Supramolecular interfacial architectures for optical biosensing with surface plasmons
Introduction
The performance of any (bio-)medical device crucially depends on the proper functionalization of its surface. This is obviously true for prosthetic implants, for scaffolds used in cell and tissue engineering, or for biosensors and alike. The challenge is not only to generate a passive biocompatibility, i.e. to engineer the surface of the device such that any biomaterial––from proteins to cells and whole living tissues––tolerate the ‘foreign’ object, the proper design and synthesis of surface functional groups also control the desired active or interactive communication between the device and its bioenvironment [1].
For the case of biosensors these criteria reduce to the seemingly ‘simple’ requirements which the sensor surface needs to fulfill: an optimized density of highly selective and specific biofunctional groups for the recognition (and binding) of the analyte molecule of interest must be combined with a matrix that passivates the sensor surface for any unspecific and, hence, undesired interaction between the many other components in the analyte solution and the sensor surface [2].
Although this set of objectives in the general definition is the same for all detection principles employed in biosensing (e.g. electrical, electrochemical, optical, via mass detection, etc.), each of these techniques define additional boundary conditions that need to be met by the functional interfacial layer at the sensor surface, e.g., if redox-reactions are to be involved in the sensing process, obviously the surface architecture must additionally allow for an efficient electron transfer process between the (bound) analyte, any mediator possibly involved, and the electrode [3].
We will concentrate on interfacial architectures that are employed in a novel, recently introduced optical detection scheme based on the well-known surface plasmon excitation but involving, in particular, the recording of fluorescence emitted from suitable chromophores excited by the surface plasmon wave [4]. In this scheme, one needs to take into account the fact that the evanescent character of the surface plasmon mode leads to an excitation probability that is exponentially decaying away from the interface. The chromophores, hence, should be placed as close to the sensor surface as possible. However, the metallic character of a substrate that is able to carry a surface mode at the same time constitutes a broad band acceptor for energy transfer (Förster) processes between an excited chromophore and that noble metal substrate. The relevant distance dependence, however, for this coupling scheme is governed by the Förster radius for energy transfer and leads to a significant loss of fluorescence intensity by quenching only at separation distances smaller than ca. 30 nm [5]. Hence, optimized fluorescence observation is possible for chromophores that are placed within a matrix layer between ca. 30 and 100 nm above the (Au) sensor surface.
We will document in the following that sensor surface coatings with this property profile can be built allowing for the detection of (bio-)analytes with unprecedented sensitivities and selectivities.
The first set of experiments that we want to describe concerns the hybridization of DNA strands from solution to surface-attached PNA-oligonucleotide sequences [6]. These catcher matrices are assembled at the sensor surface via a generic biotin–streptavidin binding matrix schematically given in Fig. 1 [7]. The first layer chemisorbed to the Au-substrate via thiol groups is composed of a binary mixture of 5 mol% of a biotinylated thiol derivative dispersed in a matrix of 95 mol% mercaptohexanol used as diluent molecules. This results in a functional layer to which streptavidin binds with high affinity through 1 or 2 of its 4 binding sites, thus generating a binding layer to which other biotinylated species can be coupled. For our experiments we choose the uncharged PNA analogues of DNA oligonucleotides that were linked to a biotin group via an ethylene oxide spacer. This architecture ensures a moderate catcher probe density of ca. 2.5 × 1012 cm−2 thus minimizing any cross-talk between individual reaction sites during the hybridization reactions. In earlier experiments with short oligonucleotides this has been shown to then allow for the quantitative evaluation of the association (hybridization) and dissociation process of targets binding from solution with kon and koff values that were coverage-independent and represented the individual binding process well described by a simple Langmuir model [8].
