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Dynamic Electrical Switching of DNA Layers on a Metal Surface
Abstract
We report on the dynamic control over the orientation of short oligonucleotide strands which are tethered to gold surfaces in electrolyte solution. By applying alternating electrical bias potentials to the supporting electrodes we are able to induce a switching of the layer conformation between a “lying” and a “standing” state, simultaneously monitored in a contactless mode by fluorescence techniques. We demonstrate that our electrooptical experiments allow for an in-depth investigation of the intriguing molecular dynamics of DNA at surfaces and, moreover, how the dynamic response of these switchable biomolecular layers opens new prospects in label-free biosensing.
... The main work of this thesis has been performed on a device called DRX 2 , which uses a technology named "switchSENSE". Originally, the technique was developed based on research on electrical manipulation of DNA strands [39,40]. ...
... When the electrode surface carries a positive charge, the DNA is attracted to the surface and lies down horizontally. An applied alternating voltage therefore results in an oscillatory behavior of the DNA according to the charge on the surface [39]. ...
... When the double-stranded DNA is in an upright position, the maximal amount of light reaches the photodetector. The fluorescence (F ) scales with the distance (z) of the fluorescent dye above the metal surface to the third power [39]: ...
In this work, binding interactions between biomolecules were analyzed by a technique that is based on electrically controllable DNA nanolevers. The technique was applied to virus-receptor interactions for the first time. As receptors, primarily peptides on DNA nanostructures and antibodies were utilized. The DNA nanostructures were integrated into the measurement technique and enabled the presentation of the peptides in a controllable geometrical order. The number of peptides could be varied to be compatible to the binding sites of the viral surface proteins. Influenza A virus served as a model system, on which the general measurability was demonstrated. Variations of the receptor peptide, the surface ligand density, the measurement temperature and the virus subtypes showed the sensitivity and applicability of the technology. Additionally, the immobilization of virus particles enabled the measurement of differences in oligovalent binding of DNA-peptide nanostructures to the viral proteins in their native environment. When the coronavirus pandemic broke out in 2020, work on binding interactions of a peptide from the hACE2 receptor and the spike protein of the SARS-CoV-2 virus revealed that oligovalent binding can be quantified in the switchSENSE technology. It could also be shown that small changes in the amino acid sequence of the spike protein resulted in complete loss of binding. Interactions of the peptide and inactivated virus material as well as pseudo virus particles could be measured. Additionally, the switchSENSE technology was utilized to rank six antibodies for their binding affinity towards the nucleocapsid protein of SARS-CoV-2 for the development of a rapid antigen test device. The technique was furthermore employed to show binding of a non-enveloped virus (adenovirus) and a virus-like particle (norovirus-like particle) to antibodies. Apart from binding interactions, the use of DNA origami levers with a length of around 50 nm enabled the switching of virus material. This proved that the technology is also able to size objects with a hydrodynamic diameter larger than 14 nm. A theoretical work on diffusion and reaction-limited binding interactions revealed that the technique and the chosen parameters enable the determination of binding rate constants in the reaction-limited regime. Overall, the applicability of the switchSENSE technique to virus-receptor binding interactions could be demonstrated on multiple examples. While there are challenges that remain, the setup enables the determination of affinities between viruses and receptors in their native environment. Especially the possibilities regarding the quantification of oligo- and multivalent binding interactions could be presented.
... When a highly charged molecule like DNA is tethered to a transducer, its properties (e. g., orientation, stability) can be effectively manipulated by electrically polarizing the transducer surface. [6,[36][37][38][39][40] Applying voltage to the electrode results in building up the electric field which engages DNA electrostatically. The energetics of interactions primarily depend on the magnitude of the applied voltage and the ion concentration of the surrounding solution, which influence the potential distribution ðF ¼ Fðd; F 0 ; C ion )), where d is a distance, F 0 is the applied potential, and C ion is the ion concentration). ...
... [39,63] The extent of the manipulation of DNA depends on the relation between the statistical gyration due to the thermal agitation and the total electrostatic interactions. [38] Rant and coworkers studied the influence of an external applied potential and ionic strength on the conformation of tethered ss-and dsDNA ( Figure 12). [41] In high ionic strength solutions, Brownian motion prevails due to a quickly decaying potential profile, and neither singlenor double-stranded DNA layers respond to applied potentials, as discussed before. ...
Numerous DNA biosensor platforms developed in the last decades rely on DNA electrostatics as the basis for detection. However, the overwhelming number of theoretical studies and computational models of DNA electrostatics poses a barrier to leveraging our deeper mechanistic understanding in this area for the development of new technologies that will push the field towards more sensitive, quantitative, and reliable DNA‐based sensors. In this review, we will bridge the gap between the theory and applications of DNA charge and electrical surfaces to overcome this barrier. We will introduce key theories such as Manning’s counterion condensation theory, the Poisson‐Boltzmann equation, and the Gouy‐Chapman model, and provide examples of technological applications that rely on these theories.
... In the last 20 years, with the advent of DNA as a building block for complex assembly, a consequent amount of research has been done on assemblies of thiol-modified DNA on the surface of metals, especially on gold [94,95,96,97,98,99,100,101]. In particular, the group of Ulrich Rant in Munich has done a lot of research on electrical switching of DNA monolayers on gold surfaces [102,103,104,105,106,107,108,109,110]. The basic idea behind their first papers is depicted in Fig. 4. 7. DNA is fluorescently labeled on one end and modified with a thiol group on the other end. ...
... Right: Negatively biased electrodes repel the likewise charged DNA strands, bright fluorescence is emitted from the dye attached to the DNA's top end. Positive surface charge attracts the strands and due to the close proximity to the metal efficient energy transfer from the excited dye to the Au results in a substantial quenching of fluorescence, from[103] ...
This thesis manuscript deals with the study of two distinct projects. The first deals with the experimental design as well as the theoretical study of a brand new type of engine based on polymer fibers: the fiberdrive. The operation of this engine is based on a new physical concept described here, deformation modes with zero elastic energy (ZEEMs). These deformation modes are driven by a flow of energy that causes elastic deformation within the material. This manuscript develops a theoretical model that is confronted with the first experimental realization of this type of engine. The realized system is the simplest engine in the world, a stator without rotor.The second part of the manuscript introduces the concept of confotronics, the study of system composed of individually switchable units cooperating on a large scale. This concept is implemented in the realization of a DNA-based confotronic fiber as well as in the design of a DNA-based molecular engine.
... For instance, relatively modest positive potentials attract the negatively charged DNA toward the electrode surface. 17,18 Positive potentials can also enhance hybridization efficiency by decreasing the electrostatic penalty of the approaching target strand and its negatively charged phosphate backbone. 19 These results suggest electrostatic effects play an important role in the hybridization and orientation of surface-bound DNA, offering routes to electrochemically control DNA assembly. ...
... Moderate DNA surface densities are used to minimize steric hindrance, facilitate diffusion of melted oligomers away from the surface, and increase responsiveness to the electric field. 17,28 Moderate to low applied potentials are applied to minimize thiol desorption. We demonstrate that under these conditions, this method is sensitive to duplex stability and capable of detecting a single, centrally located mismatch in a 34-base pair duplex. ...
Here we demonstrate a purely electrochemical approach for monitoring the electric field-induced melting of surface-bound DNA duplexes tethered to gold surfaces using standard surface attachment chemistry, i.e. single thiol-Au bonds. The sensitivity of square wave voltammetry is combined with the electric fields generated during programmed sequences of chronoamperometric pulses to enable a method for DNA analysis that can be carried out at room temperature without need for parallel spectroscopic monitoring. Electrochemical melting curves are obtained using both scanning potential pulses and constant potential pulses, which are analyzed to assess duplex stability and the extent of thiol desorption. Themelting behavior is found to depend on the pulse potential and pule time. Under optimized conditions, thiol reduction is minimized and DNA duplexes can be discriminated based on the presence of a single base pair mismatch. The method is found to be less sensitive to the duplex length, presumably due to the rapid decay of the electric field away from the electrode surface. Based on these results, a simple model is proposed in which maintenance of the double-layer charge by accumulation of ions from the bulk of solution competes with electric field-induced loss of the negatively-charged DNA at a given applied potential.
... Similar dynamic DNA modulation has also been demonstrated in optical systems employing DNA nanolevers. [9][10][11] While MPs function well for proteins and larger analytes, their dependence on hydrodynamic radius changes makes them difficult to implement for smaller, less bulky targets. Here, we present a new approach that allows for reagentless analysis of electroactive small molecules. ...
The detection of small molecules beyond glucose remains an ongoing challenge in the field of biomolecular sensing owing to their small size, diverse structures, and lack of alternative non-enzymatic sensing methods. Here, we present a new reagentless electrochemical approach for small molecule detection that involves directed movement of electroactive analytes through a self-assembled monolayer to an electrode surface. Using this method, we demonstrate detection of several physiologically relevant small molecules as well as the capacity for the system to operate in several biological fluids. We anticipate that this mechanism will further improve our capacity for small molecule measurement and provide a new generalizable monolayer-based technique for electrochemical assessment of various electroactive analytes.
