Optimization of label-free DNA detection with electrochemical impedance spectroscopy using PNA probes

DNA biosensors, especially those based upon detection of the intrinsic negative charge of target DNA, can be greatly improved by the use of uncharged peptide nucleic acid (PNA) probes. Hybridization causes an increased electrostatic barrier for the negatively charged ferri/ferrocyanide redox couple, resulting in an increase in charge transfer resistance R(ct) that is measured using electrochemical impedance spectroscopy. We report on the optimization of PNA probe surface density by the simultaneous co-immobilization of thiol-modified probes and mercaptohexanol, with the PNA surface density controlled by the thiol mole ratio in solution. Maximum R(ct) change upon hybridization is obtained with 10% PNA mole fraction. The effect of the measurement buffer ionic strength is investigated. The electrostatic barrier for charge transfer to the ferri/ferrocyanide redox couple is approximately independent of ionic strength with PNA probes, but greatly increases with decreasing ionic strength, after hybridization with target DNA. This significantly enhances the R(ct) change upon hybridization. The optimization of PNA surface density and measurement buffer ionic strength leads to a 385-fold increase in R(ct) upon hybridization, a factor of 100 larger than previously reported results using either PNA or DNA probes.     

Detection of DNA recognition events using multi-well field effect devices

We proposed the multi-well field effect device for detection of charged biomolecules and demonstrated the detection principle for DNA recognition events using quasi-static capacitance-voltage (QSCV) measurement. The multi-well field effect device is based on the electrostatic interaction between molecular charges induced by DNA recognition and surface electrons in silicon through the Si(3)N(4)/SiO(2) thin double-layer. Since DNA molecules and DNA binders such as Hoechst 33258 have intrinsic charges in aqueous solutions, respectively, the charge density changes due to DNA recognition events at the Si(3)N(4) surface were directly translated into electrical signal such as a flat band voltage change in the QSCV measurement. The average flat band shifts were 20.7 mV for hybridization and -13.5 mV for binding of Hoechst 33258. From the results of flat band voltage shifts due to hybridization and binding of Hoechst 33258, the immobilization density of oligonucleotide probes at the Si(3)N(4) surface was estimated to be 10(8) cm(-2). The platform based on the multi-well field effect device is suitable for a simple and arrayed detection system for DNA recognition events.     

Conformational states of the cell-penetrating peptide penetratin when interacting with phospholipid vesicles: effects of surface charge and peptide concentration

The most commonly studied of the cell-penetrating peptides (CPP) is "penetratin" (pAntp), which functions as a carrier (vector), even for large hydrophilic (cargo) molecules. pAntp originates from the third helix of the Antennapedia homeodomain protein. The peptide is known to interact with negatively charged phospholipid vesicles, which leads to induction of secondary structure. In the present study, circular dichroism (CD) spectroscopy has been used to characterize the different secondary structures induced upon interaction with small unilamellar vesicles (SUVs) from mixtures of zwitterionic 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG). The interaction was monitored using an electron paramagnetic resonance (EPR) spin probe attached to the peptide, and the intrinsic fluorophore (tryptophan). We measured the secondary structure as a function of surface charge density, total lipid-to-peptide (L/P) molar ratio, and salt concentration, for completely bound peptide. With vesicles from POPG/POPC in a molar ratio below 30:70, at a high L/P, the peptide adopts a mainly helical conformation. Increasing the charge density, at the same L/P, promotes a higher degree of beta-structure. At a fixed charge density, reducing the L/P also results in an alpha-->beta structure conversion. Hence, low membrane surface charge density and low pAntp concentration both favor a mainly helical conformation, while high charge density and pAntp concentration promote a dominating beta-structure. We conclude that pAntp, when residing at the surface of a membrane, is chameleon-like in terms of its induced structure.     

