SPR-fluorescence

Fig. 11. A layer-by-layer model built by biotin-streptavidin interaction. (A) Angular SPR curves of each layer. The inset shows the SPR minimum position as a function of layer number. (B) SPR, fluorescence angular scans for layers a , b , i , and j .

One of the most exciting developments with SPR is the development of coupled analytical techniques with surface plasmon resonance. While many other coupled analytical techniques such as LC-MS and GC-MS are well practiced in routine work, little research has been done coupling SPR to other analytical tools until very recently. The most promising coupling technique continues to be SPR-mass spectrometry (MS) and LC-SPR-MS. LC-SPR and SPR-fluorescence (SPFS) have gained considerable attention in recent years. We briefly review these hyphenated techniques and discuss their prospects in future SPR measurements. 

Biomolecule detectors incorporate a biorecognition device capable of selectively recognizing the analyte of interest in connection with a signal transducer and a suitable output device. Transduction methods include a variety of optical (surface plasmon resonance [SPR], fluorescence), electrochemical (voltammetry, impedance, field effect), mechanical (cantilever, surface probe microscopy), and mass-based systems (quartz crystal microgravimetry [QCM], mass spectrometry). Selection of the appropriate transduction system is partially determined by the nature of information sought (quantitative or qualitative), the analyte (concentration, molecular weight), the sample size, and assay timeline. 

Based on the preliminary understanding of ICT mechanism of coumarin core skeleton, the first systematic application of combinatorial approach toward the field of fluorescence chemistry was reported by Bauerle and co-workers in 2001 [44]. In their study, the structure-photophysical property relationships (SPR) of coumarin fluorophore were revealed by means of a combinatorial approach. 

When cells are suspended in a biological fluid or culture medium, both serum proteins and cells interact with the surface substrate. Serum protein adsorption behavior on SAMs has been examined with various analytical methods, including SPR [58-61], ellipsometry [13, 62, 63], and quartz QCM [64—66]. These methods allow in situ, highly sensitive detection of protein adsorption without any fluorescence or radioisotope labeling. SPR and QCM are compatible with SAMs that comprise alkanethiols. In our laboratory, we employed SPR to monitor protein adsorption on SAMs. 

As well as fluorescence-based assays, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In a commercial BIA-core system [231] a hydrophobic SPR sensor with an alkane thiol surface was incubated with vesicles of defined size distribution generating a hybrid membrane by fusion of the lipid vesicles with the alkane thiol layer [232]. If the vesicles contain biotinylated lipopeptides their membrane anchoring can be analyzed by incubation with streptavidine. Accordingly, experiments with lipopeptides representing the C-terminal sequence of N-Ras show clear differences between single and double hydrophobic modified peptides in their ability to persist in the lipid layer [233]. 

The advantages of SPR experiments are that only small amounts of sample are required,72 often hundreds of microliters of solutions with nanomolar to micromolar concentration of reactants and the substrate attached to the surface can oftentimes be reused after washing in buffer. The fact that changes in the refractive index values are measured avoids the need to use absorption or fluorescence markers to follow the binding kinetics. 

These preliminary experimental results illustrate the power of the OFRR to detect specifically biomolecules while using low-cost photonics and simple protocols. The OFRR is a label-free Rl-based sensor, and thus it avoids the extra steps and costly photonics of fluorescence-based techniques. Even compared with other Rl-based techniques, such as SPR, the OFRR is able to use low-cost simple photonics because of its high Q-factor and thus minimal bandwidth requirements. Additionally, the sensor is inherently integrated into the microfluidic sample delivery channel. 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

A typical time profile of the excited PMMA-Phe fluorescence intensity decay is shown in Figure 2. The MEK permeation commences at 24 sec. The SPR increases during the plasticization period until it becomes constant, the onset of the steady state. It is characterized by a linear relationship between the amount of solvent absorbed and time. It was determined from a linear regression analysis that the PMMA-Phe fluorescence intensity starts to deviate from linearity at 197 sec. This indicates a decrease in the SPR and/or the unquenched PMMA-Phe. The decrease in SPR is unexpected at this film thickness since the SPR in thicker PMMA-Phe films show no anomaly at 1 /tm. A more plausible explanation is the reduction in available PMMA-Phe, which is expected when the front end of the SCP reaches the substrate. 

SPR biosensors are label-free detection devices - binding between the biomolecular recognition element and analyte can be observed directly without the use of radioactive or fluorescent labels. In addition, the binding event can be observed in real-time. SPR affinity biosensors can, in principle, detect any analyte for which an appropriate biomolecular recognition element is available. Moreover, analyte molecules do not have to exhibit any special properties such as fluorescence or characteristic absorption or scattering bands. 

