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1.  Quantitative Analysis of Small Molecule–Nucleic Acid Interactions With a Biosensor Surface and Surface Plasmon Resonance Detection 
Surface plasmon resonance (SPR) technology with biosensor surfaces has become a widely–used tool for the study of nucleic acid interactions without any labeling requirements. The method provides simultaneous kinetic and equilibrium characterization of the interactions of biomolecules as well as small molecule–biopolymer binding. SPR monitors molecular interactions in real time and provides significant advantages over optical or calorimetic methods for systems with strong binding coupled with small spectroscopic signals and/or reaction heats. A detailed and practical guide for nucleic acid interaction analysis using SPR–biosensor methods is presented. Details of the SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips and samples as well as extensive information on experimental design, quantitative and qualitative data analysis and presentation. A specific example of the interaction of a minor–groove–binding agent with DNA is evaluated by both kinetic and steady–state SPR methods to illustrate the technique. Since the molecules that bind cooperatively to specific DNA sequences are attractive for many applications, a cooperative small molecule–DNA interaction is also presented.
doi:10.1007/978-1-60327-418-0_1
PMCID: PMC2993496  PMID: 19997874
Biosensor; surface plasmon resonance; small molecule–nucleic acid interaction; kinetics; steady–state analysis; cooperativity; Biacore; minor groove
2.  A SPR Aptasensor for Detection of Avian Influenza Virus H5N1 
Sensors (Basel, Switzerland)  2012;12(9):12506-12518.
Rapid and specific detection of avian influenza virus (AIV) is urgently needed due to the concerns over the potential outbreaks of highly pathogenic H5N1 influenza in animals and humans. Aptamers are artificial oligonucleic acids that can bind specific target molecules, and show comparable affinity for target viruses and better thermal stability than monoclonal antibodies. The objective of this research was to use a DNA-aptamer as the specific recognition element in a portable Surface Plasmon Resonance (SPR) biosensor for rapid detection of AIV H5N1 in poultry swab samples. A SPR biosensor was fabricated using selected aptamers that were biotinylated and then immobilized on the sensor gold surface coated with streptavidin via streptavidin-biotin binding. The immobilized aptamers captured AIV H5N1 in a sample solution, which caused an increase in the refraction index (RI). After optimizing the streptavidin and aptamer parameters, the results showed that the RI value was linearly related (R2 = 0.99) to the concentration of AIV in the range of 0.128 to 1.28 HAU. Negligible signal (<4% of H5N1) was observed from six non-target AIV subtypes. The AIV H5N1 in poultry swab samples with concentrations of 0.128 to 12.8 HAU could be detected using this aptasensor in 1.5 h.
doi:10.3390/s120912506
PMCID: PMC3478855  PMID: 23112728
aptamer; surface plasmon resonance; biosensor; avian influenza virus
3.  Label-Enhanced Surface Plasmon Resonance: A New Concept for Improved Performance in Optical Biosensor Analysis 
Sensors (Basel, Switzerland)  2013;13(11):15348-15363.
Surface plasmon resonance (SPR) is a well-established optical biosensor technology with many proven applications in the study of molecular interactions as well as in surface and material science. SPR is usually applied in the label-free mode which may be advantageous in cases where the presence of a label may potentially interfere with the studied interactions per se. However, the fundamental challenges of label-free SPR in terms of limited sensitivity and specificity are well known. Here we present a new concept called label-enhanced SPR, which is based on utilizing strongly absorbing dye molecules in combination with the evaluation of the full shape of the SPR curve, whereby the sensitivity as well as the specificity of SPR is significantly improved. The performance of the new label-enhanced SPR method was demonstrated by two simple model assays: a small molecule assay and a DNA hybridization assay. The small molecule assay was used to demonstrate the sensitivity enhancement of the method, and how competitive assays can be used for relative affinity determination. The DNA assay was used to demonstrate the selectivity of the assay, and the capabilities in eliminating noise from bulk liquid composition variations.
doi:10.3390/s131115348
PMCID: PMC3871110  PMID: 24217357
biosensor; surface plasmon resonance; SPR; label; sensitivity; specificity
4.  Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance 
Journal of Amino Acids  2012;2012:816032.