An Electrostatic Model for DNA Surface Hybridization

DNA hybridization at surfaces is a crucial process for biomolecular detection, genotyping, and gene expression analysis. However, hybridization density and kinetics can be strongly inhibited by electric fields from the negatively charged DNA as the reaction proceeds. Here, we develop an electrostatic model to optimize hybridization density and kinetics as a function of DNA surface density, salt concentrations, and applied voltages. The electrostatic repulsion from a DNA surface layer is calculated numerically and incorporated into a modified Langmuir scheme, allowing kinetic suppression of hybridization. At the low DNA probe densities typically used in assays (<10¹³/cm²), electrostatics effects are largely screened and hybridization is completed with fast kinetics. However, higher hybridization densities can be achieved at intermediate DNA surface densities, albeit with slower kinetics. The application of positive voltages circumvents issues resulting from the very high DNA probe density, allowing highly enhanced hybridization densities and accelerated kinetics, and validating recent experimental measurements.     

Strategies for optimizing DNA hybridization on surfaces

Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces.     

Electrochemical detection of DNA hybridization using a change in flexibility

A novel electrochemical method of detecting DNA hybridization is presented based on the change in flexibility between the single and double stranded DNA. A recognition surface based on gold nanoparticles (GNPs) is firstly modified via mixing self-assembled monolayer of thiolated probe DNA and 1,6-hexanedithiol. The hybridization and electrochemical detection are performed on the surface of probe-modified GNPs and electrode, respectively. Here in our method the charge transfer resistance (R(ct)) signal is enhanced by blocking the surface of electrode with DNA covered GNPs. The GNPs will be able to adsorb on the gold electrode when covered with flexible single stranded DNA (ssDNA). On the contrary, it will be repelled from the electrode, when covered with stiff double stranded DNA (dsDNA). Therefore, different R(ct) signals are observed before and after hybridization. The hybridization events are monitored by electrochemical impedance spectroscopy (EIS) measurement based on the R(ct) signals without any external labels. This method provides an alternative route for expanding the range of detection methods available for DNA hybridization.     

Brownian dynamics simulation of probe diffusion in DNA: effects of probe size, charge and DNA concentration

We have used Brownian dynamics simulation to study probe diffusion in solutions of short chain DNA using our previously developed simulation algorithm. We have examined the effect of probe size, charge, and DNA concentration on the probe diffusion coefficient, with the aim of gaining insight into the diffusion of proteins in a concentrated DNA environment. In these simulations, DNA was modeled as a worm-like chain of hydrodynamically equivalent spherical frictional elements while probe particles were modeled as spheres of given charge and hydrodynamic radius. The simulations allowed for both short range Lennard-Jones interactions and long ranged electrostatic interactions between charged particles. For uncharged systems, we find that the effects of probe size and DNA concentration on the probe diffusion coefficient are consistent with excluded volume models and we interpret our results in terms of both empirical scaling laws and the predictions of scaled particle theory. For charged systems, we observe that the effects of probe size and charge are most pronounced for the smallest probes and interpret the results in terms of the probe charge density. For an ionic strength of 0.1 M we find that, below a critical probe surface charge density, the probe diffusion coefficient is largely independent of probe charge and only weakly dependent on the DNA charge. These effects are discussed in terms of the interactions between the probe and the DNA matrix and are interpreted in terms of both the underlying physics of transport in concentrated solutions and the assumptions of the simulation model.     

Phase diagram, stability, and overcharging of lamellar cationic lipid-DNA self-assembled complexes.

