Carbon-on-metal films for surface plasmon resonance detection of DNA arrays

Surface plasmon resonance (SPR) imaging affords label-free monitoring of biomolecule interactions in an array format. A surface plasmon conducting metal thin film is required for SPR measurements. Gold thin films are traditionally used in SPR experiments as they are readily functionalized with thiol-containing molecules through formation of a gold-sulfur bond. The lability of this gold-thiol linkage upon exposure to oxidizing conditions and ultraviolet light renders these surfaces incompatible with light-directed synthetic methods for fabricating DNA arrays. It is shown here that applying a thin carbon overlayer to the gold surface yields a chemically robust substrate that permits light-directed synthesis and also supports surface plasmons. DNA arrays fabricated on these carbon-metal substrates are used to analyze two classes of biomolecular interactions: DNA-DNA and DNA-protein. This new strategy allows the combinatorial study of binding interactions directly from native, unmodified biomolecules of interest and offers the possibility of discovering new ligands in complex mixtures such as cell lysates.     

Kinetic discrimination of sequence-specific DNA-drug binding measured by surface plasmon resonance imaging and comparison to solution-phase measurements

We demonstrate the use of surface plasmon resonance (SPR) imaging for direct detection of small-molecule binding to surface-bound DNA probes. Using a carefully designed array surface, we quantitatively discriminate between the interactions of a model drug with different immobilized DNA binding sites. Specifically, we measure the association and dissociation intercalation rates of actinomycin-D (ACTD) to and from double-stranded 5'-TGCT-3' and 5'-GGCA-3' binding sites. The rates measured provide mechanistic information about the DNA-ACTD interaction; ACTD initially binds nonspecifically to DNA but exerts its activity by dissociating slowly from strong affinity sites. We observe a slow dissociation time of kd-1 = 3300 +/- 100 s for ACTD bound to the strong affinity site 5'-TGCT-3' and a much faster dissociation time (210 +/- 15 s) for ACTD bound weakly to the site 5'-GGCA-3'. These dissociation rates, which differ by an order of magnitude, determine the binding affinity for each site (8.8 x 10(6) and 1.0 x 10(6) M(-1), respectively). We assess the effect the surface environment has on these biosensor measurements by determining kinetic and thermodynamic constants for the same DNA-ACTD interactions in solution. The surface suppresses binding affinities approximately 4-fold for both binding sites. This suppression suggests a barrier to DNA-drug association; ACTD binding to duplex DNA is approximately 100 times slower on the surface than in solution.     

Sequence‐Specific DNA Recognition by Cyclic Pyrrole–Imidazole Cysteine‐Derived Polyamide Dimers

Pyrrole–imidazole (PI) polyamides bind to the minor groove of the DNA duplex in a sequence‐specific manner and thus have the potential to regulate gene expression. To date, various types of PI polyamides have been designed as sequence‐specific DNA binding ligands. One of these, cysteine cyclic PI polyamides containing two β‐alanine molecules, were designed to recognize a 7 bp DNA sequence with high binding affinity. In this study, an efficient cyclization reaction between a cysteine and a chloroacetyl residue was used for dimerization in the synthesis of a unit that recognizes symmetrical DNA sequences. To evaluate specific DNA binding properties, dimeric PI polyamide binding was measured by using a surface plasmon resonance (SPR) method. Extending this molecular design, we synthesized a large dimeric PI polyamide that can recognize a 14 bp region in duplex DNA.     

Surface enzyme kinetics for biopolymer microarrays: a combination of Langmuir and Michaelis-Menten concepts

Real-time surface plasmon resonance (SPR) imaging measurements of surface enzymatic reactions on DNA microarrays are analyzed using a kinetics model that couples the contributions of both enzyme adsorption and surface enzyme reaction kinetics. For the case of a 1:1 binding of an enzyme molecule (E) to a surface-immobilized substrate (S), the overall enzymatic reaction can be described in terms of classical Langmuir adsorption and Michaelis-Menten concepts and three rate constants: enzyme adsorption (k(a)), enzyme desorption (k(d)) and enzyme catalysis (k(cat)). In contrast to solution enzyme kinetics, the amount of enzyme in solution is in excess as compared to the amount of substrate on the surface. Moreover, the surface concentration of the intermediary enzyme-substrate complex (ES) is not constant with time, but goes to zero as the reaction is completed. However, kinetic simulations show that the fractional surface coverage of ES on the remaining unreacted sites does reach a steady-state value throughout the course of the surface reaction. This steady-state value approaches the Langmuir equilibrium value for cases where k(a)[E] > k(cat). Experiments using the 3' --> 5' exodeoxyribonuclease activity of Exonuclease III on double-stranded DNA microarrays as a function of temperature and enzyme concentration are used to demonstrate how this model can be applied to quantitatively analyze the SPR imaging data.     

Development of a Surface Plasmon Resonance-Based DNA Sensor and Its Application for Screening DNA-Targeted Anticancer Drugs

In this paper, we present a surface plasmon resonance (SPR)-based sensor for measuring DNA hybridization and DNA/small-molecule interactions. A mixed self-assembled monolayer (SAM) was used to optimize the biosensor sensitivity. It was observed that the mixed SAM formed by mixing 10 mM of 16-mercapto-1-hexadecanoic acid (16-MHA) and 6-mercapto-1-hexanol (6-MCH) at a 1:10 molar ratio showed the best results. Subsequently, avidin was attached to the carboxyl groups on the SAM to serve as a binding element for biotinylated single-stranded (ss)DNA. The ssDNA-coated sensor was first evaluated as a nucleic acid biosensor through a DNA-DNA hybridization assay for synthetic 28-mer ssDNA. A linear calibration curve was observed in the range of 0.25–2.5 µg/mL. Non-complementary DNA induced no significant SPR angle shift, which demonstrated the specificity of the assay. Secondly, the sensor was used to monitor the binding kinetics of DNA/small-molecule interactions in real time. The dissociation constant between immobilized DNA and sanguinarine was determined to be 8.0 × 10^?6 M. This complies with most data from the literature. In addition, the sensor could be regenerated with 0.01 M HCl and would be feasible for multiple testing. In conclusion, the experimental approach described in this study allows analysis of molecular interactions between DNA-binding drugs and selected targeted DNA sequences.     

DNA-binding small-ligand-immobilized surface plasmon resonance biosensor for detecting thymine-related single-nucleotide polymorphisms

A surface plasmon resonance (SPR) biosensor that carries DNA-binding small ligands has been developed for the detection of single-nucleotide polymorphisms (SNPs). 3,5-Diaminopyrazine derivatives, with a hydrogen-bonding profile fully complementary to the thymine base, were utilized as recognition elements on the sensor surface, and a target single-stranded DNA sequence was hybridized with a DNA probe containing an abasic site to place this site opposite a nucleobase to be detected. In a continuous flow of sample solutions buffered to pH 6.4 (0.25 M NaCl), the 3,5-diaminopyrazine-based SPR sensor can detect an orphan nucleobase in the duplex with a clear selectivity for thymine over cytosine, guanine, and adenine (5'-GTT GGA GCT GXG GGC GTA GGC-3'/3'-CAA CCT CGA CNC CCG CAT CCG-5'; X=abasic site, N=target nucleobase G, C, A, or T). The SPR response was linear in the concentration range 10-100 nM. Allele discrimination is possible based on the combination of different binding surfaces in a flow cell of the SPR system, which is demonstrated for the analysis of the thymine/cytosine mutation present in 63-meric polymerase chain reaction (PCR) amplification products (Ha-ras gene, codon 12, antisense strand). Comparison with a bulk assay based on 3,5-diaminopyrazine/DNA binding shows that the immobilization of 3,5-diaminopyrazine derivatives on the SPR sensor allows more sensitive detection of the target DNA sequence, and binding selectivity can be tuned by controlling the salt concentration of sample solutions. These features of the DNA-binding small-molecule-immobilized SPR sensor are discussed as a basis for the design of SPR biosensors for SNP genotyping.     

Binding kinetics of human cellular prion detection by DNA aptamers immobilized on a conducting polypyrrole

We developed a biosensor based on the surface plasmon resonance (SPR) method for the study of the binding kinetics and detection of human cellular prions (PrP^C) using DNA aptamers as bioreceptors. The biosensor was formed by immobilization of various biotinylated DNA aptamers on a surface of conducting polypyrrole modified by streptavidin. We demonstrated that PrP^C interaction with DNA aptamers could be followed by measuring the variation of the resonance angle. This was studied using DNA aptamers of various configurations, including conventional single-stranded aptamers that contained a rigid double-stranded supporting part and aptamer dimers containing two binding sites. The kinetic constants determined by the SPR method suggest strong interaction of PrP^C with various DNA aptamers depending on their configuration. SPR aptasensors have a high selectivity to PrP^C and were regenerable by a brief wash in 0.1 M NaOH. The best limit of detection (4 nM) has been achieved with this biosensor based on DNA aptamers with one binding site but containing a double-stranded supporting part.