Several proteins, like transcription factors, bind to certain DNA sequences, thereby regulating biochemical pathways that determine the fate of the corresponding cell. Due to these key positions, it is indispensable to analyze protein-DNA interactions and to identify their mode of action. Surface plasmon resonance is a label-free method that facilitates the elucidation of real-time kinetics of biomolecular interactions. In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides. After a description of the physical phenomenon and the instrumental realization including fiber-optic-based SPR and SPR imaging, we will continue with a survey of immobilization methods. Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published. Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.
doi:10.1155/2012/816032
PMCID: PMC3303711  PMID: 22500214
5.  Active Control of SPR by Thermoresponsive Hydrogels for Biosensor Applications 
The use of thermoresponsive poly(N-isopropylacrylamide)-based hydrogel (pNIPAAm) for rapid tuning of surface plasmon resonance (SPR) is reported. This approach is implemented by using an SPR layer architecture with an embedded indium tin oxide microheater and pNIPAAm film on its top. It takes advantage of rapid thermally induced swelling and collapse of pNIPAAm that is accompanied by large refractive index changes and leads to high thermo-optical coefficient of dn/dT = 2 × 10–2 RIU/K. We show that this material is excellently suited for efficient control of refractive index-sensitive SPR and that it can serve simultaneously as a 3D binding matrix in biosensor applications (if modified with biomolecular recognition elements for a specific capture of target analyte). We demonstrate that this approach enables modulating of the output signal in surface plasmon-enhanced fluorescence spectroscopy biosensors and holds potential for simple time-multiplexing of sensing channels for parallelized readout of fluorescence assays.
doi:10.1021/jp400255u
PMCID: PMC3677233  PMID: 23762499
6.  Nanobioengineering and Characterization of a Novel Estrogen Receptor Biosensor 
Sensors (Basel, Switzerland)  2008;8(7):4413-4428.
We constructed an original supramolecular assembly on a surface of sensor composed of an innovative combination of an engineered cytochrome b5 and a modified nucleic acid bound to a synthetic lipid hemimembrane. The protein/DNA block, called (P-DNA)2, was synthesized and purified before its immobilization onto a hybrid bilayer reconstituted on a gold surface. Surface plasmon resonance (SPR) and atomic force microscopy (AFM) were engaged in parallel on the same substrates in order to better understand dynamic events that occur at the surface of the biosensor. Good correlations were obtained in terms of specificity and reversibility. These findings allow us to present a first application of such biosensor in the study of the interaction processes between nuclear receptor and DNA.
doi:10.3390/s8074413
PMCID: PMC3697183
Nano-objects, molecular lego, AFM; SPR; Protein/DNA interaction
7.  Rapid identification of Mycobacterium tuberculosis infection by a new array format-based surface plasmon resonance method 
Nanoscale Research Letters  2012;7(1):180.
Tubercle bacillus [TB] is one of the most important chronic infectious diseases that cause millions of deaths annually. While conventional smear microscopy and culture methods are widely used for diagnosis of TB, the former is insensitive, and the latter takes up to 6 to 8 weeks to provide a result, limiting the value of these methods in aiding diagnosis and intermediate decisions on treatment. Therefore, a rapid detection method is essential for the diagnosis, prognosis assessment, and recurrence monitoring. A new surface plasmon resonance [SPR] biosensor based on an array format, which allowed immobilizing nine TB antigens onto the sensor chip, was constructed. Simultaneous determination of multiple TB antibodies in serum had been accomplished with this array-based SPR system. The results were compared with enzyme-linked immunosorbent assay, a conventional immunological method. Array-based SPR showed more advantages in providing label-free and real-time detection. Additionally, the high sensitivity and specificity for the detection of TB infection showed its potential for future development of biosensor arrays for TB diagnosis.
doi:10.1186/1556-276X-7-180
PMCID: PMC3317816  PMID: 22401500
identification; TB; SPR; biosensor; antigen
8.  Rapid label-free identification of mixed bacterial infections by surface plasmon resonance 
Background
Early detection of mixed aerobic-anaerobic infection has been a challenge in clinical practice due to the phenotypic changes in complex environments. Surface plasmon resonance (SPR) biosensor is widely used to detect DNA-DNA interaction and offers a sensitive and label-free approach in DNA research.
