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Hybridization between nucleic acid strands immobilized on a solid support with partners in solution is widely practiced in bioanalytical technologies and materials science. An important fundamental aspect of understanding these reactions is the role played by immobilization in the dynamics of duplex formation and disassembly. This report reviews and analyzes literature kinetic data to identify commonly observed trends and to correlate them with probable molecular mechanisms. The analysis reveals that while under certain conditions impacts from immobilization are minimal so that surface and solution hybridization kinetics are comparable, it is more typical to observe pronounced offsets between the two scenarios. In the forward (hybridization) direction, rates at the surface commonly decrease by one to two decades relative to solution, while in the reverse direction rates of strand separation at the surface can exceed those in solution by tens of decades. By recasting the deviations in terms of activation barriers, a consensus of how immobilization impacts nucleation, zipping, and strand separation can be conceived within the classical mechanism in which duplex formation is rate limited by preassembly of a nucleus a few base pairs in length, while dehybridization requires the cumulative breakup of base pairs along the length of a duplex. Evidence is considered for how excess interactions encountered on solid supports impact these processes.

Nanopore structures have been successfully employed in next-generation DNA sequencing. For more complicated protein which normally contains 20 different amino acids, identifying the fluctuation of ionic current caused by different amino acids appears inadequate for protein sequencing. Therefore, it is highly desirable to develop size-controllable nanopores with optical activity that can provide additional structural information. Herein, we discovered the novel nanopore properties of the self-assembled ultramicroelectrodes originally developed by Bard and co-workers. Using a slightly modified method, the self-assembly of 7 ± 1 nm gold nanoparticles (AuNPs) can be precisely controlled to form a gold nanoporous sphere (GPS) on the tip of a glass capillary. Different dithiol linker molecules (1,3-propanedithiol, C3; 1,6-hexanedithiol, C6; and 1,9-nonanedithiol, C9) reproducibly led to rather similar nanopore sizes (5.07 ± 0.02, 5.13 ± 0.02, and 5.25 ± 0.01 nm), respectively. The GPS nanostructures were found to exhibit high ionic current rectification as well as surface-enhanced Raman scattering (SERS) activity due to the presence of nanopores and numerous "hot spots" among the cross-linked AuNPs on the surface of GPS. The rectification effect of the small nanopores was observed even under high concentration of electrolyte (290 mM), along with SERS enhancement factors well above 1 × 10 5 . The GPS nanostructures were successfully applied for SERS-based detection of glutathione from a single HeLa cell.

This study demonstrates efficient electrostatic control of surface hybridization through use of morpholinos, a charge-neutral DNA mimic, as the immobilized "probes". In addition to being compatible with low ionic strengths, use of uncharged probes renders the field interaction specific to the nucleic acid analyte. In contrast to DNA probes, morpholino probes enable facile cycling between hybridized and dehybridized states within minutes. Impact of ionic strength and temperature on the effectiveness of electrostatics to direct progress of hybridization is evaluated. Optimal electrostatic control is found when stability of probe-analyte duplexes is set so that electrostatics can efficiently switch between the forward (hybridization) and reverse (dehybridization) directions.

Diagnostic applications of morpholinos take advantage of their unique properties including backbone charge neutrality, a weak impact of ionic strength on their hybridization behavior, and their resistance to enzymatic degradation. This chapter overviews how these properties have advanced transduction and other capabilities useful for the analysis of nucleic acids. In many cases, the benefits stem from electrostatic mechanisms; for example, use of low ionic strengths improves sensitivity of detection while decreasing background signals because only the nucleic acid analyte is charged. While most literature reports focus on in vitro assays in buffer, morpholinos have been also used for biodistribution measurements of species such as fungal rRNA and miRNA. After reviewing the diagnostic applications of morpholinos, the chapter describes preparation of morpholino monolayers on metal supports for electrochemical diagnostics and the procedure for performing label-free detection of DNA from changes in surface capacitance.

