Method For Covalent Immobilization Research Articles

Carboxy-Functionalized Covalent Organic Framework as a Carrier for Lipase Immobilization and Its Application in Inhibitors Screening.

Covalent organic frameworks (COFs) with large specific surface areas, high porosity, good stability, and designable structure are promising carriers for immobilized enzymes. It is important to explore lipase inhibitors from natural foods as lipase inhibitors are closely related to the treatment of obesity. In this work, a carboxyl functionalized covalent organic framework (TpBD-3COOH) was prepared by solvothermal method for covalent immobilization of porcine pancreatic lipase (PPL) and obtained the enzyme-decorated COF (PPL@COF). The immobilized lipase showed wider pH and temperature tolerance with the same optimal pH and temperature of 7.5 and 50 ℃ compared to free lipase. After 6 successive reuses, the PPL@COF maintained 53.0% of its original activity. Immobilized lipase also displayed enhanced storage stability (55.4% after 14days at 4 ℃). When p-nitrophenyl acetate was applied as the substrate, the calculated Michaelis constant was 3.57mM and the half maximal inhibitory concentration of orlistat was 3.20μM. Finally, the PPL@COF was used for enzyme inhibitors screening from natural foods combined with UV spectrophotometry, and Hawthorn was screened for excellent lipase inhibitory activity.

Continuous preparation of low-phenylalanine formulations by treatment of edible protein with immobilized phenylalanine ammonia lyase

Dietary restriction of phenylalanine is the predominant treatment for phenylketonuria. However, the currently available medicated formulations depend on an arduous and unsustainable chemical extraction procedure, which inflates the product’s price. Here, we describe a method that uses immobilized phenylalanine ammonia lyase (PAL) and offers a sustainable solution to generate a low-phenylalanine protein supplement for phenylketonuria patients. This enzyme deaminates phenylalanine to trans-cinnamic acid. Here, we optimized the high cell density cultivation of a recombinant Escherichia coli strain harboring Anabaena variabilis PAL (AvPAL) and achieved protein titers greater than 400 mg/l. This AvPAL protein was immobilized on multiwall carbon nanotubes, and the bioconjugate retained > 50 % activity after ten successive cycles with significantly improved thermostability. Batch deamination with the bioconjugate showed an 80 % reduction in the total phenylalanine content of soya protein hydrolysate. In a continuous packed bed column, > 98 % of the phenylalanine content was depleted at a feed flow rate of 0.1 ml/min, resulting in a space-time yield of cinnamate of 6.65 g/l/h. In a 72-h continuous run, the bioconjugate lost only 2 % of its activity with an operational inactivation constant of 3.9 × 10−5 min−1. This simple covalent immobilization method offers the potential to generate low-phenylalanine diet for phenylketonuria patients.

Incorporation of metal-organic framework MOFs-5 into the polymer monolith via the surface covalent immobilization method for enhanced capillary liquid chromatographic separation of benzene homologues

In this study, metal organic framework material MOFs-5 was immobilized covalently on the surface of polymer monolith to improve the separation efficiency of small molecular compounds in the mode of capillary liquid chromatography (cLC) firstly. Scanning electron microscopy, fourier transform infrared spectroscopy and flow-through property test were applied to characterize the novel [email protected] monolith, confirming that MOFs-5 was effectively immobilized on the surface of polymer monolith via covalent reactions. More importantly, Benzene homologues (benzene and phenol) which could be separated by the commercialized C18 packed chromatographic column have been well separated via the [email protected] monolith. By contrast, there wasn't any separation between benzene and phenol via the original polymer monolith. Moreover, the relative standard deviation was less than 2.2 % for run-to-run. These results showed that the [email protected] monolith is a kind of potential commercialized material for small molecular compound separation in the mode of cLC.