Different techniques have been applied to study the protein-protein, protein-ligand and, in particular, MIP-ligand interactions. They may serve to estimate or determine the binding constant and the number of independent binding sites (N) of a ligand-to-receptor (MIP or antibody) interaction. The range of affinity constants that can be calculated depends on the sensitivity of the assay and, in those cases where the separation of the bound and free species is a step of the assay, perturbation of the equilibria in the separation step will also be important [22]. Direct nonseparation techniques such as spectroscopic techniques (e.g., SPR or fluorescence polarization) can be used as well as indirect separation techniques such as radiolabeling [22]. 

In this part we will describe recent achievements in the development of biosensors based on DNA/RNA aptamers. These biosensors are usually prepared by immobilization of aptamer onto a solid support by various methods using chemisorption (aptamer is modified by thiol group) or by avidin-biotin technology (aptamer is modified by biotin) or by covalent attachment of amino group-labeled aptamer to a surface of self-assembly monolayer of 11-mercaptoundecanoic acid (11-MUA). Apart from the method of aptamer immobilization, the biosensors differ in the signal generation. To date, most extensively studied were the biosensors based on optical methods (fluorescence, SPR) and acoustic sensors based mostly on thickness shear mode (TSM) method. However, recently several investigators reported electrochemical sensors based on enzyme-labeled aptamers, electrochemical indicators and impedance spectroscopy methods of detection. 

Optical methods are based on fluorescence probe-labeled aptamers (fluorescence intensity, fluorescence anisotropy), or label-free aptamers can be used for detection of analyte using SPR or Fourier transform infrared attenuated total reflection (FTIR-ATR). 

The transducers most commonly employed in biosensors are (a) Electrochemical amperometric, potentiometric and impedimetric (b) Optical vibrational (IR, Raman), luminescence (fluorescence, chemiluminescence) (c) Integrated optics (surface plasmon resonance (SPR), interferometery) and (d) Mechanical surface acoustic wave (SAW) and quartz crystal microbalance (QCM) [4,12]. 

Nucleic acid biosensors based on optical modes of detection represent another common approach for generating analytical signals based on nucleic acid hybridization. The methods discussed herein are based on methods that are suitable for the study of materials on surfaces. There are a number of different optical methods that have been described, with the most common being attenuated total reflectance (ATR), total internal reflection fluorescence (TIRF) and surface plasmon resonance (SPR) [15]. All of these methods work... 

In this section an overview of the numerous methods and principles for the discrimination of enantiomers is given. First, the interaction principles of the polymer-based methods adapted from chromatographic procedures are illustrated. The discrimination of enantiomers was achieved some decades ago by using different types of stationary materials, like cyclodextrins or polymer-bonded amide selectors. These stationary-phase materials have successfully been appointed for label-free optical sensing methods like surface plasmon resonance (SPR) or reflectometric interference spectroscopy (RIfS). Furthermore, various successful applications to optical spectroscopy of the well-established method of fluorescence measurements for the discrimination of enantiomers are described. 

Cyclodextrins can be used as fluorescence sensors or as hosts for direct optical detection with methods like SPR. 

Abbreviations AOD, Acousto-optical deflection BCB, bisbenzyocyclobutadiene CCD, indirect contact conductivity detection CL, chemiluminescence ECD, electron capture detector FCS, fluorescence correlation spectroscopy FRET, fluorescence resonance energy transfer ICCD, integrated contact conductivity detection GMR, giant magnetoresistive LED-CFD, light emitting diode confocal fluorescence detector LIF, laser-induced fluorescence LOD, limit of detection MALDI, matrix-assisted laser desorption ionization PDMS, poly(dimethylsiloxane) PMMA, poly(methylmetha-crylate) SPR, surface plasmon resonance SVD, sinusoidal voltammetric detection TLS, thermal lens spectroscopy. 

Pharmacokinetic data analysis requires determination of the analyte in various body fluids. In the case of therapeutic antibodies, serum is the most common matrix to be analyzed. For a critical interpretation of pharmacokinetic data the chosen bioanalytical methods must be considered. The most frequently used for mAbs include enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE)/polyacrylamide gel electrophoresis (PAGE), fluorescence-activated cell sorting (FACS), and surface plasmon resonance (SPR). The challenges and limitations of bioanalytical methods used for the analysis of mAb concentrations are discussed in detail in Chapter 6. 

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