Hybridization of solution nucleic acids to DNA brushes is widely encountered in diagnostic and materials science applications. Typically, brush chain lengths of ten or more nucleotides are used to provide the needed sequence specificity and binding affinity. At these lengths, coincidental occurrence of complementary regions is expected to lead to associations between the nominally single-stranded brush chains due to intra- or interchain base pairing. This report investigates how these associations impact the brushes' hybridization activity toward complementary "target" sequences. Brushes were prepared from 20-mer chains with four-nucleotide-long "adhesive regions" through which neighboring chains could interact. The affinity and position of the adhesive region along the chain backbone were varied. DNA brushes were exposed to complementary solution targets, and the corresponding melting transitions were measured to estimate free energies of the brush-target hybridization. These results revealed that higher affinity adhesive regions more extensively suppressed brush hybridization relative to hybridization in solution. Associations near the middle of the chains were found to be more penalizing than those at the immobilized or the free end of the chains. Provided that the brush chains were close enough to associate, changes in brush density did not exert a significant effect on hybridization thermodynamics within the investigated coverage window. Comparison of the DNA brush results with those from commercial Affymetrix single-nucleotide-polymorphism (SNP) microarrays revealed agreement in the impact of chain associations on hybridization.

We describe a microfluidic concentration device to accelerate the surface hybridization reaction between DNA and morpholinos (MOs) for enhanced detection. The microfluidic concentrator comprises a single polydimethylsiloxane (PDMS) microchannel onto which an ion-selective layer of conductive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (, Pedot: PSS) was directly printed and then reversibly surface bonded onto a morpholino microarray for hybridization. Using this electrokinetic trapping concentrator, we could achieve a maximum concentration factor of ∼800 for DNA and a limit of detection of 10 nM within 15 min. In terms of the detection speed, it enabled faster hybridization by around 10-fold when compared to conventional diffusion-based hybridization. A significant advantage of our approach is that the fabrication of the microfluidic concentrator is completely decoupled from the microarray; by eliminating the need to deposit an ion-selective layer on the microarray surface prior to device integration, interfacing between both modules, the PDMS chip for electrokinetic concentration and the substrate for DNA sensing are easier and applicable to any microarray platform. Furthermore, this fabrication strategy facilitates a multiplexing of concentrators. We have demonstrated the proof-of-concept for multiplexing by building a device with 5 parallel concentrators connected to a single inlet/outlet and applying it to parallel concentration and hybridization. Such device yielded similar concentration and hybridization efficiency compared to that of a single-channel device without adding any complexity to the fabrication and setup. These results demonstrate that our concentrator concept can be applied to the development of a highly multiplexed concentrator-enhanced microarray detection system for either genetic analysis or other diagnostic assays.

Hybridization thermodynamics on solid supports are compared with those in solution for two types of hybridization probe, DNA and uncharged morpholino oligonucleotides of identical sequences. Trends in hybridization affinity are discussed with respect to ionic strength, temperature, and surface behavior.

Electrokinetic methods that conveniently concentrate charged analytes by orders of magnitude are highly attractive for nucleic acid assays where they can bypass the complexity and costs of enzyme-based amplification. The present study demonstrates an electrokinetic concentration device incorporating charge-neutral morpholino (MO) probes: as DNA analyte is concentrated in a microfluidic channel using ion concentration polarization (ICP) it is simultaneously hybridized to spots of complementary MO probes immobilized on the channel floor. This approach is uniquely favored by the match between the optimum buffer ionic strength of approximately 10mM for both MO-DNA surface hybridization and electrokinetic concentration. The simple and easily scalable poly(dimethylsiloxane) (PDMS) microfluidic device was fabricated using soft lithography and contact printing of a conductive polymer, poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate (, Pedot: PSS) as a cation-selective membrane material. Using the microfluidic concentrator, we could increase the concentration of DNA by three orders of magnitude in less than 5 min at an electric field of 75 Vcm(-1). The 1000-fold increase in concentration of DNA led to an increase in the speed of MO-DNA hybridization by two orders of magnitude and enabled a detection sensitivity of ~1 nM within 15 min of concentration. Using the proposed microfluidic concentrator, we also demonstrated a rapid hybridization with a binary DNA mixture, containing a fully complementary and a non-complementary sequence to mimic molecular backgrounds present in real DNA samples., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Interactions of proteins with nucleic acids arise at all levels of cellular function, from chromosomal packing to biological regulation. These interactions can be analyzed in a high-throughput fashion by immobilizing the DNA sequences of interest, possibly numbering in the thousands, at discrete locations on a solid support and identifying those sequences that a protein analyte binds. Ideally, such surface assays would use unlabeled analyte to simplify protocols and avoid the possibility of perturbing the protein/DNA interaction. The present study compares three electrochemical modalities for simultaneously detecting binding of unlabeled transcription factor proteins to immobilized DNA duplexes based on (i) changes in the duplex diffusive motions, (ii) variations in the surface potential, and (iii) variations in the interfacial charging impedance, all of which can be conveniently derived from AC voltammetry traces. Cro protein from bacteriophage lambda is used as a model transcription factor. Specific binding of protein was successfully detected through modalities (i) and (ii), but not (iii). The effectiveness of these techniques is compared as a function of sampling frequency and protein concentration. Binding of 15 kDa Cro slowed down rotational diffusion of immobilized duplexes approximately 3-fold, and induced up to 5 mV changes in the surface potential. Moreover, by assessing Cro binding to bacteriophage operators of variable affinity, the study illustrates how contrast between specific and nonspecific interactions impacts detection.