Cationic lipid-DNA (CL-DNA) complexes comprise a promising new class of synthetic nonviral gene delivery systems. When positively charged, they attach to the anionic cell surface and transfer DNA into the cell cytoplasm. We report a comprehensive x-ray diffraction study of the lamellar CL-DNA self-assemblies as a function of lipid composition and lipid/DNA ratio, aimed at elucidating the interactions determining their structure, charge, and thermodynamic stability. The driving force for the formation of charge-neutral complexes is the release of DNA and lipid counterions. Negatively charged complexes have a higher DNA packing density than isoelectric complexes, whereas positively charged ones have a lower packing density. This indicates that the overcharging of the complex away from its isoelectric point is caused by changes of the bulk structure with absorption of excess DNA or cationic lipid. The degree of overcharging is dependent on the membrane charge density, which is controlled by the ratio of neutral to cationic lipid in the bilayers. Importantly, overcharged complexes are observed to move toward their isoelectric charge-neutral point at higher concentration of salt co-ions, with positively overcharged complexes expelling cationic lipid and negatively overcharged complexes expelling DNA. Our observations should apply universally to the formation and structure of self-assemblies between oppositely charged macromolecules.     

The role of lipid charge density in the serum stability of cationic lipid/DNA complexes

To evaluate the role of lipid charge density in the serum stability of DOTAP-Chol/DNA complexes (lipoplexes), lipid-DNA interactions, extent of aggregation, supercoil content, and in vitro transfection efficiency of lipoplexes were investigated. In general, higher serum concentration destabilized, and increasing molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP(+)/DNA(-)) stabilized lipoplexes in serum as assessed by the criteria used in this study. The increase of cholesterol content led to increased serum stability, and DOTAP:Chol (mol/mol 1:4)/DNA lipoplex with DOTAP(+)/DNA(-) ratio 4 was the most serum stable formulation of all the formulations examined, and maintained lipid-DNA interactions, did not aggregate and exhibited high in vitro transfection efficiency in 50% (v/v) serum. The increased stability of this formulation could not be explained by the decreased charge density of the lipid component. Furthermore, no single parameter examined in the study could be used to consistently predict the in vitro transfection efficiency of lipoplexes in serum. Surprisingly, no correlation between the maintenance of supercoiled DNA content and in vitro transfection efficiency was found in the study.     

Detection of DNA hybridization and extension reactions by an extended-gate field-effect transistor: characterizations of immobilized DNA-probes and role of applying a superimposed high-frequency voltage onto a reference electrode

As we have already shown in a previous publication [Kamahori, M., Ihige, Y., Shimoda, M., 2007. Anal. Sci. 23, 75-79], an extended-gate field-effect transistor (FET) sensor with a gold electrode, on which both DNA probes and 6-hydroxyl-1-hexanethiol (6-HHT) molecules are immobilized, can detect DNA hybridization and extension reactions by applying a superimposed high-frequency voltage to a reference electrode. However, kinetic parameters such as the dissociation constant (K(d)(s)) and the apparent DNA-probe concentration (C(probe)(s)) on a surface were not clarified. In addition, the role of applying the superimposed high-frequency voltage was not considered in detail. In this study, the values of K(d)(s) and C(probe)(s) were estimated using a method involving single-base extension reaction combined with bioluminescence detection. The value of K(d)(s) on the surface was 0.38 microM, which was about six times that in a liquid phase. The value of C(probe)(s), which expressed the upper detection limit for the solid phase reaction, was 0.079 microM at a DNA-probe density of 2.6 x 10(12)molecules/cm(2). We found that applying the superimposed high-frequency voltage accelerated the DNA molecules to reach the gold surface. Also, the distance between the DNA-probes immobilized on the gold surface was controlled to be over 6 nm by applying a method of competitive reaction with DNA probes and 6-HHT molecules. This space was sufficient to enable the immobilized DNA-probes to lie down on the 6-HHT monolayer in the space between them. Thus, the FET sensor could detect DNA hybridization and extension reactions by applying a superimposed high-frequency voltage to the DNA-probes density-controlling gold surface.     