... kinFRET Experiments. kinFRET experiments were performed using switchSENSE proximity sensing technology 41,42 and a heliX adapter chip on the heliX + biosensor platform (Dynamic Biosensors GmbH). The optoelectronic and fluidic handling schematic can be seen in Figure S1 and additional technical details in the Supporting Information experimental section. ...
... [20][21][22] Applied potential has been used to control the orientation of fluorophore labeled DNA through electrostatic interactions. The modulated potential resulted in a change in the fluorescence intensity which was used for NA and protein detection with DNA SAMs [23][24][25] . Advanced fluorescence methods, like FRET (Förster Resonance Energy Transfer) have also been used to study the DNA SAM interface. ...
Interfaces modified by a molecular monolayer can be challenging to study, particularly in situ, requiring novel approaches. Coupling electrochemical and optical approaches can be useful when signals are correlated. Here we detail a methodology that uses redox electrochemistry to control surface-based fluorescence intensity for detecting DNA hybridization and studying the uniformity of the surface response. A mixed composition single-strand DNA SAM was prepared using potential-assisted thiol exchange with two alkylthiol-modified ssDNAs that were either labeled with a fluorophore (AlexaFluor488) or a methylene blue (MB) redox tag. A significant change in fluorescence was observed when reducing MB to colorless leuco-MB. In situ fluorescence microscopy on a single-crystal gold bead electrode showed that fluorescence intensity depended on (1) the potential controlling the oxidation state of MB, (2) the surface density of DNA, (3) the MB:AlexFluor488 ratio in the DNA SAM, and (4) the local environment around the DNA SAM. MB efficiently quenched AlexaFluor488 fluorescence. Reduction of MB showed a significant increase in fluorescence resulting from a decrease in quenching or energy transfer efficiency. Hybridization of DNA SAMs with its unlabeled complement showed a large increase in fluorescence due to MB reduction for surfaces with sufficient DNA coverage. Comparing electrochemical-fluorescence measurements to electrochemical (SWV) measurements showed an improvement in detection of a small fraction of hybridized DNA SAM for surfaces with optimal DNA SAM composition and coverage. Additionally, this coupled electrochemical redox-fluorescence microscopy method can measure the spatial heterogeneity of electron-transfer kinetics and the influence of the local interfacial environment.
... The fluorescence intensity depends on the distance between the fluorophore and the gold surface and hereby on the orientation of the nanolevers in relation to the gold surface. The distance-dependent fluorescence intensity is based on a distance-dependent, non-radiative energy transfer from the reporter fluorophores (on the nanolevers distal end) to the metal surface [48][49][50][51] . By alternating the applied surface potentials between an attractive and a repulsive potential at a frequency of 250 Hz, the DNA's intrinsic negative charge enables forced switching movement of the nanolevers. ...
Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.
... Also, compared to the measurements taken with the single-stranded pDNAs under no-flow conditions, the ionic strength of the electrolyte (0.01 × PBS), in which the sensing experiments are performed, is significantly lower (compared to 1 × PBS), such that the attractive electrostatic force caused by the positive surface potential extends more into the electrolyte without being significantly screened 57 . Given that the hybridised dsDNAs carry twice the amount of negative charge of the ssDNAs, it can be considered that the hybridised pDNA-tDNA pairs, in our case, are more strongly attracted to the graphene surface compared to pDNAs 58 . The stronger electrostatic attraction combined with the stretching effect of lateral microfluidic flow supports our argument. ...
Bio-inspired molecular communications (MC), where molecules are used to transfer information, is the most promising technique to realise the Internet of Nano Things (IoNT), thanks to its inherent biocompatibility, energy-efficiency, and reliability in physiologically-relevant environments. Despite a substantial body of theoretical work concerning MC, the lack of practical micro/nanoscale MC devices and MC testbeds has led researchers to make overly simplifying assumptions about the implications of the channel conditions and the physical architectures of the practical transceivers in developing theoretical models and devising communication methods for MC. On the other hand, MC imposes unique challenges resulting from the highly complex, nonlinear, time-varying channel properties that cannot be always tackled by conventional information and communication tools and technologies (ICT). As a result, the reliability of the existing MC methods, which are mostly adopted from electromagnetic communications and not validated with practical testbeds, is highly questionable. As the first step to remove this discrepancy, in this study, we report on the fabrication of a nanoscale MC receiver based on graphene field-effect transistor biosensors. We perform its ICT characterisation in a custom-designed microfluidic MC system with the information encoded into the concentration of single-stranded DNA molecules. This experimental platform is the first practical implementation of a micro/nanoscale MC system with nanoscale MC receivers, and can serve as a testbed for developing realistic MC methods and IoNT applications.
... 28 These changes are reversible, and are consistent with a morphology change that is triggered by electrostatic interactions: at voltages negative of the potential of zero charge (pzc), the negatively charged phosphate backbone is repelled from the surface causing the duplexes to stand straight up, while voltages positive of the pzc attract the phosphate groups causing the duplexes to lie down flat. Accordingly, a plot of the maximum film thickness vs the number of base pairs in the DNA duplexes reveals a nearly ideal slope of 3.4 Å/bp, Figure 3. Subsequent AFM 52 and STM data 23,25 have supported a similar DNA-morphology change upon application of small electric fields. ...
Over the past 25 years, collective evidence has demonstrated that the DNA base-pair stack serves as a medium for charge transport chemistry in solution and on DNA-modified gold surfaces. Since this charge transport depends sensitively upon the integrity of the DNA base pair stack, perturbations in base stacking, as may occur with DNA base mismatches, lesions, and protein binding, interrupt DNA charge transport (DNA CT). This sensitivity has led to the development of powerful DNA electrochemical sensors. Given the utility of DNA electrochemistry for sensing and in response to recent literature, we describe critical protocols and characterizations necessary for performing DNA-mediated electrochemistry. We demonstrate DNA electrochemistry with a fully AT DNA sequence using a thiolated preformed DNA duplex and distinguish this DNA-mediated chemistry from that of electrochemistry of largely single-stranded DNA adsorbed to the surface. We also demonstrate the dependence of DNA CT on a fully stacked duplex. An increase in the percentage of mismatches within the DNA monolayer leads to a linear decrease in current flow for a DNA-bound intercalator, where the reaction is DNA-mediated; in contrast, for ruthenium hexammine, which binds electrostatically to DNA and the redox chemistry is not DNA-mediated, there is no effect on current flow with mismatches. We find that, with DNA as a well hybridized duplex, upon assembly, a DNA-mediated pathway facilitates the electron transfer between a well coupled redox probe and the gold surface. Overall, this report highlights critical points to be emphasized when utilizing DNA electrochemistry and offers explanations and controls for analyzing confounding results.
... 24 First, they concluded that a low DNA surface coverage is needed to minimize electrostatic repulsions within the DNA monolayer to allow the free rotational mobility of the strands. 25 Applying a potential positive or negative from the PZC induces a redistribution of the dissolved ions within the double layer. Depending on the electrolyte concentration, strong electric gradient can exist at the interface that decays rapidly within a few nanometers depending on the electrolyte concentration, 23 leading to significantly different field strengths across the length of the DNA strands. ...
A 153-mer target DNA was amplified using ethynyl ferrocene dATP and a tailed forward primer resulting in a duplex with a single-stranded DNA tail for hybridization to a surface-tethered probe. A thiolated probe containing the sequence complementary to the tail as well as a 15 polythimine vertical spacer with a (CH2)6 spacer was immobilized on the surface of a gold electrode and hybridized to the ferrocene-modified complementary strand. Potential step chronoamperometry and cyclic voltammetry were used to probe the potential of zero charge, PZC, and the rate of heterogeneous electron transfer between the electrode and the immobilized ferrocene moieties. Chronoamperometry gives three, well-resolved exponential current-time decays corresponding to ferrocene centers located within 13 Å (4 bases) along the duplex. Significantly, the apparent standard heterogeneous electron transfer rate constant, kappo, observed depends on the initial potential, i.e., the rate of electron transfer at zero driving force is not the same for oxidation and reduction of the ferrocene labels. Moreover, the presence of ions, such as Sr2+, that strongly ion pair with the negatively charged DNA backbone modulates the electron transfer rate significantly. Specifically, kappo = 246 ± 23.5 and 14 ± 1.2 s-1 for reduction and oxidation, respectively, where the Sr2+ concentration is 10 mM, but the corresponding values in 1 M Sr2+ are 8 ± 0.8 and 150 ± 12 s-1. While other factors may be involved, these results are consistent with a model in which a low Sr2+ concentration and an initial potential that is negative of the PZC lead to electrostatic repulsion of the negatively charged DNA backbone and the negatively charged electrode. This leads to the DNA adopting an extended configuration (concertina open), resulting in a slow rate of heterogeneous electron transfer. In contrast, for ferrocene reduction, the initial potential is positive of PZC and the negatively charged DNA is electrostatically attracted to the electrode (concertina closed), giving a shorter electron transfer distance and a higher rate of heterogeneous electron transfer. When the Sr2+ concentration is high, the charge on the DNA backbone is compensated by the electrolyte and the charge on the electrode dominates the electron transfer dynamics and the opposite potential dependence is observed. These results open up the possibility of electromechanical switching using DNA superstructures.
