Mercury is a highly toxic environmental pollutant with bioaccumulative properties. Therefore, new materials are required to not only detect but also effectively remove mercury from environmental sources such as water. We herein describe a polyacrylamide hydrogel-based sensor functionalized with a thymine-rich DNA that can simultaneously detect and remove mercury from water. Detection is achieved by selective binding of Hg(2+) between two thymine bases, inducing a hairpin structure where, upon addition of SYBR Green I dye, green fluorescence is observed. In the absence of Hg(2+), however, addition of the dye results in yellow fluorescence. Using the naked eye, the detection limit in a 50 mL water sample is 10 nM Hg(2+). This sensor can be regenerated using a simple acid treatment and can remove Hg(2+) from water at a rate of approximately 1 h(-1). This sensor was also used to detect and remove Hg(2+) from samples of Lake Ontario water spiked with mercury. In addition, these hydrogel-based sensors are resistant to nuclease and can be rehydrated from dried gels for storage and DNA protection. Similar methods can be used to functionalize hydrogels with other nucleic acids, proteins, and small molecules for environmental and biomedical applications.

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Regenerable DNA-functionalized hydrogels for ultrasensitive, instrument-free mercury(II) detection and removal in water.

Microgels are colloidal gel particles that consist of chemically cross-linked three-dimensional polymer networks; these networks are able to dramatically shrink or swell by expelling or absorbing large amounts of water in response to external stimuli. 2] The large change in size can be achieved, for example, by modifying the pH, temperature, or ionic strength of the medium, or by applying electric or magnetic fields; it is this response that makes microgels desirable for applications in drug delivery, biosensing, diagnostics, bioseparations, and optical devices. 10] To further expand their range of applicability, there have been efforts to generate microgels that have been complexed with preformed functionalized materials that impart additional desirable properties to the microgel. These preformed materials range from molecules to microparticles and are typically complexed with the gel matrix through specific interactions. The resulting complexed microgels usually show a drastic decrease in their physical response to external stimuli compared to that of the original cross-linked polymer networks; this is an undesirable side effect since the microgel performance for a given application is based on its sensitivity to external stimuli. In addition to functionality, the size distribution of a population of microgels is important; it is critical to provide a homogeneous distribution of microgels applying formulations and in controlling the release kinetics of encapsulates or adsorbents. From the standpoint of performance and applicability, there is a need for methods to generate monodisperse microgels that maintain high sensitivity to external stimuli irrespective of the materials that are incorporated to add complementary functions. Here, we describe a flexible and straightforward method for generating monodisperse suspensions of new microgelbased materials using a capillary microfluidic technique. This technique enabled us to generate and precisely control the size of the microgel-based particles without sacrificing the physical response of the resulting microgels. We generated two novel microgel structures: a spherical microgel shell and spherical microgel particles that retain their full sensitivity to external stimuli after being physically complexed with preformed colloidal particles. The overall size and thickness of the microgel shells can be tuned with temperature. We generated the spherical microgel particles in a single step, which allows us to freely incorporate functional materials into the polymer network. We used quantum dots, magnetic nanoparticles, and polymer microparticles as examples of the materials that can be added to provide specific chemical, physical, or mechanical properties to the original microgels. To generate the microgel particles, we constructed a capillary-based microfluidic device that generated pre-microgel drops, which were then polymerized in situ with a redox reaction. The capillary microfluidic device was made of three separate capillary tubes. The two internal cylindrical tubes served as injection and collection tubes and were coaxially aligned, as shown in the inset in Figure 1A. These tapered tubes were made by axially heating and pulling cylindrical capillaries. In the region near both tips, the outer fluid focuses both the middle and inner fluids through the collection tube to form a fluid thread that then breaks into drops as a result of hydrodynamic instabilities, as shown in Figure 1A. We typically used silicon oil with viscosity hOF= 125 mPas as the outer, or continuous-phase liquid. The middle fluid was an aqueous monomer solution that contained N-isopropylacrylamide (NIPAm, 15.5% w/v), a crosslinker (N,N’-methylenebisacrylamide, BIS, 1.5% w/v), a reaction accelerator (N,N,N’,N’-tetramethylethylenediamine, 2 vol%), and two co-monomers [2-(methacryloyloxy) ethyl trimethyl ammonium chloride (METAC, 2 vol%) and allylamine (1 vol%)]. METAC was added to increase the coil-toglobule transition temperature of poly(NIPAm), thereby facilitating homogeneous polymerization at room temperature. The allylamine adds amine groups to the network, which can subsequently be labeled with dyes after the formation of the microgel particles. The chemical formula [*] Dr. J. W. Kim, A. S. Utada, Dr. A. Fern ndez-Nieves, Prof. D. A. Weitz DEAS and Department of Physics Harvard University Cambridge, MA 02138 (USA) Fax: (+1)617-495-2875 E-mail: weitz@deas.harvard.edu