Methods
In this study, we developed a single-stranded DNA (ssDNA) amplification technique and modified the traditional SPR detection system for rapid and simultaneous detection of mixed infections of four pathogenic microorganisms (Pseudomonas aeruginosa, Staphylococcus aureus, Clostridium tetani and Clostridium perfringens).
Results
We constructed the circulation detection well to increase the sensitivity and the tandem probe arrays to reduce the non-specific hybridization. The use of 16S rDNA universal primers ensured the amplification of four target nucleic acid sequences simultaneously, and further electrophoresis and sequencing confirmed the high efficiency of this amplification method. No significant signals were detected during the single-base mismatch or non-specific probe hybridization (P < 0.05). The calibration curves of amplification products of four bacteria had good linearity from 0.1 nM to 100 nM, with all R2 values of >0.99. The lowest detection limits were 0.03 nM for P. aeruginosa, 0.02 nM for S. aureus, 0.01 nM for C. tetani and 0.02 nM for C. perfringens. The SPR biosensor had the same detection rate as the traditional culture method (P < 0.05). In addition, the quantification of PCR products can be completed within 15 min, and excellent regeneration greatly reduces the cost for detection.
Conclusions
Our method can rapidly and accurately identify the mixed aerobic-anaerobic infection, providing a reliable alternative to bacterial culture for rapid bacteria detection.
doi:10.1186/1479-5876-9-85
PMCID: PMC3120692  PMID: 21649913
bacterial infection; biosensor; mixed infection; surface plasmon resonance
9.  New trends in instrumental design for surface plasmon resonance-based biosensors 
Biosensors & bioelectronics  2010;26(5):1815-1824.
Surface plasmon resonance (SPR)-based biosensing is one of the most advanced label free, real time detection technologies. Numerous research groups with divergent scientific backgrounds have investigated the application of SPR biosensors and studied the fundamental aspects of surface plasmon polaritons that led to new, related instrumentation. As a result, this field continues to be at the forefront of evolving sensing technology. This review emphasizes the new developments in the field of SPR-related instrumentation including optical platforms, chips design, nanoscale approach and new materials. The current tendencies in SPR-based biosensing are identified and the future direction of SPR biosensor technology is broadly discussed.
doi:10.1016/j.bios.2010.09.030
PMCID: PMC3014383  PMID: 20951566
Surface plasmon resonance; SPR imaging; Optical sensor; Biochip design; Biosensor; Label-free detection
10.  Energetic basis for selective recognition of T·G mismatched base pairs in DNA by imidazole-rich polyamides 
Nucleic Acids Research  2004;32(6):2000-2007.
To complement available structure and binding results and to develop a detailed understanding of the basis for selective molecular recognition of T·G mismatches in DNA by imidazole containing polyamides, a full thermodynamic profile for formation of the T·G–polyamide complex has been determined. The amide-linked heterocycles f-ImImIm and f-PyImIm (where f is formamido group, Im is imidazole and Py is pyrrole) were studied by using biosensor-surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) with a T·G mismatch containing DNA hairpin duplex and a similar DNA with only Watson–Crick base pairs. Large negative binding enthalpies for all of the polyamide–DNA complexes indicate that the interactions are enthalpically driven. SPR results show slower complex formation and stronger binding of f-ImImIm to the T·G than to the match site. The thermodynamic analysis indicates that the enhanced binding to the T·G site is the result of better entropic contributions. Negative heat capacity changes for the complex are correlated with calculated solvent accessible surface area changes and indicate hydrophobic contributions to complex formation. DNase I footprinting analysis in a long DNA sequence provided supporting evidence that f-ImImIm binds selectively to T·G mismatch sites.
doi:10.1093/nar/gkh515
PMCID: PMC390366  PMID: 15064359
11.  LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1 
Nucleic Acids Research  2004;32(10):3101-3107.
