Covalent Immobilization Of Proteins Research Articles

Oriental covalent immobilization of N-glycan binding protein via N-terminal selective modification

Lectin affinity chromatography is one of powerful tools for the study of protein glycosylation. Different lectin proteins can recognize different structures of monosaccharides or oligosaccharide units, allowing for the selective separation of glycopeptides or glycoproteins containing different polysaccharide structures. However, the N-glycans were only partially captured by most of common lectins, reducing the coverage rate of identifying N-glycoconjugates. Recently, it has been reported that the engineering variant of glycan binding protein Fbs1 has a high affinity for innermost Man3GlcNAc2 structure and is able to bind diverse types of N-glycans, which can be suitable for the analysis of protein N-glycosylation. However, efficient immobilization of protein to separation matrix is particularly challenging as it requires the functionality and integrity of the protein to be preserved. Herein, we describe a simple and robust strategy for oriental covalent immobilization of proteins on magnetic nanoparticles by N-terminal selective labeling techniques. We inserted the enterokinase cleavage site to produce the specific N- terminal glycine of protein. Under physiological conditions, the protein was immobilized on the surface of the MNPs by this glycine tag, and the enrichment process could be completed within 30 min. A whole enrichment and purification of glycan and glycopeptides were completed and analyzed by MALDI TOF-MS. The functional materials achieved stable enrichment of glycan structure in different enzyme digestion systems or complex samples, showing excellent anti-interference and applicability.

Tailor-Made Bioactive Papers by Site-Specific and Orthogonal Covalent Immobilization of Proteins.

A strategy for the bioorthogonal immobilization of proteins onto commercially available filter paper is presented. Recently, a two-step approach has been described that relies on covalent immobilization of a linker molecule to paper, followed by enzyme-mediated conjugation of a protein of interest containing an enzyme-recognition tag. Here, this strategy was expanded by evaluating four different chemical and chemoenzymatic reactions and investigating paper loading efficiency and orthogonality. Enhanced green fluorescent protein (EGFP) was used as a model protein to allow quantification of protein loading via fluorescence imaging. Two approaches were identified that showed significantly increased loading efficiencies compared with the previously applied conjugation strategy. Additionally, all four methods were proven orthogonal to each other, allowing simultaneous immobilization of a mixture of proteins to a premodified assembly of two paper sheets.

Construction and application of H1R ligand screening materials based on SMA stabilization and his-tag covalent immobilization of membrane proteins

The histamine H1 receptor (H1R) plays a pivotal role in allergy initiation and undergoes the necessity of devising a high-throughput screening approach centered on H1R to screen novel ligands effectively. This study suggests a method employing styrene maleic acid (SMA) extraction and His-tag covalent bonding to immobilize H1R membrane proteins, minimizing the interference of nonspecific proteins interference while preserving native protein structure and maximizing target exposure. This approach was utilized to develop a novel material for high-throughput ligand screening and implemented in cell membrane chromatography (CMC). An H1R-His-SMALPs/CMC model was established and its chromatographic performance (selectivity, specificity and lifespan) validated, demonstrating a significant enhancement in lifespan compared to previous CMC models. Subsequently, this model facilitated high-throughput screening of H1R ligands in the compound library and preliminary activity verification of potential H1R antagonists. Identification of a novel H1R antagonist laid the foundation for further development in this area.

Functionalization of Polydimethylsiloxane with Diazirine-Based Linkers for Covalent Protein Immobilization.

Biomolecule attachment to solid supports is critical for biomedical devices, such as biosensors and implants. Polydimethylsiloxane (PDMS) is commonly used for these applications due to its advantageous properties. To enhance the biomolecule immobilization on PDMS, a novel technique is demonstrated using newly synthesized diazirine molecules for the surface modification of PDMS. This nondestructive process involves a reaction between diazirine molecules and PDMS through C-H insertion with thermal or ultraviolet activation. The success of the PDMS modification is confirmed by various surface characterization techniques. Bovine serum albumin (BSA) and immunoglobulin G (IgG) are strongly attached to the modified PDMS surfaces, and the amount of protein is quantified using iodine-125 radiolabeling. The results demonstrate that PDMS is rapidly functionalized, and the stability of the immobilized proteins is significantly improved with multiple types of diazirine molecules and activation methods. Confocal microscopy provides three-dimensional images of the distribution of immobilized IgG on the surfaces and the penetration of diazirine-based linkers through the PDMS substrate during the coating process. Overall, this study presents a promising new approach for functionalizing PDMS surfaces to enhance biomolecule immobilization, and its potential applications can extend to multimaterial modifications for various diagnostic and medical applications such as microfluidic devices and immunoassays with relevant bioactive proteins.

A Simple and Versatile Strategy for Oriented Immobilization of His-Tagged Proteins on Magnetic Nanoparticles.

Oriented and covalent immobilization of proteins on magnetic nanoparticles (MNPs) is particularly challenging as it requires both the functionality of the protein and the colloidal stability of the MNPs to be preserved. Here, we describe a simple, straightforward, and efficient strategy for MNP functionalization with proteins using metal affinity binding. Our method involves a single-step process where MNPs are functionalized using a preformed, ready-to-use nitrilotriacetic acid-divalent metal cation (NTA-M2+) complex and polyethylene glycol (PEG) molecules. As a proof-of-concept, we demonstrate the oriented immobilization of a recombinant cadherin fragment engineered with a hexahistidine tag (6His-tag) onto the MNPs. Our developed methodology is simple and direct, enabling the oriented bioconjugation of His-tagged cadherins to MNPs while preserving protein functionality and the colloidal stability of the MNPs, and could be extended to other proteins expressing a polyhistidine tag. When compared to the traditional method where NTA is first conjugated to the MNPs and afterward free metal ions are added to form the complex, this novel strategy results in a higher functionalization efficiency while avoiding MNP aggregation. Additionally, our method allows for covalent bonding of the cadherin fragments to the MNP surface while preserving functionality, making it highly versatile. Finally, our strategy not only ensures the correct orientation of the protein fragments on the MNPs but also allows for the precise control of their density. This feature enables the selective targeting of E-cadherin-expressing cells only when MNPs are decorated with a high density of cadherin fragments.

Heterodimeric Protein Surface‐Coupling Platform: Immobilization of Conformation Switchable and Functional αIIbβ3 Integrin

AbstractMany biotechnologies require direct contact between biomolecules and (semi)solid substrates. However, little information is available regarding site‐directed covalent surface immobilization of heterodimeric proteins. Integrins are heterodimeric, conformationally dependent membrane adhesion‐receptors, which are important in the (patho)biology of almost all human diseases. In this study, a biomimetic‐platform is developed for covalent and site‐directed immobilization of oriented integrin heterodimers onto most hydroxyl bearing surfaces, herein demonstrated on both glass‐slides and silica‐particles. This platform consists of a self‐assembled monolayer, upon which C‐terminal modified αIIbβ3 integrin extracellular domains are oriented and covalently anchored. As with cell surface integrins, the conformation of immobilized αIIbβ3 is switchable and can be modulated to the active ligand‐binding conformation by divalent cations. Furthermore, the αIIbβ3‐coupled silica particles display platelet‐mimetic hemostat function and co‐aggregate with platelets from both wild‐type and fibrinogen/von Willebrand factor double deficient mice, facilitating αIIbβ3‐non‐classical ligand discoveries. This work provides a biomaterial platform for functional multimeric protein‐substrate coupling, which should have broad impact on multiple fields of biology, biotechnology, and clinical diagnosis/therapy.

Accessing Antibody Reactivities in Serum or Plasma to (Auto-)antigens Using Multiplexed Bead-Based Protein Immunoassays.

Antibody (AB) testing or serotesting for reactive ABs against antigenic proteins is broadly used. Parallel examination of many antigens is of high interest to identify autoantibodies (AAB) or differential antigenic reactivities in many biological settings like allergy and infectious autoimmune, cancerous, or systemic disease. The resulting AAB profiles can be used for diagnosis, prognosis, and monitoring of such conditions. Protein microarrays have been used for AB profiling over the past decade but show some significant limitations which make them unsuitable for clinical applications. Alternative multiplexing platforms such as bead arrays were shown to provide a versatile tool for the confirmation and efficient analysis of high numbers of biological samples. Luminex' bead-based xMAP technology combines advantages such as multiplexing and lower demand for sample volume and at the same time overcomes the challenges of microarrays. It works faster, shows better antigen stability, is more reproducible, and allows the analysis of up to 500 analytes in one sample well. In this chapter we introduce our established workflow for the use of the xMAP technology for AB profiling including an overview of the method principle and protocols for the covalent immobilization of proteins to the MagPlex beads, confirmation of protein coupling, the execution of a multiplexed bead-based protein immunoassay, and subsequent data handling.

Plasma Activation of Microplates Optimized for One-Step Reagent-Free Immobilization of DNA and Protein.

Activated microplates are widely used in biological assays and cell culture to immobilize biomolecules, either through passive physical adsorption or covalent cross-linking. Covalent attachment gives greater stability in complex biological mixtures. However, current multistep chemical activation methods add complexity and cost, require specific functional groups, and can introduce cytotoxic chemicals that affect downstream cellular applications. Here, we show a method for one-step linker-free activation of microplates by energetic ions from plasma for covalent immobilization of DNA and protein. Two types of energetic ion plasma treatment were shown to be effective: plasma immersion ion implantation (PIII) and plasma-activated coating (PAC). This is the first time that PIII and PAC have been reported in microwell plates with nonflat geometry. We confirm that the plasma treatment generates radical-activated surfaces at the bottom of wells despite potential shadowing from the walls. Comprehensive surface characterization studies were used to compare the PIII and PAC microplate surface composition, wettability, radical density, optical properties, stability, and biomolecule immobilization density. PAC plates were found to have more nitrogen and lower radical density and were more hydrophobic and more stable over 3 months than PIII plates. Optimal conditions were obtained for high-density DNA (PAC, 0 or 21% nitrogen, pH 3-4) and streptavidin (PAC, 21% nitrogen, pH 5-7) binding while retaining optical properties required for typical high-throughput biochemical microplate assays, such as low autofluorescence and high transparency. DNA hybridization and protein activity of immobilized molecules were confirmed. We show that PAC activation allows for high-density covalent immobilization of functional DNA and protein in a single step on both 96- and 384-well plates without specific linker chemistry. These microplates could be used in the future to bind other user-selected ligands in a wide range of applications, for example, for solid phase polymerase chain reaction and stem cell culture and differentiation.

Site-selective covalent immobilization of PPARγ using a label-free strategy for chromatographic study

The development of site-selective covalent protein immobilization strategies is crucial for many protein-based bioanalytical assays. Herein, we proposed a label-free, site-selective, covalent immobilization strategy for the functionalization of PPARγ on GW9662 modified macroporous silica gels. The gels were characterized by X-ray photoelectron spectroscopy (XPS) and scanning electron microscope (SEM). Molecular docking in combination with competitive study, adsorption energy distribution (AED) calculation, and frontal analysis were used to investigate the binding of ligands with GW9662 occupied PPARγ. Docking results indicated that rosiglitazone, pioglitazone, and gensenoside Rh1 could cooperatively bind to the receptor with GW9662. Competitive studies revealed that the three ligands bond with PPARγ through the same site in the presence of GW9662. AED calculation showed one type of binding site for the three ligands on the receptor. Due to the presence of GW9662, the affinities of the three ligands ((2.35 ± 0.05) × 105 M−1 for rosiglitazone, (1.27 ± 0.02) × 105 M−1 for pioglitazone, and (3.18 ± 0.13) × 105 M−1 for gensenoside Rh1) with PPARγ were one or two orders of magnitude smaller than the literature reported values. The good performance of the immobilized PPARγ provided an opportunity for application in receptor-ligand interactions and cooperative partner screening. Our strategy is, therefore, a promising way to realize the label-free, covalent immobilization of functional proteins.

Covalent Protein Immobilization on 3D‐Printed Microfiber Meshes for Guided Cartilage Regeneration

AbstractCurrent biomaterial‐based strategies explored to treat articular cartilage defects have failed to provide adequate physico‐chemical cues in order to guide functional tissue regeneration. Here, it is hypothesized that atmospheric‐pressure plasma (APPJ) treatment and melt electrowriting (MEW) will produce microfiber support structures with covalently‐immobilized transforming growth factor beta‐1 (TGFβ1) that can stimulate the generation of functional cartilage tissue. The effect of APPJ operational speeds to activate MEW polycaprolactone meshes for immobilization of TGFβ1 is first investigated and chondrogenic differentiation and neo‐cartilage production are assessed in vitro. All APPJ speeds test enhanced hydrophilicity of the meshes, with the slow treatment speed having significantly less CC/CH and more COOH than the untreated meshes. APPJ treatment increases TGFβ1 loading efficiency. Additionally, in vitro experiments highlight that APPJ‐based TGFβ1 attachment to the scaffolds is more advantageous than direct supplementation within the medium. After 28 days of culture, the group with immobilized TGFβ1 has significantly increased compressive modulus (more than threefold) and higher glycosaminoglycan production (more than fivefold) than when TGFβ1 is supplied through the medium. These results demonstrate that APPJ activation allows reagent‐free, covalent immobilization of TGFβ1 on microfiber meshes and, importantly, that the biofunctionalized meshes can stimulate neo‐cartilage matrix formation. This opens new perspectives for guided tissue regeneration.

Polyelectrolytes for Enzyme Immobilization and the Regulation of Their Properties.

In this review, we considered aspects related to the application of polyelectrolytes, primarily synthetic polyanions and polycations, to immobilize enzymes and regulate their properties. We mainly focused on the description of works in which polyelectrolytes were used to create complex and unusual systems (self-regulated enzyme–polyelectrolyte complexes, artificial chaperones, polyelectrolyte brushes, layer-by-layer immobilization and others). These works represent the field of “smart polymers”, whilst the trivial use of charged polymers as carriers for adsorption or covalent immobilization of proteins is beyond the scope of this short review. In addition, we have included a section on the molecular modeling of interactions between proteins and polyelectrolytes, as modeling the binding of proteins with a strictly defined, and already known, spatial structure, to disordered polymeric molecules has its own unique characteristics.

Tyrosine-Based Dual-Functional Interface for Trapping and On-Site Photo-Induced Covalent Immobilization of Proteins.

Tyrosine, a simple and well-available natural amino acid, is featured by the small size of the compound that contains multiple reactive groups. This study developed an efficient bioconjugation strategy using tyrosine-based dual-functional interfaces. When tyrosine molecules are immobilized on the surface of a supporting material through amino groups, their carboxyl groups can function as an attracting trap due to their anionic nature at neutral pH and ability to chelate nickel(II) ions (Ni2+), allowing the capture and enrichment of cationic proteins and histidine (His)-tagged proteins on the surface. The trapped proteins can be further covalently immobilized on site through ruthenium-mediated photochemical cross-linking, which has been found to be highly efficient and can be completed within minutes. This strategy was successfully applied to two different material systems. We found that tyrosine-modified agarose beads had a binding capacity of the His-tagged enhanced green fluorescent protein comparable to that of commonly used nitrilotriacetic acid-based resins, and further covalent coupling via dityrosine cross-linking achieved a yield of 85% within 5 min, without compromising much on its fluorescence activity. On the surface of tyrosine-modified 316L stainless steel, lysozyme was captured through electrostatic interaction and further immobilized. The resultant surface exhibited remarkable antibacterial activity against both Staphylococcus aureus and Escherichia coli. Such a tyrosine-based capture-then-coupling method is featured by its simplicity, high coupling efficiency, and high utilization rate of target molecules, making it particularly suitable for the proteins that are highly priced or vulnerable to general immobilization chemistry.

Biofunctionalization of Graphene-Based FET Sensors through Heterobifunctional Nanoscaffolds: Technology Validation toward Rapid COVID-19 Diagnostics and Monitoring.

The biofunctionalization of graphene field‐effect transistors (GFETs) through vinylsulfonated‐polyethyleneimine nanoscaffold is presented for enhanced biosensing of severe acute respiratory‐related coronavirus 2 (SARS‐CoV‐2) spike protein and human ferritin, two targets of great importance for the rapid diagnostic and monitoring of individuals with COVID‐19. The heterobifunctional nanoscaffold enables covalent immobilization of binding proteins and antifouling polymers while the whole architecture is attached to graphene by multivalent π–π interactions. First, to optimize the sensing platform, concanavalin A is employed for glycoprotein detection. Then, monoclonal antibodies specific against SARS‐CoV‐2 spike protein and human ferritin are anchored, yielding biosensors with limit of detections of 0.74 and 0.23 nm, and apparent affinity constants (KDGFET) of 6.7 and 8.8 nm, respectively. Both biosensing platforms show good specificity, fast time response, and wide dynamic range (0.1–100 nm). Moreover, SARS‐CoV‐2 spike protein is also detected in spiked nasopharyngeal swab samples. To rigorously validate this biosensing technology, the GFET response is matched with surface plasmon resonance measurements, exhibiting linear correlations (from 2 to 100 ng cm−2) and good agreement in terms of K D values. Finally, the performance of the biosensors fabricated through the nanoscaffold strategy is compared with those obtained through the widely employed monopyrene approach, showing enhanced sensitivity.

