Surface Probe Density Research Articles

Quantitation of surface coverage of oligonucleotides bound to chip surfaces: a fluorescence-based approach using alkaline phosphatase digestion

Silanized chip surfaces provide a reliable substrate for immobilization of oligonucleotides. The ability for rapid and sensitive detection of oligonucleotide surface coverage on these chips is crucial for their wide and effective applications in biotechnology. In this paper, two different silanization procedures were used to covalently bind fluorescent-labeled single-stranded DNA onto silicon dioxide or nitride chip surfaces. Effects of surface functionalization techniques for different surfaces, and immobilization conditions, including buffers and solution ionic strength, on surface probe coverage were investigated, quantifying the endpoint probe density by fluorescent measurement upon digestion with alkaline phosphatase (ALP). Digestion of surface-immobilized oligonuleotides with ALP released the fluorophore-tagged probe fragments back into the solution. The detection of DNA was accomplished by laser-induced fluorescence detection of the solution containing those cleaved fragments. The probe surface density on gold thin film, determined by ALP-digestion, was found to coincide well with that measured using the conventional alkanethiol-based fluorescence-displacement technique for the same system. The developed method has important implications for evaluating the performance of different oligonucleotide immobilization strategies. Also, it has the potential to serve as a sample-thrifty, time saving, and therefore routine tool to realize more realistic, practical quantification of the surface coverage of oligonucleotides immobilized on any solid surfaces.

Optimization of DNA immobilization on gold electrodes for label-free detection by electrochemical impedance spectroscopy

The ability to immobilize DNA probes onto gold substrates at an optimum surface density is key in the development of a wide range of DNA biosensors. We present a method to accurately control probe DNA surface density by the simultaneous co-immobilization of thiol modified probes and mercaptohexanol. Probe surface density is controlled by the thiol molar ratio in solution, with a linear relationship between thiol molar ratio and probe density spanning (1–9) × 10 12/cm 2. The probe surface density per microscopic surface area was determined using chronocoulometry, and a detailed analysis of the method presented. Using this sample preparation method, the effect of probe density and hybridization on the charge transfer resistance with the negatively charged ferri/ferrocyanide redox couple was determined. Above a threshold probe surface density of 2.5 × 10 12/cm 2, electrostatic repulsion from the negatively charged DNA modulates the charge transfer resistance, allowing hybridization to be detected. Below the threshold density no change in charge transfer resistance with probe density or with hybridization occurs. The probe surface density was optimized to obtain the maximum percentage change in charge transfer resistance with hybridization.

Computer simulation study of probe-target hybridization in model DNA microarrays: Effect of probe surface density and target concentration

We use lattice Monte Carlo simulations to study the thermodynamics of hybridization of single-stranded "target" genes in solution with complementary "probe" DNA molecules immobilized on a microarray surface. The target molecules in our system contain 48 segments and the probes tethered on a hard surface contain 8-24 segments. The segments on the probe and target are distinct, with each segment representing a sequence of nucleotides that interacts exclusively with its unique complementary target segment with a single hybridization energy; all other interactions are zero. We examine how surface density (number of probes per unit surface area) and concentration of target molecules affect the extent of hybridization. For short probe lengths, as the surface density increases, the probability of binding long stretches of target segments increases at low surface density, reaches a maximum at an intermediate surface density, and then decreases at high surface density. Furthermore, as the surface density increases, the target is less likely to bind completely to one probe; instead, it binds simultaneously to multiple probes. At short probe lengths, as the target concentration increases, the fraction of targets binding completely to the probes (specificity) decreases. At long probe lengths, varying the target concentration does not affect the specificity. At all target concentrations as the probe length increases, the fraction of target molecules bound to the probes by at least one segment (sensitivity) increases while the fraction of target molecules completely bound to the probes (specificity) decreases. This work provides general guidelines to maximizing microarray sensitivity and specificity. Our results suggest that the sensitivity and specificity can be maximized by using probes 130-180 nucleotides long at a surface density in the range of 7 x 10(-5)- 3 x 10(-4) probe molecules per nm(2).

Structure and DNA hybridization properties of mixed nucleic acid/maleimide-ethylene glycol monolayers.

The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s --> pi* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the "high-density" probe surface than on the "high-efficiency" probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.