One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera–TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets.
doi:10.1093/nar/gkh636
PMCID: PMC434442  PMID: 15181175
12.  Broadband Plasmon Waveguide Resonance Spectroscopy for Probing Biological Thin Films 
Applied spectroscopy  2009;63(9):1062-1067.
A commercially available spectrometer has been modified to perform plasmon waveguide resonance (PWR) spectroscopy over a broad spectral bandwidth. When compared to surface plasmon resonance (SPR), PWR has the advantage of allowing measurements in both s- and p-polarizations on a waveguide surface that is silica or glass rather than a noble metal. Here the waveguide is a BK7 glass slide coated with silver and silica layers. The resonance wavelength is sensitive to the optical thickness of the medium adjacent to the silica layer. The sensitivity of this technique is characterized and compared with broadband SPR both experimentally and theoretically. The sensitivity of spectral PWR is comparable to that of spectral SPR for samples with refractive indices close to that of water. The hydrophilic surface of the waveguide allows supported lipid bilayers to be formed spontaneously by vesicle fusion; in contrast, the surface of an SPR chip requires chemical modification to create a supported lipid membrane. Broadband PWR spectroscopy should be a useful technique to study biointerfaces, including ligand binding to transmembrane receptors and adsorption of peripheral proteins on ligand-bearing membranes.
doi:10.1366/000370209789379295
PMCID: PMC2912159  PMID: 19796490
Planar waveguide; Plasmon waveguide resonance; PWR; Planar supported lipid bilayer; Surface plasmon resonance; SPR
13.  SURFACE PLASMON RESONANCE BIOSENSOR FOR RAPID LABEL-FREE DETECTION OF MICRORNA AT SUBFEMTOMOLE LEVEL 
Analytical chemistry  2010;82(24):10110-10115.
MicroRNAs (miRNAs) have been linked with various regulatory functions and disorders, such as cancers and heart diseases. They therefore present an important target for detection technologies for future medical diagnostics. We report here a novel method for rapid and sensitive miRNA detection and quantitation using surface plasmon resonance (SPR) sensor technology and a DNA*RNA antibody-based assay. The approach takes advantage of a novel high-performance portable SPR sensor instrument for spectroscopy of surface plasmons based on a special diffraction grating called a surface plasmon coupler and disperser (SPRCD). The surface of the grating is functionalized with thiolated DNA oligonucleotides which specifically capture miRNA from a liquid sample without amplification. Subsequently, an antibody that recognizes DNA*RNA hybrids is introduced to bind to the DNA*RNA complex and enhance sensor response to the captured miRNA. This approach allows detecting miRNA in less than 30 minutes at concentrations down to 2 pM with an absolute amount at high attomoles. The methodology is evaluated for analysis of miRNA from mouse liver tissues and is found to yield results which agree well with those provided by the quantitative polymerase chain reaction (qPCR).
doi:10.1021/ac102131s
PMCID: PMC3021551  PMID: 21090631
microRNA; surface plasmon resonance; biosensor; liver toxicity; cancer diagnostics
14.  Use of Biosensors as Alternatives to Current Regulatory Methods for Marine Biotoxins 
Sensors (Basel, Switzerland)  2009;9(11):9414-9443.