A near-field scanning microwave microscope (NSMM) is used to readout and visualize homemade 10-mer oligonucleotide microarrays and an Agilent 60-mer DNA microarray as a realistic test of NSMM applicability to multiplexed sequence analysis. Sensitive characterization of DNA coverage and high resolution mapping of DNA spots in the microarray were realized by measuring the change of microwave reflection coefficient (S₁₁) at about 4 GHz operating frequency. Hybridization between target (free) and capture (immobilized) sequences leads to changes in the microwave reflection coefficient, which were measured by the NSMM. These changes are caused by hybridization-induced modification of the dielectric permittivity profile of the DNA film. The dynamic range based on analysis of the 10-mer microarrays is over 3 orders of magnitude with the detection limit estimated below 0.01 strands/μm². The NSMM method should be readily capable of detecting target coverages down to 98% of probe coverage. We also directly image the patterned DNA microarray by NSMM with a 2 μm resolution. The complementary optical image of the DNA microarray visualized by using a relative fluorescent intensity metric agrees well with the NSMM results., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A principal challenge in microarray experiments is to facilitate hybridization between probe strands on the array with complementary target strands from solution while suppressing any competing interactions that the probes and targets may experience. Synthetic DNA analogs, whose hybridization to targets can exhibit qualitatively different dependence on experimental conditions than for nucleic acid probes, open up an attractive alternative for improving selectivity of array hybridization. Morpholinos (MOs), a class of uncharged DNA analogs, are investigated as microarray probes instead of DNA. MO microarrays were fabricated by contact printing of amino-modified probes onto aldehyde slides. In addition to covalent immobilization, MOs were found to efficiently immobilize through physical adsorption; such physically adsorbed probes could be removed by post-printing washes with surfactant solutions. Hybridization of double-stranded DNA targets to MO microarrays revealed a hybridization maximum at intermediate ionic strengths. The decline in hybridization at lower ionic strengths was attributed to an electrostatic barrier accumulated from hybridized DNA targets, whereas at higher ionic strengths it was attributed to stabilization of target secondary structure in solution. These trends, which illustrate ionic strength tuning of forming on-array relative to solution secondary structure, were supported by a stability analysis of MO/DNA and DNA/DNA duplexes in solution., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Thermal denaturation, or melting, measurements are a classic technique for analysis of thermodynamics of nucleic base driven associations in solution, as well as of interactions between nucleic acids and small molecule ligands such as drugs or carcinogens. Performed on surface-immobilized DNA films, this well-established technique can help understand how energetics of surface hybridization relate to those in solution, as well as provide high-throughput platforms for screening of small molecule ligands. Here we describe methods for measuring DNA melting transitions at solid/liquid interfaces with focus on the role of immobilization chemistry, including a common "immobilization-through-self-assembly" approach that is effective at moderate temperatures, and a thermo-stable approach based on polymer-supported DNA monolayers that can be used at elevated temperatures. We also discuss conditions necessary for reversible measurements, as signified by superimposition of the association (cooling) and dissociation (heating) transitions of immobilized DNA strands.