... These effects have been reported previously and are reasonable estimates of the congestion of the local environment around the tethered DNA. 32,37,41,42 In addition, the potential induced reorientation measurements can be useful to ensure that the fluorescence originated from covalently bound DNA, not from DNA that were non-specifically adsorbed to the electrode surface. For bound DNA, the change in surface charge electrostatically repels or attracts the charged DNA backbone to the surface which results in a consistent modulation in fluorescence intensity. ...
The thermal stability of thiol based DNA SAMs prepared on gold surfaces is an important parameter that is correlated to sensor lifetime. The thermal stability of DNA SAMs was evaluated in aqueous buffer through the use of fluorophore labeled DNA, a single crystal gold bead electrode, and microscopy. The stability of different crystallographic regions on the electrode was studied for thermal treatments up to 95 °C for 90 min. Using a in situ combinatorial surface analytical measurement showed that the crystallography of the underlying gold surface played a significant role, with the square or rectangular lattices (e.g., 110, 100, 210) having the highest stability. Surfaces with hexagonal lattices (e.g., 111, 311, 211) were less stable toward thermal treatments. These crystallographic trends were observed for both high and low coverage DNA SAMs. High coverage DNA SAMs were the most stable, with stability decreasing with decreasing coverage on average. Increasing DNA SAM coverage appears to slow the kinetics of thermal desorption, but the coordination to the underlying surface determined their relative stability. Preparing the DNA SAMs under nominally similar conditions were found to create surfaces that were similar at room temperature, but had significantly different thermal stability. Optimal DNA sensing with these surfaces most often requires low coverage DNA SAMs which results in poor thermal stability, which is predictive of a poor shelf life, making optimization of both parameters challenging. Furthermore, the crystallographically specific results should be taken into account when studying the typically used polycrystalline substrates since the underlying surface crystallography maybe different for different samples. It appears that preparing DNA SAMs with low coverage and significant thermal stability will be challenging using the current SAM preparation procedures.
... Given that the hybridised dsDNAs carry twice the amount of negative charge of the ssDNAs, it can be considered that the hybridised pDNA-tDNA pairs, in our case, are more strongly attracted to the graphene surface compared to pDNAs [50]. Each working concentration of tDNAs were propagated in the channel until I ds reaches a plateau. ...
Bio-inspired molecular communications (MC), where molecules are used to transfer information, is the most promising technique to realise the Internet of Nano Things (IoNT), thanks to its inherent biocompatibility, energy-efficiency, and reliability in physiologically-relevant environments. Despite a substantial body of theoretical work concerning MC, the lack of practical micro/nanoscale MC devices and MC testbeds has led researchers to make overly simplifying assumptions about the implications of the channel conditions and the physical architectures of the practical transceivers in developing theoretical models and devising communication methods for MC. On the other hand, MC imposes unique challenges resulting from the highly complex, nonlinear, time-varying channel properties that cannot be always tackled by conventional information and communication tools and technologies (ICT). As a result, the reliability of the existing MC methods, which are mostly adopted from electromagnetic communications and not validated with practical testbeds, is highly questionable. As the first step to remove this discrepancy, in this study, we report on the fabrication of a nanoscale MC receiver based on graphene field-effect transistor biosensors. We perform its ICT characterisation in a custom-designed microfluidic MC system with the information encoded into the concentration of single-stranded DNA molecules. This experimental platform is the first practical implementation of a micro/nanoscale MC system with nanoscale MC receivers, and can serve as a testbed for developing realistic MC methods and IoNT applications.
... Using fluorescence spectroscopy, Rant et al. demonstrated that DNA surface density affects its responsiveness to applied potentials. 47 Lower density ssDNA films exhibited higher flexibility, and were therefore more responsive to applied potentials. A model based on the redistribution of counter ions in the double-layer was used to interpret the dynamics of the "switching" process, i.e. the response of the DNA to periodic switching of the potential. ...
DNA provides a powerful framework for the development of biosensors, DNA chips, bioelectronics, and other established and emerging technologies. Many of these applications involve DNA self-assembled monolayers (SAM) on conducting surfaces where the high molecular density, the two-dimensional nature of the interface, and the limited mobility of the strands significantly impact the behavior of the DNA. The unique steric and electrostatic conditions present in the SAM dominate hybridization, melting, and motion of the tethered oligonucleotides. At neutral pH the charged sugar-phosphate backbone makes the DNA sensitive to the electric fields present in the electrical double-layer. Electrode charge provides a means of modifying the reactivity of DNA monolayers; facilitating enhanced rates of hybridization, controlling orientation, and inducing melting (i.e. denaturation). Understanding the effects of electric fields on DNA monolayers is a prerequisite to the optimization of next generation DNA biosensors and other applications that take advantage of DNA’s selective self-assembly. This mini-review will give an overview of the ways in which electrochemical control can be used to manipulate DNA SAMs. In particular, the process of electric field-assisted melting of DNA, i.e. electrochemical melting, will be reviewed. Electrochemical melting has the potential for providing biophysical insights and for the development of new diagnostic applications.
... 100 mV (denoted by solid curves) as well as -100 mV (denoted by dashed curves).Rant et al. have previously shown that positive electrode potential causes condensation of DNA, whereas negative electrode potential provides a repulsive force60 . The analytical model described here does not incorporates the dynamic conformational changes in the DNA strands. ...
Experimental and computational approaches are utilized to investigate the influence of electrostatic fields on the binding force between human coagulation protein thrombin and its DNA aptamer. The thiolated aptamer was deposited onto gold substrate located in a liquid cell filled with binding buffer, then the thrombin-functionalized atomic force microscopy (AFM) probe was repeatedly brought into contact with the aptamer-coated surface under applied electrical potentials of -100, 0, and 100 mV respectively. Force drops during the pull-off process were measured to determine the unbinding forces between thrombin and aptamer in a range of loading rates spanning from ∼3×102 to ∼1×104 pN/s. The results from experiments showed that both of the binding strength and propensity of the complex are drastically diminished under positive electrode potential, whereas there is no influence on the molecular binding from negative electrode potential. We also used a theoretical analysis to explain the nature of electrostatic potential and field inside the aptamer-thrombin layer, which in turn could quantify the influence of the electrostatically repulsive force on a thrombin molecule that promotes dissociation from the aptamer due to positive electrode potential, and achieve good agreement with the experimental results. The study confirms the feasibility of electrostatic modulation upon the binding interaction between thrombin and aptamer, and implicates an underlying application perspective upon nanoscale manipulation of the stimuli responsive biointerface.
... Here, we introduce a convenient technology for the study of aptamers and their target interaction. switchSENSE (Dynamic Biosensors GmbH, Martinsried, DE) [24][25][26] is an emerging technology for biophysical quantification of binding and activity rates, in addition to a friction read-out. Adaptation of the DNA-based switchSENSE biosensors for aptamer measurements allowed us to profit from the inherent advantages of the technology for nucleic acid ligands, including quasi modification-free aptamer immobilization on the biosensor by hybridization. ...
Therapeutic and diagnostic nucleic acid aptamers are designed to bind tightly and specifically to their target. The combination of structural and kinetic analyses of aptamer interactions has gained increasing importance. Here, we present a fluorescence-based switchSENSE aptasensor for the detailed kinetic characterization of aptamer–analyte interaction and aptamer folding, employing the thrombin-binding aptamer (TBA) as a model system. Thrombin-binding aptamer folding into a G-quadruplex and its binding to thrombin strongly depend on the type and concentration of ions present in solution. We observed conformational changes induced by cations in real-time and determined the folding and unfolding kinetics of the aptamer. The aptamer’s affinity for K+ was found to be more than one order of magnitude higher than for other cations (K+ > NH4+ >> Na+ > Li+). The aptamer’s affinity to its protein target thrombin in the presence of different cations followed the same trend but differed by more than three orders of magnitude (KD = 0.15 nM to 250 nM). While the stability (kOFF) of the thrombin–TBA complex was similar in all conditions, the cation type strongly influenced the association rate (kON). These results demonstrated that protein–aptamer binding is intrinsically related to the correct aptamer fold and, hence, to the presence of stabilizing ions. Because fast binding kinetics with on-rates exceeding 108 M−1s−1 can be quantified, and folding-related phenomena can be directly resolved, switchSENSE is a useful analytical tool for in-depth characterization of aptamer–ion and aptamer–protein interactions.
... The ∆40p53 isoform is similar to the FLp53 and causes no inhibitory action leading to apoptosis (Figure 4) [47,49]. On the contrary, ∆133p53 and ∆160p53 act more like a survival factor [59,60,135] and could lead to a more resistant cancer [63]. It is unclear whether these two isoforms with truncated DBD are capable of binding to the specific DNA sequences. ...