Marine toxins are currently monitored by means of a bioassay that requires the use of many mice, which poses a technical and ethical problem in many countries. With the exception of domoic acid, there is a legal requirement for the presence of other toxins (yessotoxin, saxitoxin and analogs, okadaic acid and analogs, pectenotoxins and azaspiracids) in seafood to be controlled by bioassay, but other toxins, such as palytoxin, cyclic imines, ciguatera and tetrodotoxin are potentially present in European food and there are no legal requirements or technical approaches available to identify their presence. The need for alternative methods to the bioassay is clearly important, and biosensors have become in recent years a feasible alternative to animal sacrifice. This review will discuss the advantages and disadvantages of using biosensors as alternatives to animal assays for marine toxins, with particular focus on surface plasmon resonance (SPR) technology.
doi:10.3390/s91109414
PMCID: PMC3260648  PMID: 22291571
biosensor; seafood; shellfish poisoning; marine toxins; surface plasmon resonance
15.  Real-time and Label-free Bio-sensing of Molecular Interactions by Surface Plasmon Resonance: A Laboratory Medicine Perspective 
The Clinical Biochemist Reviews  2012;33(4):161-173.
Radioactive, chromogenic, fluorescent and other labels have long provided the basis of detection systems for biomolecular interactions including immunoassays and receptor binding studies. However there has been unprecedented growth in a number of powerful label free biosensor technologies over the last decade. While largely at the proof-of-concept stage in terms of clinical applications, the development of more accessible platforms may see surface plasmon resonance (SPR) emerge as one of the most powerful optical detection platforms for the real-time monitoring of biomolecular interactions in a label-free environment.
In this review, we provide an overview of SPR principles and current and future capabilities in a diagnostic context, including its application for monitoring a wide range of molecular markers of disease. The advantages and pitfalls of using SPR to study biomolecular interactions are discussed, with particular emphasis on its potential to differentiate subspecies of analytes and the inherent ability for quantitation through calibration-free concentration analysis (CFCA). In addition, recent advances in multiplex applications, high throughput arrays, miniaturisation, and enhancements using noble metal nanoparticles that promise unprecedented sensitivity to the level of single molecule detection, are discussed.
In summary, while SPR is not a new technique, technological advances may see SPR quickly emerge as a highly powerful technology, enabling rapid and routine analysis of molecular interactions for a diverse range of targets, including those with clinical applicability. As the technology produces data quickly, in real-time and in a label-free environment, it may well have a significant presence in future developments in lab-on-a-chip technologies including point-of-care devices and personalised medicine.
PMCID: PMC3529553  PMID: 23267248
surface plasmon resonance; biosensor; real-time; label-free
16.  Plasmonic-Based Electrochemical Impedance Spectroscopy: Application to Molecular Binding 
Analytical Chemistry  2011;84(1):327-333.
Plasmonic-based electrochemical impedance spectroscopy (P-EIS) is developed to investigate molecular binding on surfaces. Its basic principle relies on the sensitive dependence of surface plasmon resonance (SPR) signal on surface charge density, which is modulated by applying an AC potential to a SPR chip surface. The AC component of the SPR response gives the electrochemical impedance, and the DC component provides the conventional SPR detection. The plasmonic-based impedance measured over a range of frequency is in quantitative agreement with the conventional electrochemical impedance. Compared to the conventional SPR detection, P-EIS is sensitive to molecular binding taking place on the chip surface, and less sensitive to bulk refractive index changes or non-specific binding. Moreover, this new approach allows for simultaneous SPR and surface impedance analysis of molecular binding processes.
doi:10.1021/ac202634h
PMCID: PMC3299414  PMID: 22122514
SPR; Surface Plasmon Resonance; Electrochemical Impedance Spectroscopy; Plasmonic-based impedance spectroscopy; P-EIS
17.  Fabrication and Characterization of DNA Arrays Prepared on Carbon-on-Metal Substrates 
Analytical chemistry  2009;81(15):6429-6437.