A test of discreteness-of-charge effects in phospholipid vesicles: measurements using paramagnetic amphiphiles

A new series of negatively charged, paramagnetic alkylsulfonate probes was synthesized and can be used to measure both the internal and the external surface potentials of model membrane systems. We tested for discreteness-of-charge effects in lipid membranes by comparing the surface potentials, estimated by use of these negatively charged amphiphiles, with that of a series of positively charged alkylammonium nitroxides in charged membranes. From the partitioning of these probes, the membrane surface potential was estimated in phosphatidylcholine membranes containing either phosphatidylserine or didodecyldimethylammonium bromide. The surface potentials, estimated with either positive or negative probes, were identical, within experimental error, in either positive or negative membranes, and they were well accounted for by a simple Gouy-Chapman-Stern theory. This symmetry, with respect to the sign of the charge, indicates that discreteness-of-charge effects are not significant in determining the potential-sensitive phase partitioning of these probes in model membranes. Thus, despite the fact that charge on membranes is discrete, models that assume a uniform density of charge in the plane of the membrane adequately account for the potentials measured by these amphiphilic probes.     

Surface density dependence of PCR amplicon hybridization on PNA/DNA probe layers

Surface plasmon field-enhanced fluorescence spectroscopy was employed to extensively investigate the hybridization behaviors of polymerase chain reaction (PCR) amplicons on a peptide nucleic acid (PNA) or DNA probe layer that was previously attached on a streptavidin-modified gold surface via biotin/streptavidin interaction. Despite the neutral backbone of PNA, the hybridization reactions were strongly influenced by the variation of ionic strength. The association rates exhibited a monotonic decrease with ionic strength increase and the maximum hybridization signal was achieved at an intermediate sodium concentration (approximately 100 mM). These effects were mainly ascribed to the electrostatic cross talk among the hybridized DNA molecules and the secondary structure of PCR amplicons. For the negatively charged DNA probes, the hybridization reaction was subjected additionally to the DNA/DNA electrostatic barrier, particularly in lower ionic strength range (e.g., 10 approximately 150 mM Na(+)). The electrostatic cross talk was shown to be largely reduced if the PNA probe layer was sufficiently diluted by following a strategic templated immobilization method. As a consequence, a pseudo-first-order kinetic model was applicable to describe the hybridization kinetics, and affinity constants were derived for evaluating the influence of single nucleotide polymorphisms (SNPs).     

Shielding effect of monovalent and divalent cations on solid-phase DNA hybridization: surface plasmon resonance biosensor study

Solid-phase hybridization, i.e. the process of recognition between DNA probes immobilized on a solid surface and complementary targets in a solution is a central process in DNA microarray and biosensor technologies. In this work, we investigate the simultaneous effect of monovalent and divalent cations on the hybridization of fully complementary or partly mismatched DNA targets to DNA probes immobilized on the surface of a surface plasmon resonance sensor. Our results demonstrate that the hybridization process is substantially influenced by the cation shielding effect and that this effect differs substantially for solid-phase hybridization, due to the high surface density of negatively charged probes, and hybridization in a solution. In our study divalent magnesium is found to be much more efficient in duplex stabilization than monovalent sodium (15 mM Mg²⁺ in buffer led to significantly higher hybridization than even 1 M Na⁺). This trend is opposite to that established for oligonucleotides in a solution. It is also shown that solid-phase duplex destabilization substantially increases with the length of the involved oligonucleotides. Moreover, it is demonstrated that the use of a buffer with the appropriate cation composition can improve the discrimination of complementary and point mismatched DNA targets.     

Surface electrostatic effects in oligonucleotide microarrays: control and optimization of binding thermodynamics