Direct detection of genomic DNA by enzymatically amplified SPR imaging measurements of RNA microarrays

A novel surface enzymatic reaction scheme that amplifies the optical response of RNA microarrays to the binding of complementary DNA is developed for the direct detection and analysis of genomic DNA. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from DNA-RNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance (SPR) imaging, DNA oligonucleotides can be detected at a concentration of 1 fM. This enzymatically amplified SPR imaging methodology is then utilized to detect and identify the presence of the TSPY gene in human genomic DNA without PCR amplification.     

Structure-switching allosteric deoxyribozymes

DNA aptamers and DNA enzymes (DNAzymes or deoxyribozymes) are single-stranded DNA molecules with ligand-binding and catalytic capabilities, respectively. Allosteric DNA enzymes (aptazymes) are deoxyribozymes whose activity can be regulated by the binding state of an appended aptamer domain and have many potential uses in the fields of drug discovery and diagnostics. In this report, we describe a simple, yet potentially general, DNA aptazyme rational design strategy that requires no structural characterization of the constituent deoxyribozymes and aptamers. It is based on the concept originally developed in our laboratory for the design of structure-switching signaling aptamers that change structural states from a DNA-DNA duplex to a DNA-target complex upon target binding. In our new strategy, an antisense oligonucleotide is used to regulate the enzymatic activity of a linked aptamer-deoxyribozyme by annealing with a stretch of nucleotides on each side of the aptamer-DNAzyme junction. Structural reorganization of the aptamer domain upon target binding relieves the suppressive effect of this regulatory oligonucleotide on the attached DNA enzyme. Consequently, the target-binding event triggers the catalytic action of the aptazyme. We have demonstrated this concept using two RNA-cleaving deoxyribozymes, each adjoined to a DNA aptamer that binds ATP. These allosteric DNA enzymes exhibit the same ligand-binding specificity as the parental DNA aptamer and show up to 30-fold rate enhancement in the presence of ATP. The described methodology provides a convenient approach for rationally designing catalytic DNA-based biosensors.     

Effects of DNA mismatches on binding affinity and kinetics of polymerase-DNA complexes as revealed by surface plasmon resonance biosensor

In this study, surface plasmon resonance (SPR) biosensor techniques were used to obtain quantitative information on the kinetics of the DNA and polymerase I (Klenow fragment) interaction. DNA duplexes containing different base compositions at the binding site were immobilized on the SPR sensor surface via biotin-streptavidin chemistry and the subsequent binding of the polymerase was measured in real time. Various kinetic models were tested and a translocation model was shown to provide the best fit for the binding and dissociation profiles. The results revealed that the enzyme binds to DNA at both the polymerase and the exonuclease domains with different association and dissociation rates as well as affinity constants, depending on the presence of mismatches near the primer 3'-end. Introduction of unpaired bases increases the DNA binding affinity towards the exonuclease domain and promotes the translocation of DNA from the polymerase site to the exonuclease site. The results also demonstrated that SPR biosensors may be used as a sensitive technique for studying molecular recognition events such as single-base discrimination involved in protein-DNA interaction.     

Microchannel chips for the multiplexed analysis of human immunoglobulin G–antibody interactions by surface plasmon resonance imaging

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. Figure Microchannel SPR imaging for protein–protein interactions     

Nanomechanical sensing of DNA sequences using piezoresistive cantilevers

A microfabricated cantilever with an internal piezoresistive component has been sensitized with thiol tethered ss-DNA strands and utilized for an in situ, label-free, highly specific, and rapid DNA detection assay. The generation of a differential surface stress onto the functionalized cantilever surface upon target recognition has allowed nanomechanical identification of 12-nucleotide complementary DNA probes with single base mismatch discrimination (sensitivity of 0.2 microM). Interestingly, utilization of an overhang extension distal to the surface enhanced the sensitivity to the 0.01 microM level. The cantilever was functionalized by inkjet printing technology. Replacing the capture probe with locked nucleic acid (LNA) resulted in a faster target probe capture kinetics compared to DNA-DNA hybridization. The capabilities of the piezoresistive cantilever indicate future ergonomic convenience via miniaturization alternative to the conventional laser-based detection method for portable on-site applications.     