A variety of solution methods exist for analysis of interactions between small molecule ligands and nucleic acids; however, accomplishing this task economically at the scale of hundreds to thousands of sequences remains challenging. Surface assays offer a prospective solution through array-based multiplexing, capable of mapping out the full sequence context of a DNA/ligand interaction in a single experiment. However, relative to solution assays, accurate quantification of DNA/ligand interactions in a surface format must contend with limited understanding of molecular activities and interactions at a solid-liquid interface. We report a surface adaptation of a solution method in which shifts in duplex stability, induced by ligand binding and quantified from melting transitions, are used for thermodynamic analysis of DNA/ligand interactions. The results are benchmarked against solution calorimetric data. Equilibrium operation is confirmed through superposition of denaturation/hybridization transitions triggered by heating and cooling. The antibiotic compound netropsin, which undergoes electrostatic and sequence-specific minor groove interactions with DNA, is used as a prototypical small molecule. DNA/netropsin interactions are investigated as a function of ionic strength and drug concentration through electrochemical tracing of surface melt transitions. Comparison with solution values finds excellent agreement in free energy, though reliable separation into enthalpic and entropic contributions proves more difficult. The results establish key guidelines for analysis of DNA-ligand interactions via reversible melting denaturation at surfaces.

Monolayer films of nucleic acids on solid supports are encountered in a range of diagnostic and bioanalytical applications. These applications often rely on elevated temperatures to improve performance; moreover, studies at elevated temperatures can provide fundamental information on layer organization and functionality. To support such applications, this study compares thermostability of oligonucleotide monolayers immobilized to gold by first coating the gold with a nanometer-thick film (an "anchor layer") of a polymercaptosiloxane, to which DNA oligonucleotides are subsequently tethered through maleimide-thiol conjugation, with thermostability of monolayers formed via widely used attachment through a terminal thiol moiety on the DNA. The temperature range covered is from 25 to 90 °C. After confirming stability of immobilization and, more importantly, retention of hybridization activity even under the harshest conditions investigated, these thermostable films are used to demonstrate measurements of (1) reversible surface melting transitions and (2) temperature dependence of competitive hybridization, when fully matched and mismatched sequences compete for binding to immobilized DNA oligonucleotides. The competitive hybridization experiments reveal a pronounced impact of temperature on rates of approach to equilibrium, with kinetic freezing into nonequilibrium states close to room temperature and rapid approach to equilibrium at elevated temperatures. Modeling of competitive surface hybridization equilibria using thermodynamic parameters derived from surface melting transitions of the individual sequences is also discussed.

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.

DNA microarrays have proven extraordinarily powerful for differential expression studies across thousands of genes in a single experiment. Microarrays also have the potential for clinical applications, including the detection of infectious and immunological diseases and cancer, if they can be rendered both reliable and cost-effective. Here we report the first practical application of an active microarray based on integrated circuit technology, completely obviating the need for external measurement instrumentation while employing protocols compatible with traditional fluorescence-based surface bioassays. In a gene expression biodosimetry study, we determine the differential activity of genes from leucocytes in irradiated human blood. Quantum dots are used as fluorescence labels to realize filterless, time-gated fluorescence detection on an active complementary metal-oxide-semiconductor (CMOS) microarray with 100-pM sensitivity. Improvements in surface chemistry should allow sensitivities that approach the microarray hardware limit of less than 10 pM. Techniques for covalent attachment of DNA capture strands to the CMOS active microarrays allow integrated sensors to be placed in immediate proximity to hybridized analyte strands, maximizing photon collection efficiencies., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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.

Five electroactive ferrocene tags for labelling of biomolecules are contrasted with regard to conjugation reactivity with amine and thiol moieties, stability to loss of electrochemical activity, and impact of molecular structure on the redox potential of the free and DNA-conjugated forms.

