporation into the gel, whereby the kinetic strain of swelling will influence crosslink breaking.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1073/pnas.1109853108","ISBN":"1568715692","ISSN":"0027-8424","PMID":"21896714","abstract":"DNA bridging can be used to induce specific attractions between small particles, providing a highly versatile approach to creating unique particle-based materials having a variety of periodic structures. Surprisingly, given the fact that the thermodynamics of DNA strands in solution are completely understood, existing models for DNA-induced particle interactions are typically in error by more than an order of magnitude in strength and a factor of two in their temperature dependence. This discrepancy has stymied efforts to design the complex temperature, sequence and time-dependent interactions needed for the most interesting applications, such as materials having highly complex or multicomponent microstructures or the ability to reconfigure or self-replicate. Here we report high-spatial resolution measurements of DNA-induced interactions between pairs of polystyrene microspheres at binding strengths comparable to those used in self-assembly experiments, up to 6 k(B)T. We also describe a conceptually straightforward and numerically tractable model that quantitatively captures the separation dependence and temperature-dependent strength of these DNA-induced interactions, without empirical corrections. This model was equally successful when describing the more complex and practically relevant case of grafted DNA brushes with self-interactions that compete with interparticle bridge formation. Together, our findings motivate a nanomaterial design approach where unique functional structures can be found computationally and then reliably realized in experiment.","author":[{"dropping-particle":"","family":"Rogers","given":"W. B.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Crocker","given":"J. C.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Proceedings of the National Academy of Sciences","id":"ITEM-1","issue":"38","issued":{"date-parts":[["2011"]]},"page":"15687-15692","title":"Direct measurements of DNA-mediated colloidal interactions and their quantitative modeling","type":"article-journal","volume":"108"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1529/biophysj.107.108449","ISBN":"0006-3495","ISSN":"00063495","PMID":"17921214","abstract":"We probe DNA hybridization kinetics by measuring the lifetime distribution of single 16-bp duplexes under thermal dissociation. Our unique approach, based on two DNA-coated microspheres in an extended optical tweezer, allows the study of single duplex DNA molecules under negligible molecular tension. In contrast to earlier experiments, we find a stretched exponential lifetime distribution, which is likely due to dissociation proceeding via a number of competing pathways between highly force-sensitive intermediate states. Similar measurements of microspheres linked by multiple DNA bridges find they have unexpected short bound lifetimes, also consistent with force sensitivity. ? 2008 by the Biophysical Society.","author":[{"dropping-particle":"","family":"Biancaniello","given":"Paul L.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kim","given":"Anthony J.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Crocker","given":"John C.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biophysical Journal","id":"ITEM-2","issue":"3","issued":{"date-parts":[["2008"]]},"page":"891-896","publisher":"Elsevier","title":"Long-time stretched exponential kinetics in single DNA duplex dissociation","type":"article-journal","volume":"94"},"uris":[""]}],"mendeley":{"formattedCitation":"^46,47","plainTextFormattedCitation":"46,47","previouslyFormattedCitation":"^46,47"},"properties":{"noteIndex":0},"schema":""}46,47 The importance of this temperature study is significant, as there is the possibility of a false positive result being obtained from a DNA crosslinked hydrogel due to elevated temperatures. Furthermore, thermal stability will be vital to the manufacturing, storage and transportation of a developed sensor and may be used to reduce response time.ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/bm900218c","ISBN":"1525-7797","ISSN":"15257797","PMID":"19425572","abstract":"Development of an oligonucleotide functionalized hydrogel integrated on a high resolution interferometric readout platform capable of determining changes in optical length of the hydrogel with 2 nm resolution is described. The hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network making up a network junction point in addition to the covalent cross-links. The hybridized dioligonucleotide network junctions were made with a 10 basepair complementary region flanked by additional basepairs that could aid in destabilizing the junction points in competitive displacement hybridization by the added probe oligonucleotides. The probe oligonucleotide destabilizing the junction point thus induces swelling of the functionalized hydrogel that is sensitive to the concentration of the probe, the sequence, and matching length between the probe and sensing oligonucleotide. This design yields a molecular amplification of the change in the optical length of the gel at least 5-fold compared to a hydrogel where sensing functionality is based on hybridization with a grafted oligonucleotide that is not a part of a network junction. Concentration sensitivity applied for specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. Applications of the resulting oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequences designed with aptamer functionalities for determination of other types of molecules are discussed.","author":[{"dropping-particle":"","family":"Tierney","given":"Sven","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stokke","given":"Bj?rn