Investigation of adsorption and biosorption features of bio-functionalized poly(GMA-Co-EGDMA) polymer beads in the treatment of nicotine from tobacco industry

The investigation of multifunctional materials for modern enzyme immobilization is an attractive subject in advanced adsorption and biosorption applications. In the present study, the feasibility of immobilization of Lipozyme TL 100L (LPZM) on 3-aminopropyltriethoxysilane (APTES) modified poly-(GMA-co-EGDMA) (PEGDMA) was investigated for adsorption and biosorption of nicotine from aqueous solution. Characterization tests confirmed successful immobilization of lipozyme which significantly altered thermal behavior, surface characteristics, and surface morphology of PEGDMA and PEGDMA/APTES. In addition, the immobilization yields were calculated as 85.0% and 72.0% onto PEGDMA/APTES using physical adsorption and covalent immobilization methods, respectively. The nicotine removal efficiencies were calculated to be 66.4%, 79.0%, 98.9%, and 85.7%, using raw PEGDMA, PEGDMA/APTES, PEGDMA/APTES@LPZM, and PEGDMA/APTES/GU@LPZM, respectively. For the raw PEGDMA, the Langmuir isotherm was best fitted to the adsorption data, while Langmuir-Freundich model described well the adsorption process on PEGDMA/APTES and PEGDMA/APTES@LPZM. The maximum adsorption capacities of Langmuir-Freundlich model increased from 8.118 to 17.32mg/g after enzyme immobilization. The negative enthalpy value, ΔH° (- 10.37kJ/mol), revealed that the nicotine adsorption on PEGDMA/APTES@LPZM was exothermic in nature, which was corroborated by the decrease observed in the number of adsorbed molecules with increasing temperature. In the kinetic experiments, the adsorption on PEGDMA and PEGDMA/APTES@LPZM reached equilibrium with the removal percentages as 66.4% and 98.9% at the end of 3h, respectively. The nicotine adsorption performances in real water matrices were also investigated, and PEGDMA/APTES@LPZM showed satisfactory reusability with removal percentage decreased from 98.9% (1st cycle) to 83.0% (6th cycle).

Isocyanide-based multi-component reactions for carrier-free and carrier-bound covalent immobilization of enzymes.

Strategies for the covalent immobilization of enzymes depend on the type of functional group selected to perform the coupling reaction, and on the relative importance of selectivity, loading capacity, immobilization time and activity/stability of the resulting immobilized preparation. However, no single strategy is applicable for all covalent immobilization methods or can meet all these criteria, exemplifying the challenge of introducing a versatile process broadly compatible with the residues on the surface of proteins and the functional groups of common linkers. Here, we describe the use of isocyanide-based multi-component reactions for the carrier-bound and carrier-free covalent immobilization of enzymes. Isocyanide-based multi-component reactions can accept a wide variety of functional groups such as epoxy, acid, amine and aldehyde, as well as many commercially available bi-functional linkers, and are therefore suitable for either covalent coupling of enzymes on a solid support or intermolecular cross-linking of enzymes. In this strategy, an isocyanide is directly added to the reaction medium, the enzyme supplies either the exposed amine or carboxylic acid groups, and the support (in carrier-bound immobilization) or the bi-functional cross-linking agent (in carrier-free immobilization) provides another reactive functional group. The protocol offers operational simplicity, high efficiency and a notable reduction in time over alternative strategies, and can be performed by users with expertise in chemistry or biology. The immobilization reactions typically require 1-24 h.

Electrochemical DNA Biosensor Based on Immobilization of a Non-Modified ssDNA Using Phosphoramidate-Bonding Strategy and Pencil Graphite Electrode Modified with AuNPs/CB and Self-Assembled Cysteamine Monolayer

The present paper describes an alternative approach to the traditionally used covalent immobilization methods that require cost-intensive and complicated chemistry modification of a single-stranded DNA (ssDNA) capture probe. The low-cost pencil graphite electrode (PGE) modified with carbon black (CB) and gold nanoparticles (AuNPs) was used as an electrochemical platform and the non-modified ssDNA was immobilized on a self-assembled cysteamine modified AuNPs/CB-PGE through a phosphoramidate bond between the 5'-terminal phosphate group of ssDNA and the primary amine group of cysteamine. The microRNA-21 was used as a target model in the fabrication of this electrochemical DNA biosensor and the hybridization process with the complementary probe was monitored by differential pulse voltammetry using methylene blue (MB) as an electrochemical hybridization indicator. The decreased reduction peak current of MB shows a good linear correlation with the increased concentration of microRNA-21 target sequences because the MB signal is determined by the amount of exposed guanine bases. The linear range of the fabricated DNA biosensor was from 1.0 × 10-8 to 5.0 × 10-7 M with a detection limit of 1.0 × 10-9 M. These results show that the covalent immobilization of a non-modified ssDNA capture probe through a phosphoramidate-bonding strategy could serve as a cost-effective and versatile approach for the fabrication of DNA biosensors related to a wide range of applications that cover the fields of medical diagnostic and environmental monitoring. The fabricated electrochemical DNA biosensor was used to analyze microRNA-21 in a (spiked) human serum sample and it showed satisfactory and encouraging results as an electrochemical DNA biosensor platform.