The next series of investigations involves the binding of proteins to a corresponding surface layer. Here, we employed a commercial substrate from Biacore, the CM5 chip whose modified surface architecture is schematically depicted in Fig. 2 [9]. The essential element is the dextran matrix (brush architecture) to which the ligand system of interest can be covalently bound, thus allowing for the analyte and the fluorescently labeled antibody to bind within the optimal range of separation distances from the Au-substrate (ca. 30–100 nm) [10]. This way, a limit of detection (LOD) of proteins from solutions of 500 aM concentration could be established in a direct binding assay [11]. Here, we will report a sandwich approach equivalent to the well-known ELISA protocol [12].
The last example that we present concerns a novel platform for the investigation of biomembrane related processes––the tethered bimolecular lipid membrane (tBLM) [13]. Its basic structural elements are schematically shown in Fig. 3: a spacer system composed of polymers, oligosaccharides, peptides, proteins, etc. is covalently linked on one side to the substrate and, at the other end, to a number of anchor lipids integrated into the proximal monolayer of the tethered membrane. This tethering system leads to a stable and robust coupling of the bilayer to the sensor surface and at the same time decouples the membrane sufficiently from the substrate to allow for the lipid matrix to exist in a fluid (liquid–crystalline) state as it is required for a number of membrane proteins (receptors, channels, carriers, etc.) for their proper function. We will describe in the following some experiments with integrins reconstituted into a peptide-tethered membrane and employed for collagen binding studies.
Section snippets
Surface plasmon fluorescence spectroscopy
The optical detection principle that will be used throughout this work is based on surface plasmon optics [14]. The basic scheme for the excitation of such a surface bound evanescent mode in the so-called Kretschmann configuration is shown in Fig. 4(a). The excitation light beam, typically from a HeNe laser at λ = 633 nm incident at an angle θ, is reflected off the metal coated base of a high index glass prism (n = 1.85 at 633 nm) and monitored by a photodiode detector. The metal layer thickness of
Surface hybridization studies
Surface plasmon fluorescence spectroscopy has been shown in numerous examples to allow for a quantitative evaluation of surface hybridization reactions with short oligonucleotides [15].
Here, we describe experiments with long PCR products that were from a genetically modified soybean species, the Round-up Ready® GMO from Monsanto. Since we had found that double-stranded PCR DNA does not bind to surface probe oligonucleotides we developed a melt–quench protocol that resulted in sufficiently
Protein binding assay
The sandwich PSA assay in SPFS is essentially the analogue of the widely applied ELISA technique. Therefore, two detection modes are available, namely a two-step and a one-step mode, respectively. For the two-step mode (cf. panel (I) in Fig. 11), PSA was captured first by M37230M during a contact time of 30 min. After washing away the unbound PSA, LM86506M was introduced (as a 50 nM solution) and bound to the captured PSA (for 10 min), forming a capture/PSA/detection ‘sandwich’. The PSA
Tethered bimolecular lipid membranes for ligand–receptor binding studies
Tethered membranes provide an ideal platform for the investigation of membrane proteins with a variety of methods [17]. Whereas structural and functional properties of soluble proteins have been investigated in detail, membrane proteins and the associated biochemical and signaling pathways are still largely unknown. Few examples are described in the literature, such as the stable protein bacteriorhodopsin and few smaller membrane related proteins.
We have characterized a suitable membrane model
Conclusions
The presented examples document that supramolecularly-controlled interfacial architectures can be designed and experimentally realized allowing for a highly specific recognition and binding of certain analyte system from solution to their surface-attached complementary interaction partners (probes/targets, antigens/antibodies, ligands/receptors). This then enables us to tailor surface coatings on sensor surfaces that reach unprecedented sensitivities and selectivities in bioassays.
However,
Acknowledgments
We are grateful to a number of friends and colleagues for providing valuable advice and specific molecules needed for the presented experiments. Among others, we thank, in particular, Roberto Corradini, J. Eble, Andrea Germini, Stefan Lofas, Roseangela Marchelli, Peter Nielsen, Björn Persson, A. Scheller.
The streptavidin was kindly provided by Roche Diagnostics, Penzberg. Financial support came from the Volkswagen Foundation (I/77 708-10) and from the European Union (Project QLK1-2000-01658,
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