Torger","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Biomacromolecules","id":"ITEM-1","issue":"6","issued":{"date-parts":[["2009"]]},"page":"1619-1626","title":"Development of an oligonucleotide functionalized hydrogel integrated on a high resolution interferometric readout platform as a label-free macromolecule sensing device","type":"article-journal","volume":"10"},"uris":[""]}],"mendeley":{"formattedCitation":"¹⁶","plainTextFormattedCitation":"16","previouslyFormattedCitation":"¹⁶"},"properties":{"noteIndex":0},"schema":""}16Optimizing Sensitivity. In this work, we use a simple optical means of volume measurement. Were these MO functionalized polymers to be applied to biosensing, using a more sensitive transduction mechanism may further increase sensitivity and would be the likely basis of a sensor technology built from MOCHs. One simple way to improve sensitivity would be to reduce the volume of material, thereby reducing the number of MO crosslinks required to break to achieve a measurable response. To investigate this, we reduced the hydrogel volume to 1 ?L and synthesized and tested with no NaCl to theoretically maximize any charge related swelling from ssDNA binding (Figure 4), achieving an LoD of 10 pM. As the difference in % is similar to that of larger gels (2 ?L) at 1 ?M (Figure 3a), we expect a similar signal reduction at higher NaCl concentrations. Further reduction in volume is expected to improve sensitivity even more but requires development of different deposition (i.e. inkjet printing) or transduction methods that are beyond the scope of this contribution.Figure 4 Comparison of swelling kinetics showing lower LoD of 1 ?L MOCHs in A1 or R1 at 1-100 pM or buffer. Error bars show SEM. Figure 5 (a) Analyzed image taken using a mobile phone of 1 ?L MOCH (black “gel area”) on silicon wafer (7.6 mm wide) swollen in A1 or R1 (10 pM) for 30 minutes. Wafer edges were used for scale and gel area was manually identified, ignoring leached carbon and the reflection on the wafer. (b) Swollen area at 30 minutes in A1 or R1 (10 pM) from mobile images. Error bars show SEM.Mobile Measurements. To showcase the potential portability and accessibility of MOCHs, we have also developed a simplified gel analysis methodology. Measurements can be taken with a smartphone, basic magnifier attachment, and free image processing software. Whilst ensuring vertical and horizontal sample position is consistent is essential, the smartphone can quickly gather the required data of swelling kinetics. A typical cropped and analyzed image is shown in Figure 5a, while the analysis after 30 minutes in 10 pM A1 or R1 is shown in Figure 5b. While sensitivity is not as high as with the more complex photography and image processing set-up, the detection of oligonucleotide sequences using MOCHs through a simple, portable method is ideal for outreach or demonstration, and offers potential for incorporation into future point-of-care diagnostic devices. ConclusionsA novel MO crosslinked responsive polymer hydrogel system reported here exhibited several improvements over comparative DNA systems. These include; increased sensitivity, improved thermal stability and removal of salt requirements. Selective swelling is observed in the presence of an analyte ssDNA sequence, caused by competitive displacement of MO crosslink, to a sensitivity of 100 pM. This was further improved through halving the hydrogel volume and removing salt from both synthesis and testing, resulting in a limit of detection of 10 pM. The improved thermal stability coupled with the diminished salt sensitivity suggest significant promise for MOCHs as a more stable and controllable alternative to DNA-based responsive hydrogel systems and facilitate alternative synthesis methods previously unavailable to DNA-crosslinked materials. These MOCHs, and their swelling response, can be further optimized to increase sensitivity and into developed into biosensor systems. Simultaneously, the MO crosslink can be easily translated into current DNA crosslinked systems such as nanoparticles, electrochemical sensors or DNA nanotechnology for similar improvements.ASSOCIATED CONTENT Supporting InformationFurther data and analysis (PDF) is available free of charge on the ACS Publications website. Full MO modification structure, predicted Tm of ssDNA or MO crosslinks, swelling kinetics of A1 or R1 1 nM - 1 ?M, salt concentration kinetics with error bars, swelling kinetics of hydrogels with S1 strand only in A1 or R1 100pM.AUTHOR INFORMATIONCorresponding Author* michael.shaver@ed.ac.uk, philip.hands@ed.ac.ukPresent AddressesAuthor ContributionsThe manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We would like to thank Dr Jaclyn Raeburn for early experiments in morpholino functionalisation and Dr David Ferrier for helpful discussions and MatLab coding. We would like to thank the Engineering and Physical Sciences Research Council (EPSRC) and Axis-Shield Diagnostics Ltd. for a CASE studentship for GL and the MRC CiC programme for earlier financial support of this project.ABBREVIATIONSMOs, Morpholino Oligonucleotides; ssDNA, single-stranded DNA; MBA, N,N′-methylenebis(acrylamide); miRNA, microRNA; RT-qPCR, reverse transcription quantitative polymerase chain reaction; LoD, limit of detection; MEMS, microelectromechanical systems; NEMS, nanoelectromechanical systems; PNA, peptide nucleic acid; MOCHs, Morpholino Oligonucleotide Crosslinked Hydrogels; AAm, acrylamide; HPK, 1-hydroxycyclohexyl phenyl ketone.REFERENCESADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY (1) Ahn, S. K.; Kasi, R. M.; Kim, S. C.; Sharma, N.; Zhou, Y. Stimuli-Responsive Polymer Gels. Soft Matter 2008, 4, 1151-1157.(2) Wei, M.; Gao, Y.; Li, X.; Serpe, M. J. Stimuli-Responsive Polymers and Their Applications. 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