We present a theoretical thermodynamic framework for the design of more efficient oligonucleotide microarrays. A general thermodynamic relation is derived to describe the electrostatic surface effects on the binding of the assayed biomolecule to a surface-tethered molecular probe. The relation is applied to analyze how the nucleic acid target, the oligonuleotide probe, and their DNA duplex electrostatic interactions with the surface affect the hybridization on DNA arrays. Taking advantage of a closed form exact solution of the linear Poisson-Boltzmann equation for a charged ion-penetrable sphere in electrolyte solution interacting with a plane wall, we study the effects of the surface and solution conditions. Binding free energy is found as a function of the surface material, dielectric or metal, the surface charge density, linker molecule length, temperature, and added salt content. The charge or electric potential of the dielectric or metal surface, respectively, is shown to dominate the hybridization, especially at low added salt or short linker length. We predict that substantial enhancement of sensitivity, selectivity, and reliability of microarrays can be achieved by control of the surface conditions. As examples, we discuss how to overcome two limitations of current technologies: nonequal sensitivity of the probes with different GC and AT bases content, and poor match/mismatch discrimination. In addition, we suggest the design of microarray conditions where the tested nucleic acid is unfolded, thus making possible the screening of a larger sequence with single nucleotide resolution. These promising findings are discussed and further experimental tests suggested.     

Imaging immobilised ssDNA and detecting DNA hybridisation by means of the repelling mode of scanning electrochemical microscopy (SECM)

The supposed repelling mode of scanning electrochemical microscopy (SECM) allows truly label-free electrochemical recognition of the presence and hybridisation of nucleic acids that are immobilised on conducting DNA chips. Basically, the SECM-based detection of single- and double-stranded DNA profits from the electrostatic repulsion between deprotonated phosphate groups at the backbone of the oligonucleotides and a free-diffusing negatively charged redox mediator (e.g. [Fe(CN)(6)](3-/4-)). In electrolytes of proper pH and ionic strength, this coulomb interaction is heavily influencing the diffusion properties of the mediator in the vicinity of the surface-anchored DNA strands. This charge interaction modulates the diffusional mass transport for the charged redox species in the DNA modified regions, and thus locally decreases the positive feedback currents measured with a SECM tip placed within the electrochemical nearfield of the chip surface. This approach was used to study arrays of synthetic 20-base oligonucleotide probes that were immobilised on monolayer-modified gold surfaces. Evidence is provided that the density of probes, the ionic strength of solution and the tip-to-sample distance have a strong impact on the capability of the repelling mode of SECM to visualise probe spots and hybridisation while the concentration of the chosen mediator did not significantly affect detection.     

Surface potential determination in planar lipid bilayers: a simplification of the conductance-ratio method

One of the methods available for the measurement of surface potentials of planar lipid bilayers uses the conductance ratio between a charged and a neutral bilayer doped with ionophores to calculate the surface potential of the charged bilayer. We have devised a simplification of that method which does not require the use of an electrically neutral bilayer as control. The conductance of the charged bilayer is measured before and after the addition of divalent cations (Ba(2+)) to the bathing solution. Ba(2+) ions screen fixed surface charges, decreasing the surface potential. If the membrane is negatively charged the screening has the effect of decreasing the membrane conductance to cations. The resulting conductance ratio is used to calculate the surface potential change, which is fed into an iterative computer program. The program generates pairs of surface potential values and calculates the surface charge density for the two conditions. Since the surface charge density remains constant during this procedure, there is only one pair of surface potentials that satisfies the condition of constant charge density. Applying this method to experimental data from McLaughlin et al. [McLaughlin, S.G.A., Szabo, G. and Eisenman, G., Divalent ions and the surface potential of charged phospholipid membranes, J. Gen. Physiol., 58 (1971) 667-687.] we have found very similar results. We have also successfully used this method to determine the effect of palmitic acid on the surface potential of asolectin membranes.     

