Analysis of Oligonucleotides by HPLC-Electrospray Ionization Mass Spectrometry

A new interface procedure has been developed that allows, for the first time, the high-efficiency analysis of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and on-line electrospray ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resolution can be obtained with low levels of cation adduct formation in the negative ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compounds such as phosphorothioates and their metabolites, as well as of materials used in clinical trials.     

Kinetic measurements of DNA hybridization on an oligonucleotide-immobilized 27-MHz quartz crystal microbalance

A highly sensitive 27-MHz quartz-crystal microbalance, on which a 10-30-mer oligonucleotide was immobilized as a probe molecule, was employed to detect hybridization of complementary oligonucleotides in aqueous solution. From frequency decreases (mass increases due to the hybridization) with passage of time, kinetic parameters such as association constants (K(a)) and binding and dissociation rate constants (k(1) and k(-1)) could be obtained, as well as binding (hybridization) amount at the nanogram level (delta m). Kinetic studies were carried out by changing various parameters: (i) the immobilization method of a probe oligonucleotide on Au electrode, (ii) number of mismatching bases in sequences of target oligonucleotides, (iii) length of both probe and target oligonucleotides, (iv) hybridization temperature, and (v) ionic strength in solution. The obtained results were compared with those obtained by a surface plasmon resonance method using a BIAcore system.     

Studies of protein--DNA interactions by capillary electrophoresis/laser-induced fluorescence polarization

Protein-DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 x 10(6) M(-1)) and the 37-mer (23 x 10(6) M(-1)). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein-DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA.     

A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles

Using a fluorescence-based method, we have determined the number of thiol-derivatized single-stranded oligonucleotides bound to gold nanoparticles and their extent of hybridization with complementary oligonucleotides in solution. Oligonucleotide surface coverages of hexanethiol 12-mer oligonucleotides on gold nanoparticles (34 +/- 1 pmol/cm2) were significantly higher than on planar gold thin films (18 +/- 3 pmol/cm2), while the percentage of hybridizable strands on the gold nanoparticles (1.3 +/- 0.3 pmol/cm2, 4%) was lower than for gold thin films (6 +/- 2 pmol/cm2, 33%). A gradual increase in electrolyte concentration over the course of oligonucleotide deposition significantly increases surface coverage and consequently particle stability. In addition, oligonucleotide spacer sequences improve the hybridization efficiency of oligonucleotide-modified nanoparticles from approximately 4 to 44%. The surface coverage of recognition strands can be tailored using coadsorbed diluent oligonucleotides. This provides a means of indirectly controlling the average number of hybridized strands per nanoparticle. The work presented here has important implications with regard to understanding interactions between modified oligonucleotides and metal nanoparticles, as well as optimizing the sensitivity of gold nanoparticle-based oligonucleotide detection methods.     

Electrochemically modulated liquid chromatography coupled on-line with electrospray mass spectrometry

Electrochemically modulated liquid chromatography (EMLC) has been coupled to an electrospray mass spectrometer. This combination takes advantage of the ability of EMLC to manipulate retention and enhance separation efficiency solely through changes in the potential applied to a conductive stationary phase, thereby minimizing complications because of possible changes in analyte ionization efficiencies when gradient elution techniques are used. Three examples are presented that demonstrate the attributes of this EMLC/electrospray mass spectrometry (ES-MS) coupling. The first two examples involve the separation of mixtures of corticosteroids or of benzodiazepines, showing the general utility of the union for eluent identification and low-level detection. The ability to identify products from on-column redox transformations is also demonstrated using the benzodiazepine mixture. The third example investigates the electrooxidation of aniline by utilizing an EMLC column as an on-line electrochemical reactor and product separator and ES-MS for detection and product identification.     

Interfaces to connect thin-layer chromatography with electrospray ionization mass spectrometry

This study has developed two interfaces to connect small-size thin-layer chromatography (TLC) with electrospray ionization mass spectrometry (ES-MS) for the continuous analysis of organic mixtures. The interfaces are (1) two bound optical fibers inserted into the C18-bonded particles at the exit of a small TLC channel and (2) a small commercial TLC strip with a sharpened tip. A reservoir continuously supplied a makeup solution to the tip of the TLC channel. The high voltage required for electrospray ionization was introduced into the makeup solution or mobile phase through a Pt wire, and electrospray was generated at the tip of the bonded optical fibers and at the sharp end of the TLC strip. Since small-size TLC channels were used, the elution time was short and less than 0.2 microL of the sample solution and 200 microL of the eluting solvent were required. Organic mixtures were separated successfully and detected on-line using the TLC/ES-MS techniques.     

Analysis of nucleic acids by capillary ion-pair reversed-phase HPLC coupled to negative-ion electrospray ionization mass spectrometry

Ion-pair reversed-phase high-performance liquid chromatography was successfully coupled to negative-ion electrospray ionization mass spectrometry by using 60 × 0.20 mm i.d. capillary columns packed with 2.3-μm micropellicular, octadecylated poly(styrene/divinylbenzene) particles as stationary phase and gradients of acetonitrile in 50 mM aqueous triethylammonium bicarbonate as mobile phase. Systematic variation of the eluent composition, such as concentration of ion-pair reagent, anion in the ion-pair reagent, solution pH, and acetonitrile concentration led to the conclusion that most parameters have opposite effects on chromatographic and mass spectrometric performances. The use of acetonitrile as sheath liquid enabled the rapid and highly efficient separation and detection of phosphorylated and nonphosphorylated oligonucleotides ranging in size from 8 to 40 nucleotides. High-quality full-scan mass spectra showing little cation adduction were acquired from which the molecular masses of the separated oligonucleotides were calculated with an accuracy of 0.011%. With calibration curves being linear over at least 2 orders of magnitude, the lower limits of detection for a oligodeoxythymidine 16-mer were 104 fmol with full scan and 710 amol with selected-ion-monitoring data acquisition. The potential of ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry was demonstrated for mixed-sequence oligomers by the characterization of a reaction mixture from solid-phase synthesis of a 40-mer oligonucleotide.     

Improvement of resolution, mass accuracy, and reproducibility in reflected mode DE-MALDI-TOF analysis of DNA using fast evaporation--overlayer sample preparations

DNA analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is hindered by two processes: alkali metal adduction and fragmentation of the intact ionized molecule. The adverse effects of both processes can be reduced by adding ammonium ion salts or compounds such as fructose to the sample preparations. Matrix additives improve sensitivity and resolution of DNA analysis by MALDI. In addition, spot-to-spot reproducibility, resolution, and mass accuracy for DNA oligonucleotides (< or = 12 mer) can be improved by the use of overlayer sample preparations with matrixes that have low aqueous solubilities, such as alpha-cyano-4-hydroxycinnamic acid, ferulic acid, and 2,4,6-trihydroxyacetophenone. For example, resolution for 5-12-mer oligonucleotides is greater than 7000 using overlayer matrix preparations and mass accuracy values are well below 20 ppm. In addition to these methods, a new method for analyzing DNA in positive ion mode is reported using acidified 3-hydroxypicolinic acid. This method does not lose sensitivity for higher mass oligonucleotides as quickly as overlayer methods, and spectra retain > 6000 resolution and mass accuracies of approximately 20 ppm between different overlayer depositions.     

Voltammetric determination of underivatized oligonucleotides on graphite electrodes based on their oxidation products

A new electrochemical method to determine underivatized oligonucleotides is developed. The electro-oxidation of the adenine moieties of adsorbed oligonucleotides at elevated potentials on pyrolytic graphite electrodes (PGE) in neutral or alkaline media gives rise to electroactive products strongly adsorbed on the electrode surface. The extent of the redox processes of these products, with formal potential close to 0 V (vs Ag /AgCl) at pH 10, correlates well with the amount of parent oligonucleotide. Various electrochemical techniques have been compared and applied to the detection of specific DNA sequences and synthetic homopolynucleotides. Detection limits of 2 and 10 ng for (dA)20 and a 21-mer sequence of HIV-1, respectively, have been achieved using sample volumes of 10 microL. Moreover, the adsorbed oxidized oligonucleotide shows electrocatalytic activity toward the oxidation of NADH. The capability of the new method to detect DNA hybridization is discussed.     

Binding stoichiometry of DNA adducts with antibody studied by capillary electrophoresis and laser-induced fluorescence

Four oligonucleotides (fluorescently labeled and unlabeled 16- and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated. The stoichiometry depended upon the relative concentrations of the antibody and the DNA adducts. A new approach examining the binding of the antibody with a mixture of a tetramethylrhodamine (TMR)-labeled and unlabeled BPDE-16-mer revealed insights on ligand redistribution and exchange between the labeled and unlabeled BPDE-16-mer oligonucleotides in the complexes. The observation of this unique behavior has not been possible previously with other binding studies. A mixture of the antibody with the TMR-labeled BPDE- 16-mer and an unlabeled BPDE-90-mer further revealed the formation of three fluorescent complexes: antibody with one TMR-BPDE-16-mer molecule, antibody with two TMR-BPDE- 16-mer molecules, and antibody with one TMR-BPDE-16-mer and one BPDE-90-mer. The three complexes clearly demonstrated binding stoichiometry and ligand redistribution/exchange.     