Fabrication of gentamicin loaded Col-I/HA multilayers modified titanium coatings for prevention of implant infection.

In this study, gentamicin loaded collagen I/hyaluronic acid multilayers modified titanium coating (TC-AA(C/H)6-G) was fabricated via a layer-by-layer (LBL) covalent immobilization method. The drug releasing properties of collagen I/Hyaluronic acid (Col-I/HA) multilayers and the effect of loaded gentamicin on the antibacterial properties and cytocompatibility of modified TC were investigated. The gentamicin release assay indicated that the Col-I/HA multilayers modified TC exhibited agreeable drug-loading amount (537.22 ± 29.66µg of gentamicin) and controlled-release performance (240h of sustained release time). TC-AA(C/H)6-G revealed satisfactory antibacterial activity and inhibited the colonization and biofilm formation of S. aureus. Fortunately, the functions of hMSCs on TC-AA(C/H)6-G did not affected by the loaded gentamicin, and TC-AA(C/H)6-G could improve the adhesion, proliferation and osteogenic differentiation of cells, as well as TC-AA(C/H)6. In vivo animal study indicated that TC-AA(C/H)6-G could effectively control intramedullary cavity infection caused by S. aureus and prevent bone destruction.

Site-Specific Covalent Immobilization of Methylobacterium extorquens Non-Blue Laccse Melac13220 on Fe3O4 Nanoparticles by Aldehyde Tag

In the present study, the non-blue laccase Melac13220 from Methylobacterium extorquens was immobilized using three methods to overcome problems related to the stability and reusability of the free enzyme: entrapment of the enzyme with sodium alginate, crosslinking of the enzyme with glutaraldehyde and chitosan-, and site-specific covalent immobilization of the enzyme on Fe3O4 nanoparticles by an aldehyde tag. The site-specific covalent immobilization method showed the highest immobilization efficiency and vitality recovery. The optimum temperature of Melac13220 was increased from 65 °C to 80 °C. Immobilized Melac13220 showed significant tolerance to some organic solvents and maintained approximately 80% activity after 10 cycles of use. Differential scanning calorimetry (DSC) indicated that the melting temperature of the enzyme was increased (from 57 °C to 79 °C). Immobilization of Melac13220 also led to improvement in dye decolorization such that Congo Red was completely decolorized within 10 h. The immobilized enzyme can be easily prepared without purification, demonstrating the advantages of using the aldehyde tag strategy and providing a reference for the practical application of different immobilized laccase methods in the industrial field.

Roles of Self-Assembly and Secondary Structures in Antimicrobial Peptide Coatings

Antimicrobial peptide (AMP) coatings are promising alternatives to conventional antibiotics for the prevention of medical device- and implant-associated infections. Compared to covalent immobilization methods, coatings relying on physical interactions are more versatile but usually less stable. Previous work has developed stable noncovalent coatings on titanium and hydroxyapatite with a model AMP, GL13K, leveraging the strong hydrogen bonding between β-sheet-formed self-assemblies and polar substrates. In this work, a different GL13K self-assembly process was triggered with the formation of α-helices in ethanol/water cosolvent. We compared three different coatings on titanium to investigate the roles of self-assembly and secondary structures, including free GL13K in unordered structures, self-assembled GL13K with the formation of α-helices, and self-assembled GL13K with the formation of β-sheets. X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and water contact angle results confirmed the successful coatings of all three physiosorbed GL13K conditions. Self-assembled GL13K, either in α-helices or β-sheets, formed more effective antimicrobial coatings in killing Gram-positive Staphylococcus aureus than free GL13K. These findings could help design more stable and effective antimicrobial coatings using self-assembled AMPs.

Affinity monolith chromatography: A review of general principles and recent developments.

Affinity monolith chromatography (AMC) is a liquid chromatographic technique that utilizes a monolithic support with a biological ligand or related binding agent to isolate, enrich, or detect a target analyte in a complex matrix. The target-specific interaction exhibited by the binding agents makes AMC attractive for the separation or detection of a wide range of compounds. This article will review the basic principles of AMC and recent developments in this field. The supports used in AMC will be discussed, including organic, inorganic, hybrid, carbohydrate, and cryogel monoliths. Schemes for attaching binding agents to these monoliths will be examined as well, such as covalent immobilization, biospecific adsorption, entrapment, molecular imprinting, and coordination methods. An overview will then be given of binding agents that have recently been used in AMC, along with their applications. These applications will include bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, and dye-ligand or biomimetic affinity chromatography. The use of AMC in chiral separations and biointeraction studies will also be discussed.

Exploring the structural and catalytic features of lipase enzymes immobilized on g-C3N4: A novel platform for biocatalytic and photocatalytic reactions

The exploration of multifunctional supporting materials for modern enzyme immobilization is an attractive subject for advanced catalytic applications. In the present study, lipase enzyme types, namely palatase 20,000 L, lipozyme TL100L, and lipozyme CALB, were immobilized on graphitic carbon nitride (g-C3N4) using physical and covalent immobilization methods in order to obtain biohybrids for utilization in biocatalytic and photocatalytic reactions. Characterization tests confirmed successful immobilization of enzymes without changing the crystal phase of g-C3N4. The immobilization yields were calculated as 71.0% and 93.4% for C3N4@PLTS; 81.3% and 95.4% for C3N4@LPZYM; and 79.4% and 91.7% for C3N4@CALB biohybrids using physical adsorption and covalent bonding methods, respectively. The kinetic constant values of Km and Vmax were significantly higher for immobilized lipases than for free forms. The photocatalytic efficiencies of biohybrid catalysts were also greater than raw g-C3N4. Among the lipase types, lipozyme TL100L attached on g-C3N4 exhibited the best catalytic performance in both biocatalytic and photocatalytic experiments, which was attributed to its open lid structure. As a result, this study opens the door to utilizing g-C3N4 as a support material for the immobilization of different enzymes and establishing catalyzed reactions.

Enhanced Stability of Long-Living Immobilized Recombinant β-d-N-Acetyl-Hexosaminidase A on Polylactic Acid (PLA) Films for Potential Biomedical Applications.

β-d-N-acetyl-hexosaminidase (Hex, EC 3.2.1.52) is an acid hydrolase that catalyzes the cleavage of the β-1,4 bond in N-acetyl-d-galactosamine (Gal-NAc) and N-acetyl-d-glucosamine (Glc-NAc) from the non-reducing end of oligosaccharides and glycoconjugates. It is widely expressed in both the prokaryotic and eukaryotic world, where it performs multiple and important functions. Hex has antifungal activity in plants, is capable of degrading many biological substrates, and can play an important role in the biomedical field for the treatment of Tay-Sachs and Sandhoff diseases. With the aim being able to obtain a device with a stable enzyme, a method of covalent immobilization on polylactic acid (PLA) films was developed for the A isoform of the β-d-N-acetyl-hexosaminidase enzyme (HexA), produced in a recombinant way from Human Embryonic Kidney-293 (HEK-293) cells and suitably purified. An in-depth biochemical characterization of the immobilized enzyme was carried out, evaluating the optimal temperature, thermal stability, pH parameters, and Km value. Moreover, the stability of the enzymatic activity over time was assessed. The results obtained showed an improvement in terms of kinetic parameters and stability to heat for the enzyme following immobilization and the presence of HexA in two distinct immobilized forms, with an unexpected ability for one of them to maintain its functionality for a long period of time (over a year). The stability and functionality of the enzyme in its immobilized form are therefore extremely promising for potential biotechnological and biomedical applications.

Carboxyethyl-functionalized 3D porous polypyrrole synthesized using a porogen-free method for covalent immobilization of urease

The immobilization of enzymes onto porous supports is a common strategy for obtaining improved stability, fast product separation, enzyme reusability, and, ultimately, lower operating costs. Therefore, the development of new supports with specific surface functionalities that enable the covalent attachment of enzymes is of significant interest. Herein, stable three-dimensional (3D) porous materials were synthesized from polypyrrole by a simple template approach and used as an immobilization support for urease. The template method entails the use of polypyrrole nanoparticle building blocks, onto which a carboxylic acid-functionalized pyrrole monomer was polymerized, forming a 3D porous structure with tunable pore size distribution. Scanning electron microscopy (SEM) images, together with static light scattering (SLS), revealed the 3D porous nature of the materials. The properties of both the supports and the immobilized enzyme were characterized using a combination of techniques. The ability of the bioconjugated urease to catalyze the hydrolysis of urea into carbon dioxide and ammonia was then tested. The immobilized enzyme exhibited good catalytic activity, stability and reusability. Overall, these results suggest that such 3D porous materials with chemically accessible surfaces have considerable potential for use as biocatalyst supports.

A graphene oxide-molybdenum disulfide composite used as stationary phase for determination of sulfonamides in open-tubular capillary electrochromatography.

A graphene oxide-molybdenum disulfide (GO-MoS2) composite was synthesized and utilized as the highly efficient stationary phase of open-tubular capillary electrochromatography (OT-CEC). The characterization results indicated that GO-MoS2 composite was successfully synthesized. The GO-MoS2-coated capillary column was prepared by covalent immobilization method for the determination of seven sulfonamides. The baseline separation of seven sulfonamides was achieved by GO-MoS2-coated capillary column. The linear range was 0.05-100 μg/mL for sulfisomidine, sulfathiazole, sulfamerazine, phthalylsulfathiazole and sulfacetamide, 0.1-100 μg/mL for sulfamonomethoxine and sulfachloropyridazine with a satisfactory correlation coefficients (R2) >0.9994. This developed OT-CEC method was successfully applied to determinate of seven sulfonamides in environmental water and milk samples with good recoveries of 85.77% - 109.10% and 80.03% - 109.97%, respectively. These results indicated that GO-MoS2-coated capillary column possessed good stability and repeatability.

Детонационный наноалмаз как перспективный носитель лекарственных веществ

Обобщены сведения о применении детонационного наноалмаза (ДНА) в биомедицинских приложениях. Приведены литературные и собственные данные по получению ДНА с биологически активными веществами и распределении частиц наноалмаза in vivo. Рассмотрены методы адсорбционной и ковалентной иммобилизации биологически активных веществ на поверхности ДНА. Выделены основные направления развития в области создания на основе ДНА систем доставки биологически активных и лекарственных веществ (ЛВ). Привлекательность биомедицинских приложений ДНА обусловлена совокупностью его свойств: биосовместимостью, возможностью конструирования функциональной поверхности частиц, сильной и специфической адсорбцией/конъюгированием с ЛВ и биомолекулами, большой удельной площадью поверхности, наличием стабильной флуоресценции.

Surface density dependent orientation and immunological recognition of antibody on silicon: TOF-SIMS and surface analysis of two covalent immobilization methods

Antibodies are commonly used as detection elements in biosensors. Antibody orientation on transducer surface determines immunological recognition and biosensor performance. Although a relation between antibody orientation and adsorbed amount Γ was predicted, assuming close packing of proteins, and even applied to infer the preferred orientation from indirect surface analysis, the issue has not been so far examined experimentally with the surface techniques probing directly antibody orientation for a wide Γ range covering all possible antibody orientations. In this work, time-of-flight secondary ion mass spectrometry combined with Principal Component Analysis probes the orientation of immunoglobulin G (aIgG) immobilized on silane-modified silicon surface with surface density 0 ≤ Γ ≤ 3 mg/m2, determined with ellipsometry. Two covalent immobilization methods: with NHS-silane and with amino-silane followed by glutaraldehyde (GA) activation, are compared. Atomic Force Microscopy reveals surface density dependent nanostructure of aIgG layers. AFM and PCA of TOF-SIMS clearly distinguish between the Γ ranges of flat-on, side-on and vertical aIgG orientation, that accord with random molecular packing. For vertical aIgG arrangement, a dominant head-on orientation and a mixed tail-on/head-on orientation is concluded from PCA for GA- and NHS-surface modification, respectively. In addition, molar binding ratio of antigen to antibody accords with the determined surface density dependent aIgG orientation.

Understanding gold nanoparticles interactions with chitosan: Crosslinking agents as novel strategy for direct covalent immobilization of biomolecules on metallic surfaces

The development of a new method for covalent immobilization of biomolecules on the surface of bare gold nanoparticles (AuNPs) by using crosslinking agents in one step is presented. A very compatible biopolymer such as chitosan has been used as a target molecule to probe the viability of the proposed methodology. Click chemistry, based on biocompatible reactions and coupling with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide allow to analyze the covalent interactions between the metal nanoparticles and the biopolymer. Spectra deconvolution technique and Zeta potential measurements confirm the covalent interaction. The present study allows to quantify the proportion of AuNPs covalently adhered to the chitosan, which depends on the solution pH. The obtained results indicates that covalent interactions can be increased up to 25% in relation to total system interactions, which are mostly electrostatic. The proposed strategy opens up a new pathway for biomedical applications because the control of the chemical linkage can be directly performed on the nanoparticle surface without using any molecular intermediate, which may improve the encapsulation efficiency on drug delivery therapies.

Surface treatments and functionalization of metal‐ceramic membranes for improved enzyme immobilization performance

AbstractBACKGROUNDEnzyme immobilization in porous membranes often improves enzyme performance. This work reports the preparation and characterization of robust and scalable asymmetric metal‐ceramic microfiltration membrane. The surface of the porous metal‐ceramic membrane was treated by impregnation with a ceramic oxide for enzyme adsorption and corrosion protection. Finally, enzyme immobilization in the support was investigated.RESULTSThe bilayer membrane was successfully fabricated by combining a ceramic microfiltration layer with a metal support by tape casting, lamination and co‐sintering. A pore size in the ceramic microfiltration layer of 0.4 μm resulted in high water permeability (12 000 L/(m2 h bar)). Two different surface treatments were compared: heat treatment and yttrium(III) oxide (Y2O3) impregnation. Corrosion stability tests under enzyme‐relevant conditions gave no detectable chemical or structural changes. Alcohol dehydrogenase (EC 1.1.1.1) was immobilized in the membrane by physical adsorption and by two covalent immobilization methods. Covalent immobilization significantly improved enzyme loading, activity, and recyclability. Membrane reuse by heat treatment removed fouling, but decreased immobilization performance.CONCLUSIONThe improved microstructure obtained by Y2O3‐impregnation had a significant effect on enzyme loading yield and activity. This indicates the potential of this surface modification method and of these metal‐supported ceramic membranes in enzyme immobilization. Covalent immobilization was superior. © 2019 Society of Chemical Industry

A facile route to preparation of immobilized cellulase on polyurea microspheres for improving catalytic activity and stability

Abstract The input-output ratio of the immobilization process is crucial to determining whether the immobilized cellulase can truly participate in industrial production. This study reports a simple, efficient and environmentally friendly method for covalent immobilization of cellulase on uniform, monodisperse polyurea microspheres. The reported method offers several benefits including (i) polyurea microspheres can be obtained after one step reaction without any shaking or stirring at room temperature, (ii) Without any modification, the surface of polyurea microspheres contains abundant amine groups for immobilized cellulase, (iii) Compared with porous materials, cellulase was immobilized on the surface of the support, leading to less mass transfer resistance during immobilization and catalytic reaction. The support and immobilized cellulase were characterized by SEM, TEM, AFM, FT-IR, TGA. The immobilized cellulase demonstrated a high recovery of activity (77.9%) and showed improved thermostability, pH tolerance, reusability and lifetime. Notably, the immobilized cellulase exhibited a superior performance after 36 h in the continuous process. More interestingly, trace amounts of dimethyl sulfoxide was found to promote cellulase activity. This work provides a simple and practically method for promising applications as biocatalyst in cellulose hydrolysis and an effective strategy for the immobilization of other enzymes in green chemical industry.

A new biosensor for osteoporosis detection

Osteoporosis is a disease that is characterized by deterioration of bone tissue and increased risk of fracture as it leads to a decrease in bone mineral density, which is an important public health problem. Today, bone mineral density is measured by radiological techniques. Alternative techniques are needed because of the disadvantages such as excessive radiation intake, the cost of radiological techniques, and the necessity for specialist personnel for the devices. The quantitative determination of biochemical markers that play a role in bone mineralization may be a good alternative for the osteoporosis diagnosis and especially in the follow-up of treatment.In this study, a specific and sensitive immunological biosensor, which quantitatively determines the osteocalcin molecule, has been developed to be used in the early osteoporosis diagnosis and to evaluate the response to the drug treatment. Anti-osteocalcin antibody was immobilized onto gold electrode surface via covalent immobilization method by using 6-mercaptohexanol, 1,4-butanedioldiglycidyl ether, ethanolamine, and glutaraldehyde. