Development of surface chemistry for SPR based sensors for the detection of proteins and DNA molecules’ (original) (raw)
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Analytica Chimica Acta, 2012
The immobilisation of biological recognition elements onto a sensor chip surface is a crucial step for the construction of biosensors. While some of the optical biosensors utilise silicon dioxide as the sensor surface, most of the biosensor surfaces are coated with metals for transduction of the signal. Biological recognition elements such as proteins can be adsorbed spontaneously on metal or silicon dioxide substrates but this may denature the molecule and can result in either activity reduction or loss. Self assembled monolayers (SAMs) provide an effective method to protect the biological recognition elements from the sensor surface, thereby providing ligand immobilisation that enables the repeated binding and regeneration cycles to be performed without losing the immobilised ligand, as well as additionally helping to minimise non-specific adsorption. Therefore, in this study different surface chemistries were constructed on SPR sensor chips to investigate protein and DNA immobilisation on Au surfaces. A cysteamine surface and 1%, 10% and 100% mercaptoundeconoic acid (MUDA) coatings with or without dendrimer modification were utilised to construct the various sensor surfaces used in this investigation. A higher response was obtained for NeutrAvidin immobilisation on dendrimer modified surfaces compared to MUDA and cysteamine layers, however, protein or DNA capture responses on the immobilised NeutrAvidin did not show a similar higher response when dendrimer modified surfaces were used.
Recognition Receptors in Biosensors, 2009
The quality of a biosensing system relies on the interfacial properties where bioactive species are immobilized. The design of the surface includes both the immobilization of the bioreceptor itself and the overall chemical preparation of the transducer surface. Hence, the sensitivity and specificity of such devices are directly related to the accessibility and activity of the immobilized molecules. The inertness of the surface that limits the nonspecific adsorption sets the background noise of the sensor. The specifications of the biosensor (signal-to-noise ratio) depend largely on the surface chemistry and preparation process of the biointerface. Lastly, a robust interface improves the stability and the reliability of biosensors. This chapter reports in detail the main surface coupling strategies spanning from random immobilization of native biospecies to uniform and oriented immobilization of site-specific modified biomolecules. The immobilization of receptors on various shapes of solid support is then introduced. Detection systems sensitive to surface phenomena require immobilization as very thin layers (two-dimensional biofunctionalization), whereas other detection systems accept thicker layers (threedimensional biofunctionalization) such as porous materials of high specific area that lead to large increase of signal detection. This didactical overview introduces each step of the biofunctionalization with respect to the diversity of biological molecules, their accessibility and resistance to nonspecific adsorption at interfaces. Keywords Functionalization Á Biofunctionalization Á Surface chemical modification Á Native biomolecules Á Modified biomolecules Á Staudinger ligation Á Clickchemistry Á Native chemical ligation Á Expressed protein ligation Á Silanization Á Self-assembled monolayer Á Entrapment Á Nanoparticles Á Sol-gel process Á Adsorption Á Chemisorption Á Silica Á Silicon Á Gold layer Á Streptavidin Á Biotin Á
Biofunctionalised surfaces for molecular sensing
2013
In many application fields, like in biosensors, the sensing biomolecules are immobilized on solid surfaces to enable measuring of very small concentrations of molecules to be analysed. Such application fields are, for example, diagnostics, detection of abused drugs, environmental monitoring of toxins and tissue engineering. This thesis studies the immobilization of biomolecules (antibodies and Fab’fragments, avidins and oligonucleotide sequences) on gold surfaces in biosensors. In order to achieve high nanomolar sensitivity even in difficult sample matrices, the effect of the sensing molecule immobilization type and concentration within these biomolecular surfaces were studied in detail. The suitability of these surfaces for neuronal stem cell attachment was also one of the topics. Real-time label-free detection was performed with surface plasmon resonance (SPR). The molecular surfaces in this study were constructed of biomolecules and repellent molecules, which formed self-assemble...