Carbon-on-metal substrates consist of a surface plasmon-conducting metal substrate with a thin amorphous carbon overlayer. Recently, oligonucleotide arrays were in situ synthesized on carbon-on-gold substrates and DNA hybridization experiments were monitored with surface plasmon resonance (SPR) imaging. We describe here the thorough characterization of these substrates and arrays as they progress through the fabrication process. Two surface plasmon conducting metals, gold and silver, were utilized in the carbon-on-metal substrate preparation and their SPR responses compared. Oligonucleotide arrays synthesized on the carbon-on-metal substrates were analyzed with fluorescence- and SPR-based measurements. The stability of the carbon-on-metal substrates when exposed to prolonged incubations and/or serial hybridizations was also determined.
doi:10.1021/ac900807q
PMCID: PMC2797128  PMID: 20020675
18.  Sensitivity Comparison of Surface Plasmon Resonance and Plasmon-Waveguide Resonance Biosensors 
Sensors and actuators. B, Chemical  2011;156(1):169-175.
Plasmon-waveguide resonance (PWR) sensors are particularly useful for investigation of biomolecular interactions with or within lipid bilayer membranes. Many studies demonstrated their ability to provide unique qualitative information, but the evaluation of their sensitivity as compared to other surface plasmon resonance (SPR) sensors has not been broadly investigated. We report here a comprehensive sensitivity comparison of SPR and PWR biosensors for the p-polarized light component. The sensitivity of five different biosensor designs to changes in refractive index, thickness and mass are determined and discussed. Although numerical simulations show an increase of the electric field intensity by 30–35 % and the penetration depth by four times in PWR, the waveguide-based method is 0.5 to 8 fold less sensitive than conventional SPR in all considered analytical parameters. The experimental results also suggest that the increase in the penetration depth in PWR is made at the expense of the surface sensitivity. The physical and structural reasons for PWR sensor limitations are discussed and a general viewpoint for designing more efficient SPR sensors based on dielectric slab waveguides is provided.
doi:10.1016/j.snb.2011.04.008
PMCID: PMC3111218  PMID: 21666780
optical biosensors; surface plasmon resonance; plasmon-waveguide resonance; surface sensitivity; planar waveguide
19.  Surface plasmon resonance: a useful technique for cell biologists to characterize biomolecular interactions 
Molecular Biology of the Cell  2013;24(7):883-886.
Surface plasmon resonance (SPR) is a powerful technique for monitoring the affinity and selectivity of biomolecular interactions. SPR allows for analysis of association and dissociation rate constants and modeling of biomolecular interaction kinetics, as well as for equilibrium binding analysis and ligand specificity studies. SPR has received much use and improved precision in classifying protein–protein interactions, as well as in studying small-molecule ligand binding to receptors; however, lipid–protein interactions have been underserved in this regard. With the field of lipids perhaps the next frontier in cellular research, SPR is a highly advantageous technique for cell biologists, as newly identified proteins that associate with cellular membranes can be screened rapidly and robustly for lipid specificity and membrane affinity. This technical perspective discusses the conditions needed to achieve success with lipid–protein interactions and highlights the unique lipid–protein interaction mechanisms that have been elucidated using SPR. It is intended to provide the reader a framework for quantitative and confident conclusions from SPR analysis of lipid–protein interactions.
doi:10.1091/mbc.E12-10-0713
PMCID: PMC3608497  PMID: 23533209
20.  The Effects of Hairpin loops on Ligand-DNA Interactions 
The journal of physical chemistry. B  2009;113(43):14329-14335.
Hairpin nucleic acids are frequently used in physical studies due to their greater thermal stability compared to their equivalent duplex structures. They are also good models for more complex loop-containing structures such as quadruplexes, i-motifs, cruciforms, and molecular beacons. Although a connecting loop can increase stability, there is little information on how the loop influences the interactions of small molecules with attached base-paired nucleic acid regions. In this study, the effects of different hairpin loops on the interactions of A/T specific DNA minor groove binding agents with a common stem sequence have been investigated by spectroscopic and surface plasmon resonance (SPR) biosensor methods. The results indicate that the hairpin loop has little influence on the specific site interactions on the stem but significantly affects nonspecific binding. The use of a non-nucleotide loop (with a reduced negative charge) not only enhances the thermal stability of the hairpin but also reduces the non-specific binding at the loop without compromising the primary binding affinity on the stem.