Separation and detection of phosphorylated and nonphosphorylated peptides in liquid chromatography-mass spectrometry using monolithic columns and acidic or alkaline mobile phases

Efficient chromatographic separation is a prerequisite for the sensitive analysis of complex peptide mixtures using liquid chromatography-mass spectrometry. This is especially true for the analysis of mixtures of unmodified and posttranslationally modified peptides, for example, phosphorylated peptides in the presence of their unmodified analogues. Applying monolithic capillary columns based on poly(styrene/divinylbenzene), the influence of acidic eluents based on trifluoroacetic and heptafluorobutyric acid as well as an alkaline eluent based on triethylamine-acetic acid (pH 9.2) on the separation of synthetic phosphopeptides was evaluated. Heptafluorobutyric acid offered the longest retention times and highest selectivities and, hence, the most effective separation. Application of the alkaline eluent in conjunction with detection in negative ion mode electrospray ionization mass spectrometry, on the other hand, allowed the detection of phosphorylated peptides with significantly lower limits of detection, as compared to acidic eluents in combination with detection in positive ion mode. Pairs of phosphorylated and nonphosphorylated synthetic peptides, ranging from 7- to 16-mers, as well as phosphorylated peptides form a tryptic protein digest could be separated both at acidic and alkaline pH. Utilizing a 60 x 0.20-mm-i.d. capillary column, the limit of detection in negative ion detection mode for a 4-fold phosphorylated peptide in a beta-casein digest was 10 fmol. Together with the capability for fast separation of protein digests, monolithic columns, thus, facilitate the effective and sensitive analysis of this important posttranslational modification.     

High-mass accuracy of product ions produced by SORI-CID using a dual electrospray ionization source coupled with FTICR mass spectrometry

High-mass accuracy is demonstrated using internal calibration for product ions produced by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) of a 15-mer oligonucleotide, 5'-(CTG)5-3'. Internal calibration for this tandem MS experiment was accomplished using a dual electrospray ionization (ESI) source coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) utilizing hexapole accumulation and gated trapping. The pulse sequence entails injection, trapping, and gas-phase isolation of the precursor ion of interest followed by the SORI-CID of this ion and, subsequently, injection and trapping of the internal mass calibrant (i.e., poly(ethylene glycol) with a 1000 Da average mass). The product ions and the poly(ethylene glycol) ions are then simultaneously excited by a broadband frequency chirp excitation waveform and detected. This technique corrects for space-charge effects on the measurement of an ion's cyclotron frequency experienced when externally calibrated data are used. While external calibration for FTICR-MS can result in mass errors of greater than 100 ppm, this internal standardization method demonstrated significantly more consistent accurate mass measurements with average mass errors ranging from -1.2 to -3.2 ppm for the 15-mer oligonucleotide used in this study. This method requires limited modifications to ESI-FTICR mass spectrometers and is applicable for both positive and negative modes of ionization as well as other sample types (e.g., pharmaceuticals, proteins, etc.).     

Stepwise mobilization of focused proteins in capillary isoelectric focusing mass spectrometry

A stepwise mobilization strategy has been developed for the elution of complex protein mixtures, separated by capillary isoelectric focusing (CIEF) for detection using on-line electrospray ionization mass spectrometry (ESI-MS). Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-silica capillaries. Elution of focused protein zones is achieved by controlling the mobilization pressure and voltage, leaving the remaining protein zones focused inside the capillary. Protein zones are stepwise eluted from the capillary by changing the mobilization conditions. Stepwise mobilization improves separation resolution and simplifies coupling with multistage MS (i.e., MSn) analysis since it allows more effective temporal control of protein elution from the CIEF capillary. We also describe a modified configuration for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI-MS analyses. The stepwise mobilization strategy is demonstrated for the analysis of standard protein mixtures and soluble E. coli lysate proteins using CIEF-ESI-MS. These results indicate that inlet pressure or voltage programming to control the elution of the protein zones from the capillary (i.e., gradient mobilization) may allow for the optimization of the mobilization conditions and provide higher resolution for CIEF separation of complex mixtures with on-line MS.     

