A novel phosphoramidite method for automated synthesis of oligonucleotides on glass supports for biosensor development

Two protocols for functionalization of glass supports with hexaethylene glycol (HEG)-linked oligonucleotides were developed. The first method (standard amidite protocol) made use of the 2-cyanoethyl-phosphoramidite derivative of 4,4′-dimethoxytrityl-protected HEG. This was first coupled to the support by standard solid-phase phosphoramidite chemistry followed by extension with a thymidylic acid icosanucleotide. Stepwise addition of the linker phosphoramidite graduated at 1% (relative to the total sites available) perstep at 50°C resulted in an optimal yield of immobilized oligonucleotides at a density of 2.24 × 10¹⁰ strands/mm². This observed loading maximum lies well below the theoretical maximum loading owing to nonspecific adsorption of HEG on the glass and subsequent blocking of reactive sites. Surface loadings as high as 3.73 × 10¹⁰/mm² and of excellent sequence quality were achieved with a reverse amidite protocol. The support was first modified into a 2-cyanoethyl-N,N-diisopropylphosphoramidite analog followed by coupling with 4,4′-dimethoxytrityl-protected HEG. This protocol is conveniently available when using a conventional DNA synthesizer. The reverse amidite protocol allowed for control of the surface loading at values suitable for subsequent analytical applications that make use of immobilized oligonucleotides as probes for selective hybridization of sample nucleic acids of unknown sequence and concentration.     

Influences of non-selective interactions of nucleic acids on response rates of nucleic acid fiber optic biosensors

The immobilization of oligonucleotides to solid surfaces can provide a platform of chemistry that is suitable for the development of biosensor and microarray technologies. Experiments were performed using a fiber optic nucleic acid biosensor based on total internal reflection fluorescence to examine the effects of the presence of non-complementary DNA on the detection of hybridization of complementary target DNA. The work has focused on the rates and extent of hybridization in the presence and absence of non-selective adsorption using fluorescein-labeled DNA. A stop-flow system of 137 microL volume permitted rapid introduction and mixing of each sample. Response times measured were on the order of seconds to minutes. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on the extent of hybridization of complementary oligonucleotides, but actually reduced the response times of sensors to cDNA oligonucleotides.     

Alteration of the selectivity of hybridization of immobilized oligonucleotide probes by co-immobilization with charged oligomers of ethylene glycol

A significant challenge exists in the creation of an environment for immobilized probe oligonucleotides that offer good structural regularity and reproducibility, where nearest neighbour interactions provide for control of selectivity, yet where the degree of hybridization does not alter nearest neighbour interactions. This new work explores whether a “matrix isolation” method will produce the desired environment for the probe molecules. The DNA oligonucleotide probes are polyelectrolytes with charged backbones and significant flexibility. It is possible to isolate the probe molecules by surrounding each, on average, with a sheath of immobilized oligomer that is not based on complementary nucleic acid, yet that is a polyelectrolyte in order to control the surface density and charge within the mixed film. Preliminary work investigates a mixture of dT₂₀ as the probe oligonucleotide, and a 20-mer oligomer primarily containing ethylene glycol phosphate, as a matrix isolation material in a 1:20 mole ratio, respectively. Melt temperature (T_m) measurements indicate that the thermodynamic stability of the probe molecules can be adjusted using the oligomer matrix to achieve lower T_m values by up to 5 °C, with full retention of selectivity for discrimination of single base pair mismatches even under conditions where the probes at a surface are saturated with complementary target.     

Influences of non-selective interactions of nucleic acids on response rates of nucleic acid fiber optic biosensors

The immobilization of oligonucleotides to solid surfaces can provide a platform of chemistry that is suitable for the development of biosensor and microarray technologies. Experiments were performed using a fiber optic nucleic acid biosensor based on total internal reflection fluorescence to examine the effects of the presence of non-complementary DNA on the detection of hybridization of complementary target DNA. The work has focused on the rates and extent of hybridization in the presence and absence of non-selective adsorption using fluorescein-labeled DNA. A stop-flow system of 137 μL volume permitted rapid introduction and mixing of each sample. Response times measured were on the order of seconds to minutes. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on the extent of hybridization of complementary oligonucleotides, but actually reduced the response times of sensors to cDNA oligonucleotides. Received: 26 September 2000 / Revised: 24 November 2000 / Accepted: 30 November 2000     

Trends in the development of nucleic acid biosensors for medical diagnostics

Some of the recent advances in the field of biosensors for nucleic acid analysis in medical diagnostic applications are highlighted. Particular attention is paid in this review to the progress made in two key areas of development: (i) enhancements achieved in device selectivity, and (ii) enhancements achieved in device sensitivity.     