doi:10.1021/jp904830m
PMCID: PMC2766006  PMID: 19778070
DNA hairpin loops; DNA – small molecule interactions; surface plasmon resonance biosensor; binding affinity
21.  Conclusions from the MIRG 2010 Benchmark Study: Molecular Interactions in a Three Component System and Presentation of 2011 Survey Results on Label-Free Technologies 
Characterizing the assembly of multi-protein complexes and the competition between multiple protein ligands for a given target are common challenges faced by core facilities. The MIRG2010 Benchmark study was designed to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies such as surface Plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B and C) and asked to determine if a ternary A-B-C complex can form, or if ligands B and C bind competitively to protein A. This presentation will summarize the conclusions from the 2010 Benchmark Study, and provide perspective on the potential for future application of this system as a reference standard for quantitative characterization of protein-protein interactions using biosensor technologies. The field of label-free biophysical technologies like surface plasmon resonance (SPR) and isothermal calorimetry (ITC) are becoming indispensable in translational research and in the discovery phase of biotherapeutics. Investigators are much more aware about the developments in biomolecular interaction analysis using SPR and ITC and usefulness of these technologies in designing better drugs based on biomolecules and vaccines. The Molecular Interaction Research Group (MIRG) of ABRF has conducted an on-line survey to capture the recent explosive developments in these technologies. The survey was targeted to both academia and pharmaceutical industry and the survey data will be presented during the meeting.
PMCID: PMC3186663
22.  Surface plasmon resonance phase imaging measurements of patterned monolayers and DNA adsorption onto microarrays 
Analytical chemistry  2011;83(7):2801-2806.
The optical technique of surface plasmon resonance phase imaging (SPR-PI) is implemented in a linear microarray format for real-time measurements of surface bioaffinity adsorption processes. SPR-PI measures the phase shift of p-polarized light incident at the SPR angle reflected from a gold thin film in an ATR Kretschmann geometry by creating an interference fringe image on the interface with a polarizer-quartz wedge depolarizer combination. The position of the fringe pattern in this image changes upon the adsorption of biomolecules to the gold thin film. By using a linear array of 500 μm biosensor element lines that are perpendicular to the interference fringe image, multiple bioaffinity adsorption measurements can be performed in real time. Two experiments were performed to characterize the sensitivity of the SPR-PI measurement technique; first, a ten line pattern of a self-assembled monolayer of 11-mercaptoundecamine (MUAM) was created via photopatterning to verify that multiple phase shifts could be measured simultaneously. A phase shift difference (Δφ) of Δφ = 182.08 ± 0.03° was observed for the 1.8-nm MUAM monolayer; this value agrees with the phase shift difference calculated from a combination of Fresnel equations and Jones matrices for the depolarizer. In a second demonstration experiment, the feasibility of SPR-PI for in situ bioaffinity adsorption measurements was confirmed by detecting the hybridization and adsorption of single stranded DNA (ssDNA) onto a six component DNA line microarray patterned monolayer. Adsorption of a full DNA monolayer produced a phase shift difference of Δφ = 28.80 ± 0.03° at the SPR angle of incidence and the adsorption of the ssDNA was monitored in real time with the SPR-PI. These initial results suggest that SPR-PI should have a detection limit roughly 100 times lower than traditional intensity-based SPR imaging measurements.
doi:10.1021/ac200157p
PMCID: PMC3108464  PMID: 21355546
23.  Application of Optical Biosensors in Small-Molecule Screening Activities 
Sensors (Basel, Switzerland)  2012;12(4):4311-4323.