Impedance and cyclic voltammetry methods, complemented by Poisson-Boltzmann (PB) modeling, are used to study hybridization of DNA analyte strands to monolayers of morpholino oligomers (MOs) immobilized by one end to mercaptopropanol-passivated gold electrodes. MOs, like peptide nucleic acids (PNAs), are uncharged molecules that recognize nucleic acids following conventional base-pairing rules. The capacitive response to hybridization, determined from real-time impedance measurements, is analyzed with emphasis on understanding the underlying structural changes and on providing a foundation for label-free diagnostics. The capacitive response is correlated with the instantaneous surface molecular populations by labeling DNA and MO strands with ferrocene tags and using cyclic voltammetry to monitor their respective coverages in real-time. This approach allows analysis of hybridization-induced changes in interfacial capacitance as a function of duplex coverage, the DC bias used for readout, buffer molarity, and probe coverage. The results indicate that unhybridized MO layers exist in a compact state on the solid support. For hybridized layers, the intrinsic signal per hybridization event is strongly enhanced at low ionic strengths but, interestingly, does not depend on the readout bias in the sampled range negative of the capacitive minimum. A PB model incorporating an effective medium description of the hybridizing films is used to establish how hybridization-derived changes in dielectric composition and charge distribution at the surface translate into experimentally observed variations in interfacial capacitance.

Synthetic nucleic acid mimics provide opportunity for redesigning the specificity and affinity of hybridization with natural DNA or RNA. Such redesign is of great interest for diagnostic applications where it can enhance the desired signal against a background of competing interactions. This report compares hybridization of DNA analyte strands with morpholinos (MOs), which are uncharged nucleic acid mimics, to the corresponding DNA-DNA case in solution and on surfaces. In solution, MO-DNA hybridization is found to be independent of counterion concentration, in contrast to DNA-DNA hybridization. On surfaces, when immobilized MO or DNA "probe" strands hybridize with complementary DNA "targets" from solution, both the MO-DNA and DNA-DNA processes depend on ionic strength but exhibit qualitatively different behaviors. At lower ionic strengths, MO-DNA surface hybridization exhibits hallmarks of kinetic limitations when separation between hybridized probe sites becomes comparable to target dimensions, whereas extents of DNA-DNA surface hybridization are instead consistent with limits imposed by buildup of surface (Donnan) potential. The two processes also fundamentally differ at high ionic strength, under conditions when electrostatic effects are weak. Here, variations in probe coverage have a much diminished impact on MO-DNA than on DNA-DNA hybridization for similarly crowded surface conditions. These various observations agree with a structural model of MO monolayers in which MO-DNA duplexes segregate to the buffer interface while unhybridized probes localize near the solid support. A general perspective is presented on using uncharged DNA analogues, which also include compounds such as peptide nucleic acids (PNA), in surface hybridization applications.

The design and interpretation of surface hybridization assays is complicated by poorly understood aspects of the interfacial environment that cause both kinetic and thermodynamic behaviors to deviate from those in solution. The origins of these differences lie in the additional interactions experienced by hybridizing strands at the surface. In this report, an analysis of surface hybridization equilibria is provided for end-tethered, single-stranded oligonucleotide "probes" hybridizing with similarly sized, single-stranded solution "target" molecules. Theoretical models by Vainrub and Pettitt (Phys. Rev. E 2002, 66, 041905) and by Halperin, Buhot, and Zhulina (Biophys. J. 2004, 86, 718), and an "extended" model that in addition includes a solution-like salt dependence of probe-target dimerization, are compared to experiments as a function of salt concentration and probe coverage. Good agreement with experiment is observed when the DNA volume fraction at the surface remains below approximately 0.25. None of the models, however, can account for strong suppression of hybridization when the volume fraction of DNA approaches 0.3, realizable in the limit of high buffer strength and densely tethered films. Under these conditions, hybridization yields become insensitive to increases in analyte concentration even though many probes remain available to bind targets. These observations are attributed to the onset of packing constraints which, interestingly, become limiting significantly below maximum DNA coverages estimated from ideally efficient hexagonal packing. By delineating conditions under which specific hybridization behaviors are observed, the results advance fundamental knowledge in support of DNA microarray and biosensor applications.

This paper reports the application of frozen cyclohexane-in-water emulsions as sacrificial templates for the fabrication of hollow microcapsules through layer-by-layer assembly of polyelectrolytes, poly(styrenesulfonate sodium salt), and poly(allylamine hydrochloride). Extraction of the cyclohexane phase from frozen emulsions stabilized with 11 polyelectrolyte layers by compatibilization with 30% v/v ethanol leads to the formation of water-filled microcapsules while preserving the spherical geometry. The majority of microcapsules (>90%) are prepared with intact polyelectrolyte membranes as measured by their deformation induced by osmotic pressure. This work provides a new route for the synthesis of hollow multilayered microcapsules under mild operating conditions.