Influence of DNA structure on adjacent site cooperative binding

Previous NMR studies of Hoechst 33258 with the d(CTTTTGCAAAAG)2 sequence have shown very strong (K2 > K1) cooperativity between two adjacent binding sites (Searle, M. S.; Embrey, K. J. Nucleic Acids Res. 1990, 18 (13), 3753- 3762). In contrast, surface plasmon resonance (SPR) results with the hairpin analog of the same sequence show significantly reduced cooperativity. In an effort to explain the difference, two-dimensional (2-D) NMR experiments were done on both duplex and hairpin. Hoechst 33258 and an amidine analog, DB183, show very strong cooperativity with the duplex DNA but much weaker cooperativity with the hairpin. The significantly lower thermal melting temperature (Tm) of the duplex (34.8 degrees C) in comparison to its hairpin analog (62.3 degrees C) supports the idea of a dynamic difference between the two DNA structures that can influence cooperativity in binding. These results confirm the role of conformational entropy in positive cooperativity in some DNA interactions.     

Polymeric optical microscreen for high-resolution surface plasmon resonance microarray imaging

In this study, we demonstrate a simple method to fabricate surface plasmon resonance (SPR) imaging microarrays using polymer micropatterns. The use of a micrometer-scale polymeric optical screen (microPOS) passivates the region deposited with polymer by completely removing SPR signals or by saturating the SPR signal far beyond the detection range of SPR imaging. Two schemes were suggested to create a surface microPOS by either micropatterning a thick insulating layer before deposition of a metal layer (complete removal of SPR) or after deposition of a metal layer (saturation of SPR signal). The two schemes were successfully applied for the imaging of biological adsorption with a high imaging resolution of approximately 100 microm/pattern and 10 microm separation. The validity of the system was verified with a biotin-streptavidin system as a model for the systematic binding of biomolecules. Further, binding of prostate-specific antigen (PSA) onto the anti-PSA SPR microarray was demonstrated as a useful method for detecting a cancer marker.     

Microcontact printed antibodies on gold surfaces: function, uniformity, and silicone contamination

The function of microcontact printed protein was investigated using surface plasmon resonance (SPR) imaging, X-ray photoelectron spectroscopy spectroscopy (XPS), and XPS imaging. We chose to analyze a model protein system, the binding of an antibody from solution to a microcontact printed protein antigen immobilized to a gold surface. SPR imaging experiments indicated that the microcontact printed protein antigen was less homogeneous, had increased nonspecific binding, and bound less antibody than substrates to which the protein antigen had been physically adsorbed. SPR images of substrates contacted with a poly(dimethylsiloxane) stamp inked with buffer alone (i.e., no protein) revealed that significant amounts of silicone oligomer were transferred to the surface. The transfer of the silicone oligomer was not homogeneous, and the oligomer nonspecifically bound protein (BSA and IgG) from solution. XPS spectroscopy and imaging were used to quantify the amount of silicon (due to the presence of silicone oligomer), as well as the amounts of other elements, transferred to the surface. The results suggest that the silicone oligomer introduced by the printing process reduces the overall binding capacity of the microcontact-printed protein compared to physically adsorbed protein.     

Dendrimer-functionalized self-assembled monolayers as a surface plasmon resonance sensor surface