In this review we focus on the major isoforms of the tumor-suppressor protein p53, dysfunction of which often leads to cancer. Mutations of the TP53 gene, particularly in the DNA binding domain, have been regarded as the main cause for p53 inactivation. However, recent reports demonstrating abundance of p53 isoforms, especially the N-terminally truncated ones, in the cancerous tissues suggest their involvement in carcinogenesis. These isoforms are ∆40p53, ∆133p53, and ∆160p53 (the names indicate their respective N-terminal truncation). Due to the lack of structural and functional characterizations the modes of action of the p53 isoforms are still unclear. Owing to the deletions in the functional domains, these isoforms can either be defective in DNA binding or more susceptive to altered 'responsive elements' than p53. Furthermore, they may exert a 'dominant negative effect' or induce more aggressive cancer by the 'gain of function'. One possible mechanism of p53 inactivation can be through tetramerization with the ∆133p53 and ∆160p53 isoforms-both lacking part of the DNA binding domain. A recent report and unpublished data from our laboratory also suggest that these isoforms may inactivate p53 by fast aggregation-possibly due to ectopic overexpression. We further discuss the evolutionary significance of the p53 isoforms.
... This confirms that ET from the electrode to the intercalated dye (Figure 1) occurs via the DNA π-stacked duplex and is not assisted by lateral interactions between the electrode and electrode-tethered DNA double strands. 9,10 This conclusion is consistent with the upright orientation of the negatively charged DNA strands at the electrode surface within the negative potential window used in MB studies, 10,11,30 and such an orientation was also assisted by backfilling the DNA monolayer with a short-chain alkanethiol eliminating any unspecific adsorption of DNA ( Figure S1, SI). ...
Electrical properties of DNA critically depend on the way DNA molecules are integrated within the electronics, particularly on DNA-electrode immobilization strategies. Here, we show that the rate of electron transport in DNA duplexes spacer-free tethered to gold via the adenosine terminal region (a dA10 tag) is enhanced compared to hitherto reported DNA-metal electrode tethering chemistries. The rate of DNA-mediated electron transfer (ET) between the electrode and methylene blue intercalated into the dA10-tagged DNA duplex approached 361 s-1 at a ca. half-monolayer DNA surface coverage, being 2.7- enhanced compared to phosphorothioated dA*5 tethering (6-fold for the C6-alkanethiol linker representing an additional ET barrier). XPS evidenced dA10 binding to the Au surface via the purine N, while dA*5 predominantly coordinated to the surface via sulfur atoms of phosphothioates. The latter could induce the DNA strand twist in the point of surface attachment affecting the local DNA conformation and, as a result, decreasing ET rates through the duplex. The spacer-free DNA coupling to electrodes via dA10 tags allows a perspective design of DNA electronic circuits and sensors with advanced electronic properties and no implication from more expensive synthetic linkers.
... Other ways to map diffusion layers by EC-fluorescence were also reported. Sojic and co-workers [34] have employed optical fiber bundles, positioned perpendicularly to a planar electrode, to remotely map the fluorescence Along a very different perspective, axial information in the nm range can be obtained by playing on the distancedependence of the quenching effect of bulk metal electrodes [37] , as illustrated by the work from Rant's [38,39] and Bizzotto's [19,40 • ,41] groups on the electricfield induced orientation of oligonucleotide SAMs. ...
Electrochemistry and fluorescence microscopy are two complementary transduction techniques that offer synergetic advantages when employed simultaneously. In particular, it is possible to study electrochemical reactivity by recording in-situ fluorescence images under electrochemical control. The corresponding data are time-resolved with a quantitative electrochemical signal combined with a spatial information provided by fluorescence microscopy. This topic is illustrated with various fluorogenic systems with a special focus on confocal imaging allowing to vary the longitudinal z distance when collecting planar (x-y) data. Recent and future perspectives towards super-resolution microscopy are also discussed.
... Depending on the potential that is applied to the electrodes, the DNA is either repelled from or attracted to the gold surface. The resulting movement of the DNA molecules is monitored in real time by the emission of a fluorescent dye attached to the distal end of covalently immobilized DNA strands (anchor strands) [9]. A DNA strand complementary to the anchor strands can be functionalized with a ligand molecule which principally can be any molecule that allows conjugation with standard chemical and biochemical methods. ...
Characterization of RNA-binding protein interactions with RNA became inevitable to properly understand the cellular mechanisms involved in gene expression regulation. Structural investigations bring information at the atomic level on these interactions and complementary methods such as Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) are commonly used to quantify the affinity of these RNA-protein complexes and evaluate the effect of mutations affecting these interactions. The switchSENSE technology has recently been developed and already successfully used to investigate protein interactions with different types of binding partners (DNA, protein/peptide or even small molecules). In this study, we show that this method is also well suited to study RNA binding proteins (RBPs). We could successfully investigate the binding to RNA of three different RBPs (Fox-1, SRSF1 and Tra2-β1) and obtained KD values very close to the ones determined previously by SPR or ITC for these complexes. These results show that the switchSENSE technology can be used as an alternative method to study protein-RNA interactions with KD values in the low micromolar (10⁻⁶) to nanomolar (10⁻⁷ to 10⁻⁹) and probably picomolar (10⁻¹⁰ to 10⁻¹²) range. The absence of labelling requirement for the analyte molecules and the use of very low amounts of protein and RNA molecules make the switchSENSE approach very attractive compared to other methods. Finally, we discuss about the potential of this approach in obtaining more sophisticated information such as structural conformational changes upon RBP binding to RNA.
... 6,18,23−25 First, the electric field was shown to affect the macromolecular orientation of DNA. Atomic force microscopy (AFM) 23,26 and fluorescence spectroscopy 27,28 performed under the electrochemical control demonstrated a potential dependent orientation of dsDNA helices in compact 23 and loosely packed 27 monolayers adsorbed on the gold electrode surface. Initially, it was proposed that at positive potentials DNA helices are tilted toward the Au surface due to the electrostatic attraction between negatively charged phosphate groups of the DNA backbone and positively charged electrode surfaces. ...
Unique electronic and ligand recognition properties of the DNA double helix provide basis for DNA applications in biomolecular electronic and biosensor devices. However, the relation between the structure of DNA at electrified interfaces and its electronic properties is still not well understood. Here, potential-driven changes in the submolecular structure of DNA double helices composed of either adenine-thymine (dAdT)25 or cytosine-guanine (dGdC)20 base pairs tethered to the gold electrodes are for the first time analyzed by in situ polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) performed under the electrochemical control. It is shown that the conformation of the DNA duplexes tethered to gold electrodes via the C6 alkanethiol linker strongly depends on the nucleic acid sequence composition. The tilt of purine and pyrimidine rings of the complementary base pairs (dAdT and dGdC) depends on the potential applied to the electrode. By contrast, neither the conformation nor orientation of the ionic in character phosphate–sugar backbone is affected by the electrode potentials. At potentials more positive than the potential of zero charge (pzc), a gradual tilting of the double helix is observed. In this tilted orientation, the planes of the complementary purine and pyrimidine rings lie ideally parallel to each other. These potentials do not affect the integral stability of the DNA double helix at the charged interface. At potentials more negative than the pzc, DNA helices adopt a vertical to the gold surface orientation. Tilt of the purine and pyrimidine rings depends on the composition of the double helix. In monolayers composed of (dAdT)25 molecules the rings of the complementary base pairs lie parallel to each other. By contrast, the tilt of purine and pyrimidine rings in (dGdC)20 helices depends on the potential applied to the electrode. Such potential-induced mobility of the complementary base pairs can destabilize the helix structure at a submolecular level. These pioneer results on the potential-driven changes in the submolecular structure of double stranded DNA adsorbed on conductive supports contribute to further understanding of the potential-driven sequence-specific electronic properties of surface-tethered oligonucleotides.
Engineered noble metal nanomaterials (NMN) possess adjustable optical, electrical, and biocompatible properties that make them excellent tools for probing the nano‐bio‐interface. Understanding their interactions with biomolecules, cells, and tissues at the nano‐bio‐interface is crucial in designing these nanomaterials for biomedical applications. This review summarizes the structure, properties, synthesis, and passivation methods of noble metal nanoparticles, as well as the construction strategy and detection technology of the nano‐bio‐interface to provide important information about their uptake, distribution, metabolism, and degradation in vivo and in vitro. The related action mechanisms include the kinetic and thermodynamic processes of the nano‐bio‐interface, the driving forces for its formation, and the chemical reactions at the nano‐bio‐interface. By exploring the action mechanism of the nano‐bio‐interface, the antibacterial properties and cytotoxicity of NMN could be better understood, and open up more extensive biological applications. Finally, the future trends of NMNs in the biological field and the challenges encountered in realizing these technologies are discussed.
The detection of a protein analyte and use of this type of information for disease diagnosis and physiological monitoring requires methods with high sensitivity and specificity that have to be also easy to use, rapid and, ideally, single step. In the last 10 years, a number of DNA-based sensing methods and sensors have been developed in order to achieve quantitative readout of protein biomarkers. Inspired by the speed, specificity, and versatility of naturally occurring chemosensors based on structure-switching biomolecules, significant efforts have been done to reproduce these mechanisms into the fabrication of artificial biosensors for protein detection. As an alternative, in scaffold DNA biosensors, different recognition elements (e.g., peptides, proteins, small molecules, and antibodies) can be conjugated to the DNA scaffold with high accuracy and precision in order to specifically interact with the target protein with high affinity and specificity. They have several advantages and potential, especially because the transduction signal can be drastically enhanced. Our aim here is to provide an overview of the best examples of structure switching-based and scaffold DNA sensors, as well as to introduce the reader to the rational design of innovative sensing mechanisms and strategies based on programmable functional DNA systems for protein detection.