This paper describes the design of an active, integrated CMOS sensor array for fluorescence applications which enables time-gated, time-resolved fluorescence spectroscopy. The 64-by-64 array is sensitive to photon densities as low as 8.8 × 10(6) photons/cm(2) with 64-point averaging and, through a differential pixel design, has a measured impulse response of better than 800 ps. Applications include both active microarrays and high-frame-rate imagers for fluorescence lifetime imaging microscopy.

Surface hybridization, a reaction in which nucleic acid molecules in solution react with nucleic acid partners immobilized on a surface, is widely practiced in life science research. In these applications the immobilized partner, or "probe", is typically single-stranded DNA. Because DNA is strongly charged, high salt conditions are required to enable binding between analyte nucleic acids ("targets") in solution and the DNA probes. High salt, however, compromises prospects for label-free monitoring or control of the hybridization reaction through surface electric fields; it also stabilizes secondary structure in target species that can interfere with probe-target recognition. In this work, initial steps toward addressing these challenges are taken by introducing morpholinos, a class of uncharged DNA analogues, for surface-hybridization applications. Monolayers of morpholino probes on gold supports can be fabricated with methods similar to those employed with DNA and are shown to hybridize efficiently and sequence-specifically with target strands. Hybridization-induced changes in the interfacial charge organization are analyzed with electrochemical methods and compared for morpholino and DNA probe monolayers. Molecular mechanisms connecting surface hybridization state to the interfacial capacitance are identified and interpreted through comparison to numerical Poisson-Boltzmann calculations. Interestingly, positive as well as negative capacitive responses (contrast inversion) to hybridization are possible, depending on surface populations of mobile ions as controlled by the applied potential. Quantitative comparison of surface capacitance with target coverage (targets/area) reveals a nearly linear relationship and demonstrates sensitivities (limits of quantification) in the picogram per square millimeter range.

Optical biosensing based on fluorescence detection has arguably become the standard technique for quantifying extents of hybridization between surface-immobilized probes and fluorophore-labeled analyte targets in DNA microarrays. However, electrochemical detection techniques are emerging which could eliminate the need for physically bulky optical instrumentation, enabling the design of portable devices for point-of-care applications. Unlike fluorescence detection, which can function well using a passive substrate (one without integrated electronics), multiplexed electrochemical detection requires an electronically active substrate to analyze each array site and benefits from the addition of integrated electronic instrumentation to further reduce platform size and eliminate the electromagnetic interference that can result from bringing non-amplified signals off chip. We report on an active electrochemical biosensor array, constructed with a standard complementary metal-oxide-semiconductor (CMOS) technology, to perform quantitative DNA hybridization detection on chip using targets conjugated with ferrocene redox labels. A 4 x 4 array of gold working electrodes and integrated potentiostat electronics, consisting of control amplifiers and current-input analog-to-digital converters, on a custom-designed 5 mm x 3 mm CMOS chip drive redox reactions using cyclic voltammetry, sense DNA binding, and transmit digital data off chip for analysis. We demonstrate multiplexed and specific detection of DNA targets as well as real-time monitoring of hybridization, a task that is difficult, if not impossible, with traditional fluorescence-based microarrays.

The physical behavior of polyelectrolytes at solid-liquid interfaces presents challenges both in measurement and in interpretation. An informative, yet often overlooked, property that characterizes the equilibrium organization of these systems is their membrane or rest potential. Here a general classification scheme is presented of the relationship between the rest potential and structural response of polyelectrolyte films to salt concentration. A numerical lattice theory, adapted from the polymer community, is used to analyze the rest potential response of end-tethered polyelectrolyte layers in which electrostatics and short-range contact interactions conspire to bring about different structural states. As an experimental quantity the rest potential is a readily accessible, nonperturbing metric of the equilibrium structure of a polyelectrolyte layer. A first set of measurements is reported on monolayers of end-tethered, single-stranded DNA in monovalent (NaCl) and divalent (MgCl(2)) counterion environments. Intriguingly, in NaCl electrolyte at least two different mechanisms appear by which the DNA layers can structurally relax in response to changing salt conditions. In MgCl(2) the layers appear to collapse. The possible molecular mechanisms behind these behaviors are discussed. These studies provide insight into phenomena more generally underlying polyelectrolyte applications in the chemical, environmental, and biotechnological fields.