We report here a multistep route for the immobilization of DNA and proteins on chemically modified gold substrates using fourth-generation NH(2)-terminated poly(amidoamine) dendrimers supported by an underlying amino undecanethiol (AUT) self-assembled monolayer (SAM). Bioactive ultrathin organic films were prepared via layer-by-layer self-assembly methods and characterized by fluorescence microscopy, variable angle spectroscopic ellipsometry, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR). The thickness of the AUT SAM base layer on the gold substrates was determined to be 1.3 nm from ellipsometry. Fluorescence microscopy and AFM measurements, in combination with analyses of the XPS/ATR-FTIR spectra, confirmed the presence of the dendrimer/biopolymer molecules on the multilayer sensor surfaces. Model proteins, including streptavidin and rabbit immunoglobulin proteins, were covalently attached to the dendrimer layer using linear cross-linking reagents. Through surface plasmon resonance measurements, we found that sensor surfaces containing a dendrimer layer displayed an increased protein immobilization capacity, compared to AUT SAM sensor surfaces without dendrimer molecules. Other SPR studies also revealed that the dendrimer-based surfaces are useful for the sensitive and specific detection of DNA-DNA interactions. Significantly, the multicomponent films displayed a high level of stability during repeated regeneration and hybridization cycles, and the kinetics of the DNA-DNA hybridization process did not appear to be influenced by surface mass transport limiting effects.     

DNA recognition interfaces: the influence of interfacial design on the efficiency and kinetics of hybridization

The effect of the surface chemistry of DNA recognition interfaces on DNA hybridization at a gold surface was investigated using both electrochemistry and the quartz crystal microbalance (QCM) technique. Different DNA recognition interfaces were prepared using a two-component self-assembled monolayer consisting of thiolated 20-mer probe single-stranded DNA (ss-DNA) containing either a 3'-mercaptopropyl or a 3'-mercaptohexyl linker group and an alcohol-terminated diluent layer with 2-, 6-, or 11-carbon length. The influence of the interfacial design on the hybridization efficiency, the affinity constant (Ka) describing hybridization, and the kinetics of hybridization was assessed. It was found that the further the DNA was above the surface defined by the diluent layer the higher the hybridization efficiency and Ka. The kinetics of DNA hybridization was assessed using both a QCM and an electrochemical approach to ascertain the influence of the interface on both the initial binding of target DNA to the surface and the formation of a complete duplex. These measurements showed that the length of the diluent layer has a large impact on the time taken to form a perfect duplex but no impact on the initial recognition of the target DNA by the immobilized probe DNA.     

Covalent attachment of functionalized cardiolipin on a biosensor gold surface allows repetitive measurements of anticardiolipin antibodies in serum

Antiphospholipid antibodies (aPL) are a relevant serological indicator of antiphospholipid syndrome (APS). A solid-state surface with covalently bound ω-amine-functionalized cardiolipin was established and the binding of β2-glycoprotein I (β2-GPI) was investigated either by use of surface plasmon resonance (SPR) biosensor, by electrically switchable DNA interfaces (switchSENSE) and by scanning tunneling microscopy (STM). STM could clearly visualize the attachment of β2-GPI to the cardiolipin surface. Using the switchSENSE sensor, β2-GPI as specific ligand could be identified by increased hydrodynamic friction. The binding of anti-cardiolipin antibodies (aCL) was detected against the ω-amine-functionalized cardiolipin-modified SPR biosensor (aCL biosensor) using sera from healthy donors, APS patients and syphilis patients. Our results showed that the aCL biosensor is a much more sensitive diagnostic device for APS patients compared to previous methods. The specificity between β2-GPI-dependent autoimmune- and β2-GPI-independent infection-associated types of aPLs was also studied and they can be distinguished by the different binding kinetics and patterns.     

基于氨基与表面乙烯砜基反应动力学调控配基表面密度

配基表面密度可控为定量研究生物分子相互作用提供了精准的分子基础。然而,经典混合自组装的方法控制配基密度对于不同自组装体系不具有普适性。本文报道了一种基于表面乙烯砜基反应动力学的配基表面密度调控方法。以Nα,Nα-二（羧甲基）-L-赖氨酸（ab-NTA）为生物配基模型,对该表面反应进行了催化剂筛选并利用X射线光电子能谱（XPS）和表面膜电位对该表面反应进行了表征。采用静态水接触角的方法对表面反应的动力学进行了定量表征,计算得到反应速率常数为0.0012 min^-1。采用表面等离子体共振（SPR）分析了该生物功能表面结合组氨酸标签蛋白（SA-6His）的能力,结果表明该表面比传统NHS-NTA表面具有更高的蛋白结合量和结合强度。通过控制反应时间和催化剂种类制备了四种配基密度不同的生物功能表面,并利用SPR对四种表面进行了蛋白质静态吸附实验。实验结果表明通过控制反应时间和催化剂类型均能够实现配基表面密度的调控,并且可以实现表面多价态的调控。