Mixed DNA SAMs labeled with a fluorophore (either AlexaFluor488 or AlexaFluor647) were prepared on a single crystal gold bead electrode using potential-assisted thiol exchange and studied using Förster resonance energy transfer (FRET). A measure of the local environment of the DNA SAM (e.g., crowding) was possible using FRET imaging on these surfaces since electrodes prepared this way have a range of surface densities (ΓDNA). The FRET signal was strongly dependent on ΓDNA and on the ratio of AlexaFluor488 to AlexaFluor647 used to make the DNA SAM, which were consistent with a model of FRET in 2D systems. FRET was shown to provide a direct measure of the local DNA SAM arrangement on each crystallographic region of interest providing a direct assessment of the probe environment and its influence on the rate of hybridization. The kinetics of duplex formation for these DNA SAMs was also studied using FRET imaging over a range of coverages and DNA SAM compositions. Hybridization of the surface-bound DNA increased the average distance between the fluorophore label and the gold electrode surface and decreased the distance between the donor (D) and acceptor (A), both of which result in an increase in FRET intensity. This increase in FRET was modeled using a second order Langmuir adsorption rate equation, reflecting the fact that both D and A labeled DNA are required to become hybridized to observe a FRET signal. The self-consistent analysis of the hybridization rates on low and high coverage regions on the same electrode showed that the low coverage regions achieved full hybridization 5× faster than the higher coverage regions, approaching rates typically found in solution. The relative increase in FRET intensity from each region of interest was controlled by manipulating the donor to acceptor composition of the DNA SAM without changing the rate of hybridization. The FRET response can be optimized by controlling the coverage and the composition of the DNA SAM sensor surface and could be further improved with the use of a FRET pair with a larger (e.g., > 5 nm) Förster radius.
DNA nanotechnology provides efficient methods for the sequence-programmable construction of mechanical devices with nanoscale dimensions. The resulting nanomachines could serve as tools for the manipulation of macromolecules with similar functionalities as mechanical tools and machinery in the macroscopic world. In order to drive and control these machines and to perform specific tasks, a fast, reliable, and repeatable actuation mechanism is required that can work against external loads. Here we describe a highly effective method for actuating DNA structures using externally applied electric fields. To this end, electric fields are generated with controllable direction and amplitude inside a miniature electrophoresis device integrated with an epifluorescence microscope. With this setup, DNA-based nanoelectromechanical devices can be precisely controlled. As an example, we demonstrate how a DNA-based nanorobotic system can be used to dynamically position molecules on a molecular platform with high speeds and accuracy. The microscopy setup also described here allows simultaneous monitoring of a large number of nanorobotic arms in real time and at the single nanomachine level.
This chapter explores the basic concept of DNA origami and its various types. By showing the progress made in structural DNA nanotechnology during the last 15 years, the chapter draws attention to the capability of DNA origami to construct complex structures in both 2D and 3D level. As well as looking at a few examples of dynamic DNA nanostructures, the chapter also explores the possible applications of DNA origami in different fields, such as biological computing, nanorobotics, and DNA walkers.
A variety of electrochemical (EC) biosensors play critical roles in disease diagnostics. More recently, DNA-based EC sensors have been established as promising for detecting a wide range of analyte classes. Since most of these sensors rely on the high specificity of DNA hybridization for analyte binding or structural control, it is crucial to understand the kinetics of hybridization at the electrode surface. In this work, we have used methylene blue-labeled DNA strands to monitor the kinetics of DNA hybridization at the electrode surface with square-wave voltammetry. By varying the position of the double-stranded DNA segment relative to the electrode surface as well as the bulk solution's ionic strength (0.125-1.00 M), we observed significant interferences with DNA hybridization closer to the surface, with more substantial interference at lower ionic strength. As a demonstration of the effect, toehold-mediated strand displacement reactions were slowed and diminished close to the surface, while strategic placement of the DNA binding site improved reaction rates and yields. This work manifests that both the salt concentration and DNA hybridization site relative to the electrode are important factors to consider when designing DNA-based EC sensors that measure hybridization directly at the electrode surface.
The discovery and introduction of the switchSense technique in the chemical laboratory have drawn well-deserved interest owing to its wide range of applications. Namely, it can be used to determine the diameter of proteins, alterations in their tertiary structures (folding), and many other conformational changes that are important from a biological point of view. The essence of this technique is based on its ability to study of the interactions between an analyte and a ligand in real time (in a buffer flow). Its simplicity, on the other hand, is based on the use of a signaling system that provides information about the ongoing interactions based on the changes in the fluorescence intensity. This technique can be extremely advantageous in the study of new pharmaceuticals. The design of compounds with biological activity, as well as the determination of their molecular targets and modes of interactions, is crucial in the search for new drugs and the fight against drug resistance. This article presents another possible application of the switchSense technique for the study of the binding kinetics of small model molecules such as ethidium bromide (EB) and selected sulfonamide derivatives with DNA in the static and dynamic modes at three different temperatures (15, 25, and 37 °C) each. The experimental results remain in very good agreement with the molecular dynamics docking ones. These physicochemical insights and applications obtained from the switchSense technique allow for the design of an effective strategy for molecular interaction assessments of small but pharmaceutically important molecules with DNA.
Electrically controllable deoxyribonuclic acid (DNA) nanolevers are used to investigate the binding interaction between Influenza A/Aichi/2/1968 and the peptide called “PeB”, which specifically binds the viral surface protein hemagglutinin. PeB is immobilized on gold electrodes of a “switchSENSE” biochip by conjugation to DNA‐strands that are hybridized to complementary anchors. The surface‐tethered DNA strand carries a fluorophore while the complementary strand is a multivalent arrangement carrying up to three PeB peptides. The nanolevers are kept upright (static) by applying a negative potential. Signal read‐out for this static measurement mode is the change in fluorescence intensity due to changes in the local environment of the dye upon binding. Measurements of virus‐peptide interaction show that the virus material specifically binds to the immobilized peptides and remains bound throughout the measurement time. Immobilized viruses are subsequently used as ligands to characterize oligovalent peptide binding to hemagglutinin, revealing rate constants of the interaction. Moreover, three Influenza A subtypes are compared in their binding behavior. Overall, this paper shows the ability to immobilize virus material on a sensor surface, which allows to target virus‐proteins in their native environment. The “switchSENSE” method is therefore applicable to characterize virus‐receptor interactions.
Lack of long‐time stability of dsDNA‐based supramolecular assemblies is an important issue which hinders their applications. In this work, 20 base pairs long dsDNA fragments [(dCdG) 20 ‐65%] composed of 65% dCdG and 35% dAdT nucleotides were tethered via a thiol to the surface of a gold electrode. The self‐assembled (dCdG) 20 ‐65% monolayer was immersed in solutions containing ectoine, a compatible solute. Electrochemical results showed that these monolayers were stable for one month. In situ IR spectroscopy indicated that ectoine interacts weakly with the phosphate‐ribose backbone, dehydrating the phosphate groups and stabilizing the A‐DNA conformation. This structural reorganization led to a reorientation of nucleic acid base pairs and a local disruption of the double‐helix structure. However, the conformation and orientation of the dsDNA fragment was stable in the –0.4 < E < 0.3 potential range. As a direct interaction between ectoine and dsDNA, the enzymatic reaction of exonuclease VII, hydrolyzing the ester phosphate bond in ssDNA, was blocked. We show that the addition of a compatible solute to the electrolyte solution, despite structural rearrangements, stabilized the dsDNA structure.
This paper reports on the electrostatic denaturation of double-stranded DNA monolayers in the presence of anti-cancer drug cis-Diamminedichloroplatinum(II) (cisplatin). Cisplatin binds DNA at purine bases, effecting its thermal stability and melting temperature. Here we show for the first time how cisplatin effects the stability of DNA monolayers in the presence of high electric fields generated at the electrode/solution interface, and how this behavior is effected by the method of monolayer preparation. A slight decrease in melting rate and a large decrease in the extent of melting are observed for electrodes prepared with methods that produce homogeneous DNA surface coverage where interstrand distances are maximized. On the other hand, heterogeneous and densely-packed monolayers are found to melt much slower and to a slightly greater extent with bound cisplatin. Three procedures for preparation of the mercaptohexanol/DNA mixed monolayers are compared, demonstrating the potential application of cisplatin, along with electrostatic denaturation analysis, to interrogate surface density and surface heterogeneity in self-assembled DNA monolayers.