Molecular monolayers on silicon as substrates for biosensors
Bioelectrochemistry, 2010
- silicon surfaces can be controlled down to atomic level and offer a remarkable starting point for elaborating nanostructures. Hydrogenated surfaces are obtained by oxide dissolution in hydrofluoric acid or ammonium fluoride solution. Organic species are grafted onto the hydrogenated surface by a hydrosilylation reaction, providing a robust covalent Si-C bonding. Finally, probe molecules can be anchored to the organic end group, paving the way to the elaboration of sensors. Fluorescence detection is hampered by the high refractive index of silicon. However, improved sensitivity is obtained by replacing the bulk silicon substrate by a thin layer of amorphous silicon deposited on a reflector. The development of a novel hybrid SPR interface by the deposition of an amorphous silicon-carbon alloy is also presented. Such an interface allows the subsequent linking of stable organic monolayers through Si-C bonds for a plasmonic detection. On the other hand, the semiconducting properties of silicon can be used to implement field-effect label-free detection. However, the electrostatic interaction between adsorbed species may lead to a spreading of the adsorption isotherms, which should not be overlooked in practical operating conditions of the sensor. Atomically flat silicon surfaces may allow for measuring recognition interactions with local-probe microscopy.
Stable Self-Assembled Monolayers for sensitive biosensor interfaces
2007
The controlled interaction of (bio)molecules with various types of surfaces, nanoparticles and nanostructures has become increasingly important for advanced biosensors and biochips. Our aim is to construct stable and well characterized interface chemistries based on pre-activated mixed self-assembled monolayers (SAMs) of alkane thiols. To allow a full exploration of this interface chemistry, first we have investigated the characteristics and biosensing properties of different SAMs on gold substrates. The detection of the cancer marker prostate specific antigen (PSA) was used as a model system. Secondly, we have functionalized gold nanoparticles using such SAMs. Functionalized nanoparticles can be used for enhancing the biosensor performance and constructing nano-(bio)assemblies. This study clearly demonstrated that pre-activated SAMs have advanced properties and can be used for various applications.
The Analyst, 2011
A peptide self-assembled monolayer (SAM) was designed to bind His-tagged biomolecules for surface plasmon resonance (SPR) bioanalysis, which was applied for the determination of K d for small ligand screening against CD36. Nonspecific adsorption could be minimized using penta-and hexa-peptide monolayers. In particular, monolayers consisting of 3-mercaptopropionyl-leucinyl-histidinyl-aspartylleucinyl-histidinyl-aspartic acid (3-Mpa-LHDLHD) exhibited little (12 ng cm À2 ) nonspecific adsorption in crude serum. Modification of this peptide monolayer with Na,Na-bis(carboxymethyl)-L-lysine gave a surface competent for binding His-tagged proteins, as demonstrated using enzyme (human dihydrofolate reductase), protein/antibody and receptor (CD36) examples. Immobilization featured chelation of copper and the His-tagged protein by the peptide monolayer, which could be recycled by removing the copper using imidazole washes prior to reuse.
Thiolated self-assembled monolayers (SAMs) are typically used to anchor on a gold surface biomolecules serving as recognition elements for biosensor applications. Here, the design and synthesis of N-(2-hydroxyethyl)-3-mercaptopropanamide (NMPA) in biotinylated mixed SAMs is proposed as an alternative strategy with respect to on-site multistep functionalization of SAMs prepared from solutions of commercially available thiols. In this study, the mixed SAM deposited from a 10:1 solution of 3mercaptopropionic acid (3MPA) and 11-mercaptoundecanoic acid (11MUA) is compared to that resulting from a 10:1 solution of NMPA:11MUA. To this end, surface plasmon resonance (SPR) and attenuated total reflectance infrared (ATR-IR) experiments have been carried out on both mixed SAMs after biotinylation. The study demonstrated how the fine tuning of the SAM features impacts directly on both the biofunctionalization steps, i.e., the biotin anchoring, and the biorecognition properties evaluated upon exposure to streptavidin analyte. Higher affinity for the target analyte with reduced nonspecific binding and lower detection limit has been demonstrated when NMPA is chosen as the more abundant starting thiol. Molecular dynamics simulations complemented the experimental findings providing a molecular rationale behind the performance of the biotinylated mixed SAMs. The present study confirms the importance of the functionalization design for the development of a highly performing biosensor.