Surface hybridization reactions, in which sequence-specific recognition occurs between immobilized and solution nucleic acids, are routinely carried out to quantify and interpret genomic information. Although hybridization is fairly well understood in bulk solution, the greater complexity of an interfacial environment presents new challenges to a fundamental understanding, and hence application, of these assays. At a surface, molecular interactions are amplified by the two-dimensional nature of the immobilized layer, which focuses the nucleic acid charge and concentration to levels not encountered in solution, and which impacts the hybridization behavior in unique ways. This study finds that, at low ionic strengths, an electrostatic balance between the concentration of immobilized oligonucleotide charge and solution ionic strength governs the onset of hybridization. As ionic strength increases, the importance of electrostatics diminishes and the hybridization behavior becomes more complex. Suppression of hybridization affinity constants relative to solution values, and their weakened dependence on the concentration of DNA counterions, indicate that the immobilized strands form complexes that compete with hybridization to analyte strands. Moreover, an unusual regime is observed in which the surface coverage of immobilized oligonucleotides does not significantly influence the hybridization behavior, despite physical closeness and hence compulsory interactions between sites. These results are interpreted and summarized in a diagram of hybridization regimes that maps specific behaviors to experimental ranges of ionic strength and probe coverage.

DNA monolayers are widely used in fundamental and applied genomics and are versatile experimental models for elucidating the behavior of charged polymers at interfaces. The physical behavior of these systems is to a large extent governed by their internal ionic microenvironment, which is investigated here for layers of end-tethered, single-stranded DNA oligonucleotides (DNA brushes). Retention of counterions by the DNA brush manifests as lowered susceptibility of the interfacial capacitance to external salt conditions. A physical model based on concepts adapted from polymer science was used to further elucidate the connection between monolayer organization and its charging behavior. The data indicate a reorganization of the monolayer with changes in ionic strength and strand coverage that is consistent with that expected for a polyelectrolyte brush. A method for electrochemical quantification of strand coverage, based on shift of reduction potential for redox counterions associated with the DNA monolayer, is also described. These results provide guidance for development of label-free electrochemical diagnostics employing DNA monolayers and formulate a description of monolayer behavior within a polymer science framework.

Biological assays often rely on "reporter labels" to enhance measurement sensitivity, for example, by incorporation of a fluorescent dye or a nanoparticle into a nucleic acid or a protein. Use of labels, however, complicates sample preparation, increases assay costs, and can cause experimental artifacts by interfering with assay thermodynamics or limiting label stability. We evaluate near-field microwave imaging (NFMI) as an alternative, label-free technique for molecular diagnostics. Using DNA monolayers as an experimental model, NFMI is demonstrated to achieve sensitivities comparable to conventional fluorescence bioassays. Moreover, NFMI is shown to be compatible with imaging at resolutions required by microarray applications, as demonstrated by monitoring DNA hybridization in an array format.

Detection and sequence-identification of nucleic acid molecules is often performed by binding, or hybridization, of specimen "target" strands to immobilized, complementary "probe" strands. A familiar example is provided by DNA microarrays used to carry out thousands of solid-phase hybridization reactions simultaneously to determine gene expression patterns or to identify genotypes. The underlying molecular process, namely sequence-specific recognition between complementary probe and target molecules, is fairly well understood in bulk solution. However, this knowledge proves insufficient to adequately understand solid-phase hybridization. For example, equilibrium binding constants for solid-phase hybridization can differ by many orders of magnitude relative to solution values. Kinetics of probe-target binding are affected. Surface interactions, electrostatics and polymer phenomena manifest themselves in ways not experienced by hybridizing strands in bulk solution. The emerging fundamental understanding provides important insights into application of DNA microarray and biosensor technologies.

Surface-tethered nucleic acids are widely applied in solid-phase assays in which complementary strands must be detected against a complex mixture of other sequences. In response to such needs, numerous methods have been developed for immobilizing nucleic acids on solid supports. Often, detailed analysis of associated chemical transformations and of potential side reactions is difficult to obtain. Combined use of planar and high surface area powder supports allows characterization using surface as well as bulk diagnostic techniques. This approach is followed in the present study in which X-ray photoelectron spectroscopy (XPS), transmission infrared spectroscopy (FTIR) and reactivity titrations are used to investigate siliceous supports modified with an aminosilane precursor followed by a maleimide-bearing crosslinker for attachment of nucleic acids. The supports retain maleimide activity for approximately a day when stored under buffer, but deactivation is accelerated under basic conditions or by incomplete conversion of the precursor aminosilane monolayer. Reactions involving the olefinic bond of the imide as well as its carbonyl groups are observed and analyzed. Attachment of sulfhydryl-terminated oligodeoxyribonucleotides is highly site specific, and immobilized strands exhibit excellent hybridization activity. Quantitative use of XPS for label-free determination of DNA coverage based on calibration against reference materials is also described.

Monolayers of DNA chains of polymeric dimensions, considered here to be longer than approximately 100 nucleotides, are widely encountered in biomolecular diagnostics as well as present for a model system for investigating behavior of polyelectrolytes at interfaces. A major challenge in advancing such applications is assembling the DNA on the surface in a controlled way. Although covalent immobilization is expected to produce optimal stability, the multitude of potential reactive sites along the contour of long DNA molecules requires that any chemical transformations be strictly site-specific to preserve control over attachment geometry and function. A synthetic approach to fabricating monolayers of DNA genes on gold using polymeric anchor (adhesion) films is presented that (i) possesses stringent site-specificity of surface-attachment, (ii) exhibits excellent stability to elevated temperatures, allowing denaturation of duplex chains at 90 degrees C without loss of surface-linked strands, and (iii) achieves surface coverages suitable for investigating multichain polyelectrolyte behavior in regimes of strong interchain interactions.

Reaction of N-phenyl maleimide (NPM) with silica surfaces modified with a self-assembled monolayer of (aminopropyl)triethoxysilane (APTES) was investigated using infrared spectroscopy (FTIR), elemental analysis, and titration assays. This reaction is of interest as a test case for using amine-maleimide coupling for immobilization of biomolecules. Addition of NPM to surface APTES residues was consistently sub-stoichiometric, with typical yields of about 75% on monolayers with a coverage of 1.15 APTES residues/nm2. Titration analysis found negligible presence of imide alkene C=C bonds in modified supports, indicating that addition of NPM to APTES proceeded via amine attack at the imide olefinic bond. FTIR measurements also revealed presence of amide bands which intensified over periods of 10 h. These observations were attributed to a slower secondary process in which APTES amines attack imide carbonyls to produce amide linkages. Stability of NPM-modified surfaces was examined under room temperature storage in pH 7 buffer up to 72 h and for 2 h exposure to buffer at temperatures up to 90 degrees C. It was found that stability was determined by robustness of APTES-silica attachment, with about 30% loss under the harshest conditions investigated.

This report investigates the effect of DNA length and the presence of an anchoring group on the assembly of presynthesized oligonucleotides at a gold surface. The work seeks to advance fundamental insight into issues that impact the structure and behavior of surface-immobilized DNA layers, as in, for instance, DNA microarray and biosensor devices. The present study contrasts immobilization of single-stranded DNA (ssDNA) containing a terminal, 5' hexanethiol anchoring group with that of unfunctionalized oligonucleotides for lengths from 8 to 48 bases. Qualitatively, the results indicate that the thiol anchoring group strongly enhances oligonucleotide immobilization, but that the enhancement is reduced for longer strand lengths. Interestingly, examination of the probe coverage as a function of strand length suggests that adsorbed thiol-ssDNA oligonucleotides shorter than 24 bases tend to organize in end-tethered, highly extended configurations for which the long-term surface coverage is largely independent of oligonucleotide length. For strands longer than 24 bases, the surface coverage begins to decrease notably with probe length. The decrease is consistent with a less ordered arrangement of the DNA chains, presumably reflecting increasingly polymeric behavior.