Fabrication of Monodisperse Gel Shells and Functional Microgels in Microfluidic Devices

The interaction between DNA and inorganic surfaces has attracted intense research interest, as a detailed understanding of adsorption and desorption is required for DNA microarray optimization, biosensor development, and nanoparticle functionalization. One of the most commonly studied surfaces is gold due to its unique optical and electric properties. Through various surface science tools, it was found that thiolated DNA can interact with gold not only via the thiol group but also through the DNA bases. Most of the previous work has been performed with planar gold surfaces. However, knowledge gained from planar gold may not be directly applicable to gold nanoparticles (AuNPs) for several reasons. First, DNA adsorption affinity is a function of AuNP size. Second, DNA may interact with AuNPs differently due to the high curvature. Finally, the colloidal stability of AuNPs confines salt concentration, whereas there is no such limit for planar gold. In addition to gold, graphene oxide (GO) has emerged as a new material for interfacing with DNA. GO and AuNPs share many similar properties for DNA adsorption; both have negatively charged surfaces but can still strongly adsorb DNA, and both are excellent fluorescence quenchers. Similar analytical and biomedical applications have been demonstrated with these two surfaces. The nature of the attractive force however, is different for each of these. DNA adsorption on AuNPs occurs via specific chemical interactions but adsorption on GO occurs via aromatic stacking and hydrophobic interactions. Herein, we summarize the recent developments in studying non-thiolated DNA adsorption and desorption as a function of salt, pH, temperature and DNA secondary structures. Potential future directions and applications are also discussed.

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Adsorption of DNA onto gold nanoparticles and graphene oxide: surface science and applications

Methods and apparatus for implementing microfluidic analysis devices are provided. A monolithic elastomer membrane associated with an integrated pneumatic manifold allows the placement and actuation of a variety of fluid control structures, such as structures for pumping, isolating, mixing, routing, merging, splitting, preparing, and storing volumes of fluid. The fluid control structures can be used to implement a variety of sample introduction, preparation, processing, and storage techniques.

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Fluid control structures in microfluidic devices

Microfluidic devices for DNA sequencing: sample preparation and electrophoretic analysis.

Acrylamide-modified DNA probes are immobilized in polycarbonate microfluidic channels via photopolymerization in a polyacrylamide matrix. The resulting polymeric, hydrogel plugs are porous under electrophoretic conditions and hybridize with fluorescently tagged complementary DNA. The double-stranded DNA can be chemically denatured, and the chip may be reused with a new analytical sample. Conditions for photopolymerization, hybridization, and denaturation are discussed. We also demonstrate the photopolymerization of plugs containing different DNA probe sequences in one microfluidic channel, thereby enabling the selective detection of multiple DNA targets in one electrophoretic pathway.

Immobilization of DNA hydrogel plugs in microfluidic channels.

A method and device for analyzing species in a sample includes immobilized ligands in a porous matrix disposed in a microchannel. A method of analysis is provided by loading a sample to the microchannel and establishing an analyte flow through the porous matrix. The analyte is detected as the species that binds to the ligand immobilized in the porous matrix. The present method may be adapted to identify multiple species present in a sample by immobilizing different ligands in a series in a single microfluidic channel by sequentially photopolymerization of various porous matrixes, each containing different ligands.

Kimberly G. Olsen

Papers

Immobilization of DNA hydrogel plugs in microfluidic channels.

Bio-affinity porous matrix in microfluidic channels