A “Clickable” Electrodeposited Polymer Films Based on 3‐Ethynylthiophene for the Covalent Immobilization of Proteins. Application to a Label‐free Electrochemical Immunosensor for Escherichia Coli and Staphylococcus Aureus Determination

AbstractAn original electrodeposited polymer film, based on 3‐ethynylthiophene, for covalent immobilization of proteins via “click” reaction of copper catalyzed azide‐alkyne cycloaddition was developed. The best characteristics have demonstrated the layer‐by‐layer immobilized 3,4‐ethylendioxythiophene‐3‐ethynylthiophene composite film. The bovine serum albumin and antibodies have been successfully immobilized on the surface of planar platinum electrode. The approach has been applied to label‐free electrochemical immunosensors for Escherichia coli and Staphylococcus aureus determination. The immunosensors demonstrated LODs 7.2 CFU/mL for Escherichia coli and 15.9 CFU/mL for Staphylococcus aureus, linear range 102–106 CFU/mL and could be promising for environmental and food control.

Magnetic Nanoplatforms for Covalent Protein Immobilization Based on Spy Chemistry.

Immobilization of proteins on magnetic nanoparticles (MNPs) is an effective approach to improve protein stability and facilitate separation of immobilized proteins for repeated use. Herein, we exploited the efficient SpyTag-SpyCatcher chemistry for conjugation of functional proteins onto MNPs and established a robust magnetic-responsive nanoparticle platform for protein immobilization. To maximize the loading capacity and achieve outstanding water dispersity, the SpyTag peptide was incorporated into the surface-charged polymers of MNPs, which provided abundant active sites for Spy chemistry while maintaining excellent colloidal stability in buffer solution. Conjugation between enhanced green fluorescence protein (EGFP)-SpyCatcher-fused proteins and SpyTag-functionalized MNPs was efficient at ambient conditions without adding enzymes or chemical cross-linkers. Benefiting from the excellent water dispersity and interface compatibility, the surface Spy reaction has fast kinetics, which is comparable to that of the solution Spy reaction. No activity loss was observed on EGFP after conjugation due to the site-selective nature of Spy chemistry. The immobilization process of EGFP on MNPs was highly specific and robust, which was not affected by the presence of other proteins and detergents, such as bovine serum albumin and Tween 20. The MNP platform was demonstrated to be protective to the conjugated EGFP and significantly improved the shelf life of immobilized proteins. In addition, experiments confirmed the retained magnetophoresis of the MNP after protein loading, demonstrating fast MNP recovery under an external magnetic field. This MNP is expected to provide a versatile and modular platform to achieve effective and specific immobilization of other functional proteins, enabling easy reuse and storage.

Targeting and Covalently Immobilizing the EGFR through SNAP-Tag Technology for Screening Drug Leads.

Membrane protein immobilization is particularly significant in in vitro drug screening and determining drug-receptor interactions. However, there are still some problems in the immobilization of membrane proteins with controllable direction and high conformational stability, activity, and specificity. Cell membrane chromatography (CMC) retains the complete biological structure of membrane proteins. However, conventional CMC has the limitation of poor stability, which results in its limited life span and low reproducibility. To overcome this limitation, we propose a method for the specific covalent immobilization of membrane proteins in cell membranes. We used the SNAP-tag as an immobilization tag fused to the epidermal growth factor receptor (EGFR), and Cys145 located at the active site of the SNAP-tag reacted with the benzyl group of O6-benzylguanine (BG). The SNAP-tagged EGFR was expressed in HEK293 cells. We captured the SNAP-tagged EGFR from the cell membrane suspension onto a BG-derivative-modified silica gel. Our immobilization strategy improved the life span and specificity of CMC and minimized loss of activity and nonspecific attachment of proteins. Next, a SNAP-tagged EGFR/CMC online HPLC-IT-TOF-MS system was established to screen EGFR antagonists from Epimedii folium. Icariin, magnoflorine, epimedin B, and epimedin C were retained in this model, and pharmacological assays revealed that magnoflorine could inhibit cancer cell growth by targeting the EGFR. This EGFR immobilization method may open up possibilities for the immobilization of other membrane proteins and has the potential to serve as a useful platform for screening receptor-binding leads from natural medicinal herbs.