The last two decades have seen remarkable progress and improvements in optical biosensor systems such that those are currently seen as an important and value-adding component of modern drug screening activities. In particular the introduction of microplate-based biosensor systems holds the promise to match the required throughput without compromising on data quality thus representing a sought-after complement to traditional fluidic systems. This article aims to highlight the application of the two most prominent optical biosensor technologies, namely surface plasmon resonance (SPR) and optical waveguide grating (OWG), in small-molecule screening and will present, review and discuss the advantages and disadvantages of different assay formats on these platforms. A particular focus will be on the specific advantages of the inhibition in solution assay (ISA) format in contrast to traditional direct binding assays (DBA). Furthermore we will discuss different application areas for both fluidic as well as plate-based biosensor systems by considering the individual strength of the platforms.
doi:10.3390/s120404311
PMCID: PMC3355412  PMID: 22666031
screening; fragment; ligand; biosensor; surface plasmon resonance; optical waveguide grating; drug discovery
24.  Induced Fit Conformational Changes of a “Reversed Amidine” Heterocycle: Optimized Interactions in a DNA Minor Groove Complex 
To better understand the molecular basis for recognition of the DNA minor groove by heterocyclic cations, a series of “reversed amidine” substituted heterocycles has been prepared. Amidine derivatives for targeting the minor groove have the amidine carbon linked to a central heterocyclic system while in the reverse orientation, an amidine nitrogen provides the link. The reverse system has a larger dihedral angle as well as a modified spatial relationship with the groove relative to amidines. Because of the large dihedral, the reversed amidines should have reduced binding to DNA relative to similar amidines. Such a reduction is observed in footprinting, circular dichroism (CD), biosensor-surface plasmon resonance (SPR) and isothermal titration calorimetric (ITC) experiments with DB613, which has a central phenyl-furan-phenyl heterocyclic system. The reduction is not seen when a pyrrole (DB884) is substituted for the furan. Analysis of a number of derivatives defines the pyrrole and a terminal phenyl substituent on the reversed amidine groups as critical components in the strong binding of DB884. ITC and SPR comparisons showed that the better binding of DB884 was due to a more favorable binding enthalpy and that it had exceptionally slow dissociation from DNA. Crystallographic analysis of DB884 bound to an AATT site show that the compound was bound in the minor groove in a 1:1 complex as suggested by CD solution studies. Surprisingly, unlike the amidine derivative, the pyrrole -NH of DB884 formed an H-bond with a central T of the AATT site and this accounts for the enthalpy driven strong binding. The structural results and molecular modeling studies provide an explanation for the differences in binding affinities for related amidine and reversed amidine analogs.
doi:10.1021/ja069003n
PMCID: PMC2547086  PMID: 17425312
25.  Surface Plasmon Resonance: An Introduction to a Surface Spectroscopy Technique 
Journal of chemical education  2010;87(7):742-746.
Surface plasmon resonance (SPR) has become an important optical biosensing technology in the areas of biochemistry, biology, and medical sciences because of its real-time, label-free, and noninvasive nature. The high cost of commercial devices and consumables has prevented SPR from being introduced in the undergraduate laboratory. Here we present an affordable homemade SPR device with all of its components accessible to visualization. This design allows ease of integration with electrochemistry and makes the device suitable for education. We describe a laboratory experiment in which students examine the relationship between the SPR angle and the solution refractive index at the interface and perform a coupled SPR–electrochemistry experiment. Students also study the antibody–antigen binding activity. Most of the experimental work was done as a project by a grade 12 high-school student under proper supervision. We believe that the SPR device and the SPR laboratory will enhance undergraduate chemical education by introducing students to this important modern instrumentation and will help students to learn and understand the molecular interactions occurring at interfaces.
doi:10.1021/ed100186y
PMCID: PMC3045209  PMID: 21359107
Analytical Chemistry; Bioanalytical Chemistry; Biochemistry; Biotechnology; Electrochemistry; Hands-On Learning/Manipulatives; Kinetics; Laboratory Equipment/Apparatus; Laboratory Instruction; Mechanisms of Reactions; Molecular Biology; Quantitative Analysis; Second-Year Undergraduate; Spectroscopy; Surface Science; Upper-Division Undergraduate

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