Gold-sputtered microelectrodes with built-in gold reference and counter electrodes represent a promising platform for the development of disposable DNA sensors. Pretreating gold electrode surfaces and immobilization of DNA thereon is commonly employed in biosensing applications. However, with no scientific or practical guidelines to prepare a DNA sensor using these miniature gold-sputtered microelectrodes, cleaning and immobilization steps need to be systematically optimized and updated. In this work, we present efficient cleaning and modification of miniaturized gold-sputtered microelectrodes with thiolated DNA probes for DNA detection. Additional discussions on subtleties and nuances involved at each stage of pretreating and modifying gold-sputtered microelectrodes are included to present a robust, well-founded protocol. It was evident that the insights on cleaning polycrystalline gold disk electrodes with a benchmark electrode surface for DNA sensors, cannot be transferred to clean these miniature gold-sputtered microelectrodes. Therefore, a comparison between five different cleaning protocols was made to find the optimal one for gold-sputtered microelectrodes. Additionally, two principally different immobilization techniques for gold-sputtered microelectrode modification with thiolated ssDNA were compared i.e., immobilization through passive chemisorption and potential perturbation were compared in terms of thiol-specific attachment and thiol-unspecific adsorption through nitrogenous bases. The hybridization performance of these prepared electrodes was characterized by their sensitive complementary DNA capturing ability, detected by a standard alkaline phosphatase assay. Immobilization through passive chemisorption proved to be efficient in capturing the complementary target DNA with a detection limit of 0.14 nM and sensitivity of 9.38 A M-1 cm2. In general, this work presents a comprehensive understanding of cleaning, modification and performance of gold-sputtered microelectrodes with built-in gold reference and counter electrodes for both fundamental investigations and practical DNA sensing applications.
DNA self-assembled monolayers (SAMs) were prepared using potential-assisted deposition on clean gold single crystal bead electrodes under a number of conditions (constant or square wave potential perturbations in TRIS or phosphate immobilization buffers, with and without Cl–). The local environment around the fluorophore labeled DNA tethered to the electrode surface was characterized using in-situ fluorescence microscopy during electro- chemical measurements as a function of the underlying surface crystallography. Potential- assisted deposition from a TRIS buffer containing Cl– created DNA SAMs that were uniformly distributed on the surface with little preference to the underlying crystallography. A constant (+0.4V/SCE) or a square-wave potential perturbation (+0.4 to -0.3V/SCE, 50Hz) resulted in similar DNA modified surfaces in TRIS. Deposition using a square-wave potential without Cl– resulted in a densely packed DNA SAM despite having a surface with low overall coverage. This implies the formation of clusters of densely packed DNA in the SAM. This effect was also demonstrated when depositing from a phosphate buffer. DNA clusters were significantly reduced when Cl– was present in the buffer. Clusters were most prevalent on the low index plane surfaces (e.g., {111} and {100}) and less on the higher index planes (e.g., {210} or {311}). A mechanism is proposed to rationalize the formation of DNA clustered regions for deposition using a square-wave potential perturbation. The conditions for creating clusters of DNA in a SAM or for preventing these clusters from forming provide an approach for tailoring the surfaces used for biosensing.
The concomitant detection, monitoring and analysis of biomolecules have assumed utmost importance in the field of medical diagnostics as well as in different spheres of biotechnology research such as drug development, environmental hazard detection and biodefense. There is an increased demand for the modulation of the biological response for such detection / sensing schemes which will be facilitated by the sensitive and controllable transmission of external stimuli. Electrostatic actuation for the controlled release/capture of biomolecules through conformational transformations of bioreceptors provides an efficient and feasible mechanism to modulate biological response. In addition, electrostatic actuation mechanism has the advantage of allowing massively parallel schemes and measurement capabilities that could ultimately be essential for biomedical applications.Experiments have previously demonstrated the unbinding of thrombin from its aptamer in presence of small positive electrode potential whereas the complex remained associated in presence of small negative potentials / zero potential. However, the nanoscale physics/chemistry involved in this process is not clearly understood. In this thesis a combination of continuum mechanics based modeling and a variety of atomistic simulation techniques have been utilized to corroborate the aforementioned experimental observations. It is found that the computational approach can satisfactorily predict the dynamics of the electrically excited aptamer-thrombin complex as well as provide an analytical model to characterize the forced binding of the complex.
Dynamic methods of biosensing based on electrical actuation of surface-tethered nanolevers require the use of levers whose movement in ionic liquids is well controllable and stable. In particular, mechanical integrity of the nanolevers in a wide range of ionic strength will enable to meet the chemical conditions of a large variety of applications where the specific binding of biomolecular analytes is analyzed. Herein we study the electrically induced switching behavior of different rod-like DNA origami nanolevers and compare that to the actuation of simply double-stranded DNA nanolevers. Our measurements reveal a significantly stronger response of the DNA origami to switching of electrode potential, leading to a smaller potential change necessary to actuate the origami, and subsequently, to a long-term stable movement. Dynamic measurements in buffer solutions with different Mg2+ content show that the levers do not disintegrate even at very low ion concentration and constant switching stress, and thus, provide stable actuation performance. The latter will pave the way for many new applications without largely restricting application-specific environments.
The ability to measure the levels of diagnostically relevant proteins, such as antibodies, directly at the point of care could significantly impact healthcare. Thus motivated, we explore here the E-DNA "scaffold" sensing platform, a rapid, convenient, single-step means to this end. These sensors are comprised of a rigid nucleic acid "scaffold" attached via a flexible linker to an electrode and modified on its distal end with a redox reporter and a protein binding "recognition element." The binding of a targeted protein reduces the efficiency with which the redox reporter approaches the electrode, resulting in an easily measured signal change when the sensor is interrogated voltammetrically. Previously we have demonstrated scaffold sensors employing a range of low molecular weight hap-tens and linear peptides as their recognition elements. Expanding on this here we have characterized sensors employing much larger recognition elements (up to and including full length proteins) in order to: 1) define the range of recognition elements suitable for use in the platform; 2) better characterize the platform's signaling mechanism to aid its design and optimization; and 3) demonstrate the analytical performance of sensors employing full-length proteins as recognition elements. In doing so we have enlarged the range of molecular targets amenable to this rapid and convenient sensing platform.
The characterization of bio-functionalized surfaces such as alkanethiol self-assembled monolayers (SAMs) on gold modified with DNA or other biomolecules is a challenging analytical problem and access to a routine method is desirable. Despite substantial investigation from structural and mechanistic perspectives, robust and high-throughput metrology tools for SAMs remain elusive but essential for the continued development of these devices. We demonstrate that scanning electron microscopy (SEM) can provide excellent contrast of the molecular interface at every step of SAM functionalization. The high-speed, large magnification range, and ease-of-use make this widely available technique a powerful platform for measuring the structure and composition of SAM surfaces. This increased throughput allows for a better understanding of the non-ideal spatial heterogeneity characteristic of SAMs utilized in real-world conditions. SEM image contrast is characterized through the use of fluorescently labeled DNA which enables correlative SEM and fluorescence microscopy. This allows identification of the DNA modified regions at resolutions that approach the size of the biomolecule. The effect of electron beam irradiation dose are explored, which leads to straightforward lithographic patterning of DNA SAMs with nanometer resolution and with control over the surface coverage of specifically adsorbed DNA.
DNA brushes, consisting of layers of DNA molecules attached by one end to a support, are widely used for bioanalytical applications including in microarray technologies and in next-generation sequencing platforms. This chapter overviews preparation of DNA brushes, their physicochemical properties with emphasis on their polyelectrolyte nature, and illustrative examples of their applications. Because most of these applications are based on hybridization of single-stranded brushes with complementary sequences from solution, current understanding of the hybridization response is also discussed. DNA brushes are expected to continue to be widely used in materials chemistry for creation of nanostructures, in bioanalytical methods for demonstrating new diagnostic concepts, and in commercial sequencing and microarray technologies for both research and clinical applications.
The development of electrically powered DNA origami nanomachines requires effective means to actuate moving origami parts by externally applied electric fields. Here, we demonstrate how origami nanolevers on an electrode can be manipulated (switched) at high frequency by alternating voltages. The orientation switching is long-time stable and can be induced by applying low voltages of merely 200 mV. The mechanical response time of a 100 nm long origami lever to an applied voltage step is less than 100 µs, allowing for a highly dynamic control of the induced motion. Moreover, through voltage assisted capture, the origamis can be immobilized directly from folding solution without purification, even in the presence of a large excess of staple strands. The results establish a way for interfacing and controlling DNA origamis with standard electronics, and enable their use as moving parts in electro-mechanical nanodevices.