Electric Potential Induced Dissociation of Hybridized DNA with Hairpin Motif Immobilized on Silicon Surface

DNA probes were immobilized on silicon surfaces through ester condensation between the -COOH group on the surface and the terminal -OH group in the oligonucleotide, and the surface density of DNA probes against the concentration of immobilization solution was measured by radioactive labeling. The dissociation of DNA duplex on the surface by an electric potential was studied with the scanning potential hairpin denaturation/dissociation (SPHD). The influence of the stem length in the hairpin probe on the SPHD curve was systematically investigated. It was found that the capability of discrimination on single nucleotide polymorphism (SNP) by a hairpin probe was related to the free energy of formation of the secondary structure in the probe (DeltaG(ss)). In our system, when DeltaG(ss) was around -3 kcal/mol, an optimal recognition of SNP was reached and the SPHD curve was sigmoid. In contrast, the equivalent SPHD curve from a linear probe was exponential-decay alike with a poor discrimination of SNP. The concentration dependent experiments showed good linearity between the melting potential and logarithm of target concentration in the range of 1 x 10(-9) to 5 x 10(-7) M.

Immobilization of DNA Probes onto Gold Surface and its Application to Fully Electric Detection of DNA Hybridization using Field-Effect Transistor Sensor

A field-effect transistor (FET) sensor with a gold sensing electrode (extended-gate FET sensor), on which DNA probes can be immobilized via an Au–S bond, was designed. A method of controlling the surface density of DNA probes immobilized on the gold electrode was developed using a competitive reaction between DNA probes and alkanethiols. The immobilized DNA probes were characterized using voltammetry and a single-base extension reaction combined with bioluminescence detection. The relationship between DNA probe density and hybridization efficiency was clarified, and it was found that the optimum density for FET sensors was about 2.6×1012 molecules/cm2. The fully electric detection of hybridized target DNA (about 7 fmol) was achieved by the extended-gate FET sensor with the above DNA probe density. In addition, the surface potential in proportion to the density of both single-stranded DNA and double-stranded DNA immobilized on the gold electrode was successfully obtained using the extended-gate FET sensor.

Hybridization isotherms of DNA microarrays and the quantification of mutation studies.

Diagnostic DNA arrays for detection of point mutations as markers for cancer usually function in the presence of a large excess of wild-type DNA. This excess can give rise to false positives as a result of competitive hybridization of the wild-type target at the mutation spot. Analysis of the DNA array data is typically qualitative, aimed at establishing the presence or absence of a particular point mutation. Our theoretical approach yields methods for quantifying the analysis to obtain the ratio of concentrations of mutated and wild-type DNA. The theory is formulated in terms of the hybridization isotherms relating the hybridization fraction at the spot to the composition of the sample solutions at thermodynamic equilibrium. It focuses on samples containing an excess of single-stranded DNA and on DNA arrays with a low surface density of probes. The hybridization equilibrium constants can be obtained by the nearest-neighbor method. Two approaches allow acquisition of quantitative results from the DNA array data. In one, the signal of the mutation spot is compared with that of the wild-type spot. The implementation requires knowledge of the saturation intensity of the two spots. The second approach requires comparison of the intensity of the mutation spot at two different temperatures. In this case, knowledge of the saturation signal is not always necessary. DNA arrays can be used to obtain quantitative results on the concentration ratio of mutated DNA to wild-type DNA in studies of somatic point mutations.

Unique supramolecular assembly of a redox protein with nucleic acids onto hybrid bilayer: towards a dynamic DNA chip

Highly controlled supramolecular assemblies combining a genetically engineered redox protein, cytochrome b5, and modified oligonucleotides are presented. Modified b5 and DNA are covalently assembled through a hetero bifunctional cross-linker to give a unique hybrid molecular species. Moreover, the assembly includes a histidine tag head able to bind to modified phospholipids which lead to a new generation of self-assembled dynamic DNA chips. The interaction of the construction with a complementary oligonucleotide sequence can be monitored in real time by surface plasmon resonance using Biacore technology. The biochip, presented herein, features unique properties including tunable surface density of probes, very low non-specific interactions and optimization of hybridization efficiency. In addition, we demonstrated that the phase transition of the lipidic layer can modulate the dynamic of the association of the complex to the supported membrane. Potential applications of this new device are multiple including high sensitivity and high selectivity biochips, especially for studies of the DNA-ligands interactions in a biomimetic environment.

Theoretical aspects of genomic variation screening using DNA microarrays.

We present a theoretical model for typical microarray-based single nucleotide polymorphism (SNP) assay of small genomic DNA amount. We derived the adsorption isotherm expressing the on-array hybridization efficiency in terms of genomic target sequence and concentration, oligonucleotide probe sequence and surface density, hybridization buffer, and temperature. This isotherm correctly describes the surface probe density effects, the sensitivity peak, and the melting temperature depression, and is in accord with published experiments. We discuss optimization of parallel SNP genotyping. Our estimates show that SNP detection at a single temperature in aqueous hybridization buffer is restricted by DNA regions that differ by less than 20% in GC content. We predict that the variety of genotyped SNPs could be substantially extended using an assay design with high probe density and a large fraction of probes hybridized.