Oligonucleotide mixture analysis via electrospray and ion/ion reactions in a quadrupole ion trap

Electrospray ionization combined with ion/ion reactions in a quadrupole ion trap can be used for the direct analysis of oligonucleotide mixtures. Elements to the success of this approach include factors related to ionization, ion/ion reactions, and mass analysis. This paper deals with issues regarding the ion polarity combination, viz., positive oligonucleotides/negative charge-transfer agent versus negative oligonucleotides/positive charge-transfer agent. Anions derived from perfluorocarbons appear to be directly applicable to mixtures of positive ions derived from electrospray of oligonucleotides, in direct analogy with positive protein ions. Conditions for forming positive oligonucleotide ions devoid of adducts were more difficult to establish than for forming relatively clean negative oligonucleotide ions. A new approach for manipulating negative ion charge states in the ion trap is described and is based on use of the electric field of the positive charge-transfer agent for storage of high-mass negative ions formed during the ion/ion reaction period. Oxygen cations are shown to be acceptable for charge-state manipulation of mixed-base oligomers but induce fragmentation in polyadenylate homopolymers. Protonated isobutylene (C4H9+), on the other hand, is shown to induce significantly less fragmentation of polyadenylate homopolymers.     

Chip-based capillary electrophoresis/mass spectrometry determination of carnitines in human urine

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the CE separation and on-line electrospray detection of carnitine and selected acylcarnitines from mixtures of analytical standards as well as extracts of fortified human urine. Chip-based CE/MS experiments in two different laboratories were carried out using a triple-quadrupole mass spectrometer and a quadrupole time-of-flight (QTOF) mass spectrometer, respectively. The glass chips used with both systems were comparably equipped with a microfabricated capillary electrophoresis (CE) channel but with different electrosprayers. The quadrupole chip-based CE/MS experiments employed a miniature coupled microsprayer, which allowed coupling of the microelectrospray process via a micro liquid junction at the exit of the CE capillary channel. Selected ion monitoring (SIM) CE/MS experiments were employed for all of the quadrupole CE/MS work. The QTOF CE/MS full-scan single MS and MS/MS experiments were carried out in another laboratory using accurate mass measurement TOF mass spectrometry techniques. The electrospray process that was employed with the QTOF system differed in that an inserted nanoelectrospray capillary needle was carefully affixed into a flat-bottomed hole that was aligned with the CE channel exit orifice. SIM CE/MS using the described quadrupole system provided acceptable ion current electropherograms from fmole levels from analytical standard solutions of carnitine and acylcarnitines that were manually injected (loaded) onto the chip. In addition, the corresponding electropherograms for human urine fortified with the target carnitine and acylcarnitines at a 10-20 microg/mL (35-124 microM) level were obtained via SIM CE/MS techniques. The measured CE separation efficiency for the SIM CE/MS electropherograms was determined to be 2860 plates (peak width at half-height method or N = 5.54(T/WO.5(2)), and carnitine and three acylcarnitines were separated in less than 48 s. In contrast, using quadrupole-TOF technologies, the same samples could be diluted by a factor of 2-4 to obtain a comparable detector response for the target compounds. In the full-scan, single mass analyzer mode (m/z 150-500), the CE separation efficiency was measured to be 2600 plates, but mass measurement accuracy was less than 5.0 ppm for the quaternary cations. In the CE/MS/MS mode, full-scan collision-induced dissociation (CID) mass spectra were obtained with a mass accuracy of < or =10 ppm for the higher mass ions and < or =27 ppm for the lower mass product ions. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric detection for these important compounds in synthetic mixtures, as well as in human urine extracts.     

Cantilever-based optical deflection assay for discrimination of DNA single-nucleotide mismatches

Characterization of single-nucleotide polymorphisms is a major focus of current genomics research. We demonstrate the discrimination of DNA mismatches using an elegantly simple microcantilever-based optical deflection assay, without the need for external labeling. Gold-coated silicon AFM cantilevers were functionalized with thiolated 20- or 25-mer probe DNA oligonucleotides and exposed to target oligonucleotides of varying sequence in static and flow conditions. Hybridization of 10-mer complementary target oligonucleotides resulted in net positive deflection, while hybridization with targets containing one or two internal mismatches resulted in net negative deflection. Mismatched targets produced a stable and measurable signal when only a four-base pair stretch was complementary to the probe sequence. This technique is readily adaptable to a high-throughput array format and provides a distinct positive/negative signal for easy interpretation of oligonucleotide hybridization.     