Toward Fabrication of Bioactive Papers: Covalent Immobilization of Peptides and Proteins.

Herein, we report a novel two-step method for the covalent, site-directed, and efficient immobilization of proteins on lab-made paper sheets. First, paper fibers were modified with a peptidic anchor comprising enzyme recognition motifs. Four different conjugation strategies for peptide immobilization were evaluated with respect to reproducibility and fiber loading efficiency. After manufacturing of the peptide-preconditioned paper, oriented conjugation of the model protein tGFP containing a C-terminal recognition sequence for either sortase A or microbial transglutaminase was assessed semiquantitatively by fluorescence measurement and inspected by confocal laser scanning microscopy (CLSM). The two enzymes utilized for protein conjugation used the same oligoglycine peptide anchor, and both proved to be suitable for controlled oriented linkage of substrate proteins at physiological conditions.

Neurothreads: Development of supportive carriers for mature dopaminergic neuron differentiation and implantation

In this study we present the use of elastic macroporous cryogels for differentiation and transplantation of mature neurons. We develop a coating suitable for long-term neuronal culture, including stem cell differentiation, by covalent immobilization of neural adhesion proteins. In the context of cell therapy for Parkinson's disease, we show compatibility with established dopaminergic differentiation of both immortalized mesencephalic progenitors - LUHMES - and human embryonic stem cells (hESCs). We adjust structural properties of the biomaterial to create carriers - Neurothreads - favourable for cell viability during transplantation. Finally, we show feasibility of preservation of mature neurons, supported by Neurothreads, one month after in-vivo transplantation. Preliminary data suggests that the Neurothread approach could provide more mature and less proliferative cells in vivo.

Site-Specific Biofunctionalization of Cellulose and Poly(dimethylsiloxane): A Chemoenzymatic Approach for Surface Engineering.

Site-specific, covalent immobilization of protein is of great importance in the design of bioanalytical devices. User-defined covalent coupling of protein onto the surface has been primarily limited to a noncanonical amino acid or cysteine residues. It is desirable to develop a new approach for site-specific biofunctionalization. Herein, we demonstrate a robust and modular chemoenzymatic approach for site-specific, covalent grafting of proteins onto a surface. The synthetic strategy relies on the combination of surface amine functionalization, followed by sortase-mediated coupling. The developed method was validated by site-specific immobilization of two model proteins (glutathione S-transferase and green fluorescent protein) on cellulose and polydimethylsiloxane surfaces via a short recognition motif (LPETG). The covalent coupling of immobilized proteins at the interface was characterized by Fourier Transform Infrared Spectroscopy in attenuated total reflectance mode, X-ray photoelectron spectroscopy, atomic force microscope, and fluorescent microscopy. This enzymatic surface functionalization approach could permit an oriented, homogeneous, and site-specific covalent tethering of LPETG-tag proteins to other materials under mild conditions.

Covalent Protein Immobilization onto Muscovite Mica Surface with a Photocrosslinker

Muscovite mica with an amino silane-modified surface is commonly used as a substrate in atomic force microscopy (AFM) studies of biological macromolecules. Herein, the efficiency of two different protein immobilization strategies employing either (N-hydroxysuccinimide ester)-based crosslinker (DSP) or benzophenone-based photoactivatable crosslinker (SuccBB) has been compared using AFM and mass spectrometry analysis. Two proteins with different physicochemical properties—human serum albumin (HSA) and horseradish peroxidase enzyme protein (HRP)—have been used as model objects in the study. In the case of HRP, both crosslinkers exhibited high immobilization efficiency—as opposed to the case with HSA, when sufficient capturing efficiency has only been observed with SuccBB photocrosslinker. The results obtained herein can find their application in commonly employed bioanalytical systems and in the development of novel highly sensitive chip-based diagnostic platforms employing immobilized proteins. The obtained data can also be of interest for other research areas in medicine and biotechnology employing immobilized biomolecules.