Sensitive quantitative nucleic acid detection using oligonucleotide microarrays.

We report a new theoretical approach to optimize the performance and quantify the results of gene expression oligonucleotide microarrays which are widely used in biomedical research. An on-array hybridization isotherm that takes into account the screened Coulomb repulsion between the assayed nucleic acid target and the layer of surface tethered oligonucleotide probes is presented. The hybridization efficiency is found as a function of the genomic target (sequence, length, and concentration), array parameters (probe sequence and length, surface probe density), and hybridization conditions (temperature and buffer ionic strength). We present simple relations for the hybridization signal maximum and the linear dynamic detection range and show explicit criteria for optimization. The approach is based on an extension of our recently published theory (Vainrub, A.; Pettitt, B. M. Phys. Rev. E 2002, 66, art. no.-041905) which we generalize here for the cases of target depletion effects and arbitrary target length.

Coulomb blockage of hybridization in two-dimensional DNA arrays.

Experiments on DNA microarrays have revealed substantial differences in hybridization thermodynamics between DNA free in solution and surface tethered DNA. Here we develop a mean field model of the Coulomb effects in two-dimensional DNA arrays to understand the binding isotherms and thermal denaturation of the double helix. We find that the electrostatic repulsion of the assayed nucleic acid from the array of DNA probes dominates the binding thermodynamics, and thus causes the Coulomb blockage of the hybridization. The results explain, observed in DNA microarrays, the dramatic decrease of the hybridization efficiency and the thermal denaturation curve broadening as the probe surface density grows. We demonstrate application of the theory for evaluation and optimization of the sensitivity, specificity, and the dynamic range of DNA array devices.

Oligonucleotide arrays for high-throughput SNPs detection in the MHC class I genes: HLA-B as a model system.

A simple and efficient oligonucleotide array was developed to identify single nucleotide polymorphisms (SNPs) encoded within the highly polymorphic human major histocompatibility complex (MHC) using HLA-B as a model system. A total of 137 probes were designed to represent all known polymorphisms encoded in exons 2 and 3. PCR products were amplified from human genomic DNA and allowed to hybridize with the oligonucleotide array. Hybridization was detected by fluorescence scanning, and HLA-B alleles were assigned by quantitative analysis of the hybridization results. Variables known to influence the specificity of hybridization, such as oligonucleotide probe size, spacer length, surface density, hybridization conditions, and array uniformity and stability were studied. The efficiency and specificity of identifying HLA-B SNPs using the oligonucleotide arrays was evaluated by blinded analysis of 100 samples from unrelated individuals representing all HLA-B phenotypes. The oligonucleotide array method described in this paper provides unambiguous detection of complex heterozygous SNP combinations. This methodological approach may be applied to other highly polymorphic gene systems.

The effect of surface probe density on DNA hybridization.

The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we study how probe immobilization at surfaces, specifically probe density, influences the kinetics of target capture using surface plasmon resonance (SPR) spectroscopy, an in situ label-free optical method. Probe density is controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential and whether duplex or single stranded oligonucleotides are used. Independent of which probe immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for probe/target hybridization. However, hybridization depends strongly on probe density in both the efficiency of duplex formation and the kinetics of target capture. We propose that probe density effects may account for the observed variation in target-capture rates, which have previously been attributed to thermodynamic effects.

Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays.

Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique for measuring interactions between unlabeled biological molecules with arrays of surface-bound species. In this paper, SPR imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array. For the hybridization adsorption of both DNA and RNA oligonucleotides, a detection limit of 10 nM is reported; for large (1,500-base) 16S rRNA molecules, concentrations as low as 2 nM are detected. The covalent attachment of thiol-DNA probes to the gold surface leads to high surface probe density (10(12) molecules/cm2) and excellent probe stability that enables more than 25 cycles of hybridization and denaturing without loss in signal or specificity. Fresnel calculations are used to show that changes in percent reflectivity as measured by SPR imaging are linear with respect to surface coverage of adsorbed DNA oligonucleotides. Data from SPR imaging is used to construct a quantitative adsorption isotherm of the hybridization adsorption on a surface. DNA and RNA 18-mer oligonucleotide hybridization adsorption is found to follow a Langmuir isotherm with an adsorption coefficient of 1.8 x 10(7) M(-1).

The biophysics of DNA hybridization with immobilized oligonucleotide probes

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.