Surface and Interface Analysis, 2018
The goal of this study was to use a novel surface chemistry for modifying gold surfaces to decrease the steric hindrance, minimize the nonspecific bindings while providing directed immobilization of proteins for advancing the transducer property and to provide a biosensing platform for surface plasmon resonance (SPR) applications. Mixed self-assembled monolayers (mSAMs) were prepared using 3,3′-Dithiodipropionic acid di (N-hydroxysuccinimide ester) (DSP) and 6-mercapto-1-hexanol (MCH) and the selected model proteins bovine serum albumin (BSA) and lysozyme were tested for binding efficiency. First, binding of these two proteins at constant concentration to different DSP:MCH mSAMs were compared to deduce the best molar ratio for forming mSAM using a continuous flow system coupled to SPR. Coincidently the maximum protein binding DSP:MCH mSAM were the same for both proteins. The change in Response Unit (ΔRU) signal due to protein binding between DSP SAM and maximum protein binding DSP:MCH mSAM for lysozyme binding was more in comparison to BSA binding. Second, the effect of BSA and lysozyme concentration on binding efficiency to maximum protein binding DSP: MCH mSAM were compared and discussed. Lysozyme and BSA were shown to reach saturations on the same monolayer at concentrations of 5.7x10 −5 and 8.96x10 −6 [M] respectively, hence the molar ratio for limit concentrations is 6:1. The DSP SAM, MCH SAM, and DSP:MCH mSAMs where maximum and minimum protein binding occurs were also characterized with XPS and Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Blank gold surface, maximum protein binding DSP:MCH mSAM and BSA immobilized DSP:MCH mSAM on gold surface were also investigated utilizing tapping mode AFM.
The identification and quantification of biomarkers or proteins is a real challenge in allowing the early detection of diseases. The functionalization of the biosensor surface has to be properly designed to prevent nonspecific interactions and to detect the biomolecule of interest specifically. A multilayered nanoarchitecture, based on polyelectrolyte multilayers (PEM) and the sequential immobilization of streptavidin and a biotinylated antibody, was elaborated as a promising platform for the label-free sensing of targeted proteins. We choose ovalbumin as an example. Thanks to the versatility of PEM films, the platform was built on two types of sensor surface and was evaluated using both optical-and viscoelasticbased techniques, namely, optical waveguide lightmode spectroscopy and the quartz crystal microbalance, respectively. A library of biotinylated poly(acrylic acids) (PAAs) was synthesized by grafting biotin moieties at different grafting ratios (GR). The biotin moieties were linked to the PAA chains through ethylene oxide (EO) spacers of different lengths. The adsorption of the PAA-EO n -biotin (GR) layer on a PEM precursor film allows tuning the surface density in biotin and thus the streptavidin adsorption mainly through the grafting ratio. The nonspecific adsorption of serum was reduced and even suppressed depending on the length of the EO arms. We showed that to obtain an antifouling polyelectrolyte the grafting of EO 9 or EO 19 chains at 25% in GR is sufficient. Thus, the spacer has a dual role: ensuring the antifouling property and allowing the accessibility of biotin moieties. Finally, an optimized platform based on the PAA-EO 9 -biotin (25%)/streptavidin/biotinylated-antibody architecture was built and demonstrated promising performance as interface architecture for bioaffinity sensing of a targeted protein, in our case, ovalbumin.
Building Functional Surfaces for Biosensors Development
Key Engineering Materials, 2013
Polyelectrolyte multilayers (PEM) built by layer-by-layer technique have been extensively studied over the last years, resulting in a wide variety of current and potential applications. This technique can be used to construct thin films with different functionalities, or to functionalize surfaces with substantial different properties of those of the underlying substrates. The multilayering process is achieved by the alternate adsorption of oppositely charged polyelectrolytes. In this work we get advantage of the protein resistant property of the Poly (l-lysine)-graft-(polyethyleneglycol) to create protein patterns. Proteins can be immobilized on a surface by unspecific physical adsorption, covalent binding or through specific interactions. The first protein used in this work was laccase, a copper-containing redox enzyme that catalyse the oxidation of a broad range of polyphenols and aromatic substrates, coupled to the reduction of O2 to H2O without need of cofactors. Applications of...