In this article we investigated the effect of surface pretreatment procedures on electron transfer of methylene blue (MB) covalently labeled double-stranded DNA (ds-DNA)/mercaptohexanol (MCH) mixed self-assembled monolayers (SAMs) on gold by cyclic voltammetry (CV) and chronocoulometry (CC). The pretreatment procedures included M+E, M + C (piranha), M + C (dilute aqua regia), M + C + E (piranha), M + C + E (dilute aqua regia) and RM + C + E (piranha). The M was mechanical polishing, C was piranha or dilute aqua regia dipping, E was electrochemical polishing and RM was roughly mechanical polishing. Results indicated electron transfer reaction of MB was mainly adsorption controlled. The electron transfer rate (ks) values of MB labeled ds-DNA/MCH mixed SAMs on gold pretreated by M+C(piranha) and M+C(dilute aqua regia) were 0.84 ± 0.15 and 0.82 ± 0.17 s⁻¹, smaller than those (2.76 ± 0.28, 4.76 ± 2.68, 3.89 ± 2.06, 2.26 ~ 7.79 s⁻¹) by M+E, M+C+E(piranha), M+C+E(dilute aqua regia) and RM+C+E(piranha) respectively. Thus, electrochemical polishing was an important pretreatment step, which might influence the ks of MB. Furthermore, the ks values of MB did not change monotonically with increasing gold surface roughness Rf, indicating that Rf was not the key factor to make the difference of ks. We considered that the difference of elemental composition on gold surface possibly led to different ks of MB. These conclusions provided the important reference for electrochemically studying DNA electron transfer mechanism.
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 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.
We report an aptamer functionalized stimuli responsive surface that can controllably switch between binding and releasing a specific ligand under application of electrical stimuli. The high affinity of the aptamer for thrombin makes the surface undergo specific binding while electrostatic field induced actuation of aptamer is utilized to release the ligand from the surface. Atomic force microscopy (AFM) was utilized to determine the characteristic height change, associated with specific binding of thrombin, on the anti-thrombin aptamer coated surfaces. Subsequently, the thrombin/aptamer complex covered surfaces were subjected to different magnitudes of electrostatic field and height changes on the surface were measured to investigate the influence of electrical field. Application of positive electrical potential led to the removal of thrombin from the aptamer-covered surface. While under moderate magnitude of negative electrical potential the binding complexes were maintained but increasing the magnitude led to the removal of both molecules from the surface. Molecular dynamics (MD) simulations of the thrombin/aptamer complex under electrostatic fields show that thrombin dissociates from the aptamers in presence of positive electric field. These results demonstrate that aptamer covered surfaces undergo specific binding to the ligand and electrostatic field may be used to disrupt the binding and on-demand release of the ligand from the surface.
The intercalation of [Ru(bpy)2(dppz)]2 + labeled as Ru(II) (bpy = 2,2′-bipyridine and dppz = dipyrido[3,2,-a:2′,3′-c]phenazine) into herring sperm DNA leads to the formation of emissive Ru(II)-DNA dyads, which can be quenched by TiO2 nanoparticles (NPs) and sol-gel silica matrices at heterogeneous interfaces. The calcinations temperature exhibits a remarkable influence on the luminescence quenching of the Ru(II)-DNA dyads by TiO2 NPs. With increasing calcinations temperature in the range from 200 to 850 °C, the anatase-to-rutile TiO2 crystal structure transformation increases the average particle size and hydrodynamic diameter of TiO2 and DNA@TiO2. The anatase TiO2 has the stronger ability to unbind the Ru(II)-DNA dyads than the rutile TiO2 at room temperature. The TiO2 NPs and sol-gel silica matrices can quench the luminescence of the Ru(II) complex intercalated into DNA by selectively capturing the negatively DNA and positively charged Ru(II) complex to unbind the dyads, respectively. This present results provide new insights into the luminescence quenching and competitive binding of dye-labeled DNA dyads by inorganic NPs.
We have devised a supramolecular edifice involving His-tagged protein A and antibodies to yield surface immobilized, uniformly oriented, IgG-type, antibody layers with Fab fragments exposed off an electrode surface. We demonstrate here that we can affect the conformation of IgGs, likely pushing/pulling electrostatically Fab fragments towards/from the electrode surface. A potential difference between electrode and solution acts on IgGs’ charged aminoacids modulating the accessibility of the specific recognition regions of Fab fragments by antigens in solution. Consequently, antibody-antigen affinity is affected by the sign of the applied potential: a positive potential enables an effective capture of antigens; a negative one pulls the fragments towards the electrode, where steric hindrance caused by neighboring molecules largely hampers the capture of antigens. Different experimental techniques (electrochemical quartz crystal microbalance, electrochemical impedance spectroscopy, fluorescence confocal microscopy and electrochemical atomic force spectroscopy) were used to evaluate binding kinetics, surface coverage, effect of the applied electric field on IgGs, and role of charged residues on the phenomenon described. These findings expand the concept of electrical control of biological reactions and can be used to gate electrically specific recognition reactions with impact in biosensors, bioactuators, smart biodevices, nanomedicine, and fundamental studies related to chemical reaction kinetics.
In surface-based biosensors, the non-specific or undesired adsorption of the probe is an important characteristic that is typically difficult to measure and therefore to control or eliminate. A methodology for measuring and then minimizing or eliminating this problem on gold surfaces, readily applicable to many common surface modifications is presented. Combining electrochemical perturbation and fluorescence microscopy, we show that the potential at which the adsorbed species is removed can be used as an estimate of the strength of the adsorbate-surface interaction. This desorption potential can be easily measured through an increase in fluorescence intensity as the potential is manipulated. Furthermore, this method can be used to evaluate strategies for preventing or removing non-specific adsorption. This is demonstrated for a wide variety of surface modifications, from strongly chemisorbed monolayers such as thiol self-assembled monolayers (SAMs), to physisorbed monolayers, as well as for complex surface structures like peptide and DNA mixed-component SAMs. The use of a co- adsorption strategy or small magnitude potential-step cycles was shown to significantly decrease the amount of non-specifically or non-covalently bound probe, creating better defined surfaces.
Fluorescence properties of submonolayers of rhodamine 6G have been measured as a function of distance to an aluminum mirror. For distances less than 5 nm (spacer layers of 1 nm thickness were used) a broadening of the emission spectrum is observed. The fluorescence lifetime has been measured for distances up to 6 nm. The broadening of the fluorescence spectra and the shortening of the lifetime observed when the sample is close to the mirror are attributed to efficient energy transfer from the monolayer to the mirror. The experiments clearly demonstrate that both volume and surface contributions must be considered in this energy-transfer process.
In this review, recent advances in DNA microarray technology and their applications are examined. The many varieties of DNA microarray or DNA chip devices and systems are described along with their methods for fabrication and their use. This includes both high-density microarrays for high-throughput screening applications and lower-density microarrays for various diagnostic applications. The methods for microarray fabrication that are reviewed include various inkjet and microjet deposition or spotting technologies and processes, in situ or on-chip photolithographic oligonucleotide synthesis processes, and electronic DNA probe addressing processes. The DNA microarray hybridization applications reviewed include the important areas of gene expression analysis and genotyping for point mutations, single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs). In addition to the many molecular biological and genomic research uses, this review covers applications of microarray devices and systems for pharmacogenomic research and drug discovery, infectious and genetic disease and cancer diagnostics, and forensic and genetic identification purposes. Additionally, microarray technology being developed and applied to new areas of proteomic and cellular analysis are reviewed.
The basic features of DNA were elucidated during the half-century following the discovery of the double helix. But it is only during the past decade that researchers have been able to manipulate single molecules of DNA to make direct measurements of its mechanical properties. These studies have illuminated the nature of interactions between DNA and proteins, the constraints within which the cellular machinery operates, and the forces created by DNA-dependent motors.
Electrochemistry-based sensors offer sensitivity, selectivity and low cost for the detection of selected DNA sequences or mutated genes associated with human disease. DNA-based electrochemical sensors exploit a range of different chemistries, but all take advantage of nanoscale interactions between the target in solution, the recognition layer and a solid electrode surface. Numerous approaches to electrochemical detection have been developed, including direct electrochemistry of DNA, electrochemistry at polymer-modified electrodes, electrochemistry of DNA-specific redox reporters, electrochemical amplifications with nanoparticles, and electrochemical devices based on DNA-mediated charge transport chemistry.
We present a new technology that uses nanopatterned surfaces to separate DNA. This technology eliminates the need for disposable separation media (i.e., gels or polymer solutions that are susceptible to degradation and are difficult to load into small devices due to their inherent high viscosity). We demonstrate using molecular dynamics simulations and experiments that this method can simultaneously separate a broad band of DNA ranging from a few hundred base pairs (bp) through genomic size without a loss in resolution.
Inside cells, motor proteins perform a variety of complex tasks including the transport of vesicles and the separation of chromosomes. We demonstrate a novel use of such biological machines for the mechanical manipulation of nanostructures in a cell-free environment. Specifically, we show that purified kinesin motors in combination with chemically modified microtubules can transport and stretch individual λ-phage DNA molecules across a surface. This technique, in contrast to existing ones, enables the parallel yet individual manipulation of many molecules and may offer an efficient mechanism for assembling multidimensional DNA structures.
The self-diffusion coefficient of a series of DNA fragments ranging from 280 to 5386 bases has been measured by fluorescence recovery after photobleaching after thermal denaturation in 8 M urea. The total persistence length p of single-stranded DNAs and its variation in ionic strength down to 10-3 M has been deduced. The importance of the value of p versus the pore size a and contour length L of the DNA in the optimization of sequencing by gel electrophoresis is emphasized.