Oligonucleotides form a duplex with non-helical properties on a positively charged surface

The double helix is known to form as a result of hybridization of complementary nucleic acid strands in aqueous solution. In the helix the negatively charged phosphate groups of each nucleic acid strand are distributed helically on the outside of the duplex and are available for interaction with cationic groups. Cation-coated glass surfaces are now widely used in biotechnology, especially for covalent attachment of cDNAs and oligonucleotides as surface-bound probes on microarrays. These cationic surfaces can bind the nucleic acid backbone electrostatically through the phosphate moiety. Here we describe a simple method to fabricate DNA microarrays based upon adsorptive rather than covalent attachment of oligonucleotides to a positively charged surface. We show that such adsorbed oligonucleotide probes form a densely packed monolayer, which retains capacity for base pair-specific hybridization with a solution state DNA target strand to form the duplex. However, both strand dissociation kinetics and the rate of DNase digestion suggest, on symmetry grounds, that the target DNA binds to such adsorbed oligonucleotides to form a highly asymmetrical and unwound duplex. Thus, it is suggested that, at least on a charged surface, a non-helical DNA duplex can be the preferred structural isomer under standard biochemical conditions.     

Detection of hybrid formation between peptide nucleic acids and DNA by fluorescence polarization in the presence of polylysine.

A new method for the detection of PNA/DNA hybrids is presented. In this method, short PNA probes (9-13 mer) are labeled with a fluorescent dye and allowed to hybridize to target DNA molecules. A cationic polyamino acid, such as polylysine, is then added to the reaction mixture, whereupon the DNA molecules bind electrostatically to this polycation. The PNA probes, which are uncharged or may carry only a small charge due to the fluorescent dye, do not bind to polylysine unless hybridized to the negatively charged DNA target. The binding of the labeled PNA/DNA hybrid to the high-molecular-weight polymer leads to a significant change in the rotational correlation time of the fluorophore attached to the PNA. This can be conveniently detected by measuring the fluorescence polarization of the latter. The method is completely homogeneous because no separation of free from bound PNA probe is required. The hybridization and dehybridization reactions can be followed in real time. The method has been applied to the typing of single-nucleotide polymorphisms in PCR products.     

Surface potential determination in planar lipid bilayers: A simplification of the conductance-ratio method

One of the methods available for the measurement of surface potentials of planar lipid bilayers uses the conductance ratio between a charged and a neutral bilayer doped with ionophores to calculate the surface potential of the charged bilayer. We have devised a simplification of that method which does not require the use of an electrically neutral bilayer as control. The conductance of the charged bilayer is measured before and after the addition of divalent cations (Ba²⁺) to the bathing solution. Ba²⁺ ions screen fixed surface charges, decreasing the surface potential. If the membrane is negatively charged the screening has the effect of decreasing the membrane conductance to cations. The resulting conductance ratio is used to calculate the surface potential change, which is fed into an iterative computer program. The program generates pairs of surface potential values and calculates the surface charge density for the two conditions. Since the surface charge density remains constant during this procedure, there is only one pair of surface potentials that satisfies the condition of constant charge density.Applying this method to experimental data from McLaughlin et al. [McLaughlin, S.G.A., Szabo, G. and Eisenman, G., Divalent ions and the surface potential of charged phospholipid membranes, J. Gen. Physiol., 58 (1971) 667–687.] we have found very similar results. We have also successfully used this method to determine the effect of palmitic acid on the surface potential of asolectin membranes.     

Application of cationic conjugated polymers in microarrays using label-free DNA targets

A fluorescence-based microarray technique that does not require target DNA labeling is detailed. This 'label-free' approach utilizes a cationic, water-soluble conjugated polymer PFBT (poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole) dibromide]), and neutral PNA (peptide nucleic acid) hybridization probes. DNA hybridization to immobilized PNA spots results in a change in the net charge at that particular surface. Electrostatic interactions between the cationic polymer and negatively charged DNA bind the polymer to the hybrid DNA/PNA complex. By exciting the conjugated polymer at 488 nm on a commercial microarray scanner, the presence of the target is directly indicated by the fluorescence emission of the polymer. This feature eliminates the necessity of target labeling required in traditional microarray protocols. There are five steps involved in the procedure before scanning or imaging the array: (i) slide hydration, (ii) target hybridization, (iii) post-hybridization washing, (iv) polymer application and (v) polymer washing. Each step takes 20 min to 1 h. The overall protocol requires approximately 2-3 h.