Towards the optimization of an optical DNA sensor: control of selectivity coefficients and relative surface affinities

Short single-stranded DNA (ssDNA) oligonucleotides can be grown on the surface of fused silica by automated nucleic acid synthesis. The immobilized ssDNA can be deposited at a desired average density. The density of ssDNA provides a controlled parameter that in combination with temperature, ionic strength and pH, can be used to define the selectivity of hybridization. Furthermore, the density of ssDNA can be used to control the affinity of complementary DNA so that it associates with the nucleic acids on the surface rather than areas that are not coated with ssDNA. The characteristic melt temperature observed for immobilized double-stranded DNA (dsDNA) 20mer shifts by up to 10 °C when a single base pair mismatch is present in the center of a target oligonucleotide. Optimization of quantitative analysis of such single base pair mismatches requires use of select experimental conditions to maximize the formation of the fully matched target duplex while minimizing the formation of the mismatched duplex. Results based on fiber optic biosensors that are used to study binding of fluorescein-labeled complementary DNA demonstrate that it is possible to achieve a selectivity coefficient of fully matched to single base pair mismatch of approximately 85-1, while maintaining >55% of the maximum possible signal that can be obtained from the fully matched target duplex.     

Considerations for the quantitative transduction of hybridization of immobilized DNA

Immobilized single-stranded DNA (ssDNA) can be used as a selective ‘reagent’ to bind complementary DNA or RNA for applications such as the detection of pathogenic organisms, gene therapy agents and genetic mutations. The density of ssDNA on a surface will determine the charge density due to ionizable phosphate groups. Such a negatively charged interface will attract positive counter-ions from solution, which may result in a local ionic strength, pH and dielectric constant on the surface that is substantially different from that in bulk electrolyte solution. It is the local conditions which influence the thermodynamics of hybridization, and this can studied by the melt temperature (T_m) of double-stranded DNA (dsDNA). Experimental work and theoretical models have been used to examine whether hybridization reactions on a surface can cause dynamic changes in local charge density, and therefore, changes in selectivity and drift in calibration for quantitative analysis. Organosilane chemistry has been used to covalently immobilize hexaethylene glycol linkers and to control the subsequent density of dT₂₀ that was prepared by automated synthesis. Fiber-optic biosensors based on fused silica that was coated with DNA were used in a total internal reflection fluorescence instrument to determine T_m from the dissociation of duplexes of fluorescein-labeled dA₂₀ : dT₂₀. The experimental results suggest that the thermodynamic stability of duplexes that are immobilized on a surface is dependent on the density of immobilized DNA and on the extent of hybridization of DNA. The experimental results show that the thermodynamic stability of immobilized dsDNA is significantly different than that of dsDNA in bulk solution, and include observations of the variation of enthalpy at different ionic strengths, asymmetry in the melt curves, and the possibility of a reduced dielectric constant within a DNA layer relative to that in bulk solution.     

Practical physical aspects of interfacial nucleic acid oligomer hybridisation for biosensor design

A significant amount of research concerning rapid, selective biomolecular analysis has focused on development of analytical methods that make use of nucleic acid hybridisation as the basis for selective recognition. The development of biosensors based on nucleic acid hybridisation requires consideration of the thermodynamics of hybrid formation at a solid interface, because the relative thermodynamic stability can dictate the selectivity of hybridisation. Careful control of hybridisation conditions such as the density of oligonucleotides, as well as the temperature, pH, and ionic strength, may therefore enhance the selectivity, sensitivity and speed of a nucleic acid hybridisation assay that is located at an interface.