Gold surfaces modified with thiol-derivatized DNA duplexes have been investigated as a function of applied electrochemical potential via atomic force microscopy (EC-AFM). At open circuit, monolayers of well-packed DNA helices form with a film depth of 45(3) Å. On the basis of the anisotropic dimensions of these 15 base pair duplexes (20 Å in diameter versus 50 Å in length), this corresponds to an average 45° orientation of the helical axis with respect to the gold surface. Under potential control, the monolayer thickness (and therefore the orientation of the helices) changes dramatically with applied potential. At potentials negative of 0.45 V (versus a Ag wire quasi-reference electrode) film thicknesses of 55 Å are observed, whereas at more positive potentials the monolayer thickness drops to a limiting value of 20 Å. These results are consistent with a morphology change in which the helices either stand straight up or lie flat down on the metal surface, depending on the electrode potential relative to the potential of zero charge (pzc). This voltage-induced morphology change is reversible and effectively constitutes a nanoscale mechanical “switch”.
We have characterized thiol-derivatized, single-stranded DNA (5‘-HS-(CH2)6-CAC GAC GTT GTA AAA CGA CGG CCA G-3‘, abbreviated HS-ssDNA) attached to gold via a sulfur−gold linkage using X-ray photoelectron spectroscopy (XPS), ellipsometry, and 32P-radiolabeling experiments. We found that hybridization of surface-bound HS-ssDNA is dependent on surface coverage. The buffer concentration of the HS-ssDNA solution was found to have a profound effect on surface coverage, with adsorption greatly reduced at low salt concentration. More precise control over surface coverage was achieved by creating mixed monolayers of the thiol-derivatized probe and a spacer thiol, mercaptohexanol (MCH), by way of a two-step method, where first the gold substrate is exposed to a micromolar solution of HS-ssDNA, followed by exposure to a millimolar solution of MCH. A primary advantage of using this two-step process to form HS-ssDNA/MCH mixed monolayers is that nonspecifically adsorbed DNA is largely removed from the surface. Thus, the majority of surface-bound probes are accessible for specific hybridization with complementary oligonucleotides and are able to discriminate between complementary and noncomplementary target molecules. Moreover, the probe-modified surfaces were found to be stable, and hybridization reactions were found to be completely reversible and specific in a series of experiments where duplex melting was examined.
Polyelectrolyte brushes
constitute a new class of material which has recently received considerable interest. The strong segment–segment repulsions and the electrostatic interactions present in such systems bring about completely new physical properties of such monolayers compared to those consisting of either non-stretched or non-charged polymer chains. In this review some recent progress on the theory, synthesis, and swelling behavior of polyelectrolyte brush systems in different environments is discussed. The height of the polyelectrolyte brushes is studied as a function of the molecular weight and graft density on both planar and spherical surfaces. In addition it is elucidated how the brushes are affected by external conditions such as the ionic strength of the surrounding medium, the presence of multivalent or polymeric ions and in some cases by the pH of the contacting solution. Two more specific cases, the synthesis and characterization of mixed polyelectrolyte brushes and cylindrical polyelectrolyte brushes, in which charged polymer chains are attached to the backbone of other polymers, are described in more detail.
Recent analytical innovations for nucleic acid detection have revolutionized the biological sciences. Single nucleic acid sequence detection methods have been expanded to incorporate multiplexed detection strategies. A variety of nucleic acid detection formats are now available that can address high throughput genomic interrogation. Many of these parallel detection platforms or arrays, employ fluorescence as the signaling method. Fluorescence-based assays offer many advantages, including increased sensitivity, safety and multiplexing capabilities, as well as the ability to measure multiple fluorescence properties. Multiplexed microarray platforms provide parallel detection capabilities capable of measuring thousands of simultaneous responses. This review will discuss both single target detection and microarray applications with a focus on gene expression and pathogenic microorganism (PM) detection.
A statistical mechanical treatment of the wormlike chain model (WLC) is used to analyze experiments in which double-stranded DNA, tethered at one end, is stretched by a force applied directly to the free end, by an electric field, or by hydrodynamic flow. All experiments display a strong-stretching regime where the end-to-end distance approaches the DNA contour length as 1/(force)(1/2), which is a clear signature of WLC elasticity. The elastic properties of DNA become scale dependent in the presence of electrostatic interactions; the effective electric charge and the intrinsic bending elastic constant are determined from experiments at low salt concentration. We also consider the effects of spontaneous bends and the distortion of the double helix by strong forces.
We have developed an electrochemical method to quantify the surface density of DNA immobilized on gold. The surface density of DNA, more specifically the number of nucleotide phosphate residues, is calculated from the amount of cationic redox marker measured at the electrode surface. DNA was immobilized on gold by forming mixed monolayers of thiol-derivitized, single-stranded oligonucleotide and 6-mercapto-1-hexanol. The saturated amount of charge-compensation redox marker in the DNA monolayer, determined using chronocoulometry, is directly proportional to the number of phosphate residues and thereby the surface density of DNA. This method permits quantitative determination of both single- and double-stranded DNA at electrodes. Surface densities of single-stranded DNA were precisely varied in the range of (1-10) x 10(12) molecules/cm2, as determined by the electrochemical method, using mixed monolayers. We measured the hybridization efficiency of immobilized single-stranded DNA to complementary strands as a function of the immobilized DNA surface density and found that it exhibits a maximum with increasing surface density.
In this paper, we demonstrate immobilization and stretching of single lambda-phage DNA molecules within microfluidic systems using ac fields. We present a novel "thiol-on-gold"-based immobilization technique for fixing one specific end (3' end) of a DNA molecule onto a gold electrode. A polymer-enhanced medium (approximately 3.75 wt % linear polyacrylamide in Tris-HCl) is used to obtain fully stretched configurations (21 microm) of fluorescently stained lambda-DNA molecules. We also present an optimized microelectrode design with pointed electrodes and an electrode spacing of 20 microm for stretching DNA molecules with an ac field (1 MHz, 3 x 10(5) V/m). Finally, using these techniques, we immobilize a single DNA molecule at one electrode edge, stretch the molecule, and fix the other end at an adjacent electrode edge, forming a bridge between two electrodes within a microfabricated device.
We present experiments on the bias-induced release of immobilized, single-stranded (ss) 24-mer oligonucleotides from Au-surfaces into electrolyte solutions of varying ionic strength. Desorption is evidenced by fluorescence measurements of dye-labeled ssDNA. Electrostatic interactions between adsorbed ssDNA and the Au-surface are investigated with respect to 1), a variation of the bias potential applied to the Au-electrode; and 2), the screening effect of the electrolyte solution. For the latter, the concentration of monovalent salt in solution is varied from 3 to 1600 mM. We find that the strength of electric interaction is predominantly determined by the effective charge of the ssDNA itself and that the release of DNA mainly occurs before the electrochemical double layer has been established at the electrolyte/Au interface. In agreement with Manning's condensation theory, the measured desorption efficiency (etarel) stays constant over a wide range of salt concentrations; however, as the Debye length is reduced below a value comparable to the axial charge spacing of the DNA, etarel decreases substantially. We assign this effect to excessive counterion condensation on the DNA in solutions of high ionic strength. In addition, the relative translational diffusion coefficient of ssDNA in solution is evaluated for different salt concentrations.
We present optical investigations on the conformation of oligonucleotide layers on Au surfaces. Our studies concentrate on the effect of varying surface coverage densities on the structural properties of layers of 12- and 24mer single-stranded DNA, tethered to the Au surface at one end while being labeled with a fluorescent marker at the opposing end. The distance-dependent energy transfer from the marker dye to the metal surface, which causes quenching of the observed fluorescence, is used to provide information on the orientation of the DNA strands relative to the surface. Variations in the oligonucleotide coverage density, as determined from electrochemical quantification, over 2 orders of magnitude are achieved by employing different preparation conditions. The observed enhancement in fluorescence intensity with increasing DNA coverage can be related to a model involving mutual steric interactions of oligonucleotides on the surface, as well as fluorescence quenching theory. Finally, the applicability of the presented concepts for investigations of heterogeneous monolayers is demonstrated by means of studying the coadsorption of mercaptohexanol onto DNA-modified Au surfaces.
Chain-like macromolecules (polymers) show characteristic adsorption properties due to their flexibility and internal degrees of freedom, when attracted to surfaces and interfaces. In this review we discuss concepts and features that are relevant to the adsorption of neutral and charged polymers at equilibrium, including the type of polymer/surface interaction, the solvent quality, the characteristics of the surface, and the polymer structure. We pay special attention to the case of charged polymers (polyelectrolytes) that have a special importance due to their water solubility. We present a summary of recent progress in this rapidly evolving field. Because many experimental studies are performed with rather stiff biopolymers, we discuss in detail the case of semi-flexible polymers in addition to flexible ones. We first review the behavior of neutral and charged chains in solution. Then, the adsorption of a single polymer chain is considered. Next, the adsorption and depletion processes in the many-chain case are reviewed. Profiles, changes in the surface tension and polymer surface excess are presented. Mean-field and corrections due to fluctuations and lateral correlations are discussed. The force of interaction between two adsorbed layers, which is important in understanding colloidal stability, is characterized. The behavior of grafted polymers is also reviewed, both for neutral and charged polymer brushes. Comment: a review: 130 pages, 30 ps figures; final form, added references