Infrared multiphoton dissociation in an external ion reservoir.

In this work we present a novel scheme for performing infrared multiphoton dissociation (IRMPD) external to the mass analyzer in an external ion reservoir consisting of an rf-only multipole and a pair of electrostatic lens elements. Ions generated by electrospray ionization (ESI) are accumulated in an rf-only hexapole and dissociated by irradiation at 10.6 microns from a CW CO2 laser in the source region of the mass spectrometer. This scheme is unique from other IRMPD schemes as dissociation occurs in a spatially distinct region of the spectrometer and is independent of the mass spectrometry platform used to analyze the fragment ions. The effectiveness of the technique is demonstrated with ESI IRMPD FTICR mass spectrometry of a 20-mer phosphorothioate oligonucleotide. A comparison of the external IRMPD scheme with nozzle-skimmer dissociation and conventional in-cell IRMPD reveals a significant improvement in signal-to-noise ratio and fragment yield, particularly for larger, more highly charged fragment ions.     

Integration of microfabricated devices to capillary electrophoresis-electrospray mass spectrometry using a low dead volume connection: application to rapid analyses of proteolytic digests.

This report describes the development of a compact and versatile, micromachined chip device enabling the efficient coupling of capillary electrophoresis to electrospray mass spectrometry (CE-ESMS). On-chip separation provides a convenient means of achieving rapid sample cleanup and resolution of multicomponent samples (typically 2-5 min) prior to mass spectral analysis. A low dead volume connection facilitating the coupling of microfabricated devices to CE-ESMS was evaluated using two different interfaces. The first configuration used disposable nanoelectrospray emitters directly coupled to the chip device via this low dead volume junction, thereby providing rapid separation of complex protein digests. The performance of this interface was compared with that of more traditional configurations using a sheath flow CE-ESMS arrangement where a fused-silica capillary of varying length enabled further temporal resolution of the multicomponent samples. The sensitivity and analytical characteristics of these interfaces were investigated in both negative and positive ion modes using standard peptide mixtures. The separation performance for synthetic peptides using a chip coated with amine reagent ranged from 26,000 to 58,000 theoretical plates for a sheath flow CE-ESMS interface comprising a 15-cm CE column. Replicate injections of a dilution series of peptide standards provided detection limits of 45-400 nM without the use of on-line preconcentration devices. The reproducibility of migration time ranged from 0.9 to 1.5% RSD whereas RSDs of 5-10% were observed on peak areas. The application of these devices for the analysis of protein digests was further evaluated using on-line tandem mass spectrometry.     

Interaction of chemically modified antisense oligonucleotides with sense DNA: a label-free interaction study with reflectometric interference spectroscopy.

Antisense oligonucleotides (ON) are regarded as potential therapeutic agents for controlling gene expression at the mRNA level. The strength of the interaction with the target sequence is one critical factor for the therapeutic efficiency of an ON. Herein, the results of studies on antisense 15mer and 20mer ONs against mdr1b-mRNA are described. The mdr1b is a member of the group that encodes the P-glycoprotein (Pgp), responsible for the phenomenon of multidrug resistance. The effects of backbone modification (DNA, phosphorothioate (PTO)), terminal modifications (hexadecyl, cholesteryl, tocopherol, polyethylenglycol, 2'-O-methyl-modified RNA) and base sequence misalignments (1 to 3 bases) on interaction kinetics and binding strength were investigated. The interaction of an immobilized sense strand with the dissolved antisense ON was monitored with a label-free optical transducer based on thin film interference (RIfS). Association kinetics were detected at a low density of immobilized ON. Thermodynamics were investigated by homogeneous phase titration of sense and antisense ON and subsequent quantification of equilibrium concentrations of unbound ON at a transducer highly loaded with sense ON. Association rate constants varied from 3.1 (+/- 0.2) x 10(4) M-1 s-1 (poly(ethylene glycol)-modified DNA strand) to 4.3 (+/- 0.1) x 10(4) M-1 s-1 (hexadecyl-modified strand). Binding constants varied from 1.9 (+/- 0.1) x 10(8) M-1 (cholesteryl modification) to 5 (+/- 0.4) x 10(7) M-1 (tocopherol modification). Phosphorothioate ON showed a reduction in binding strength of more than 1 order of magnitude. The data presented give valuable information for the efficiency of modified antisense oligonucleotides.     

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).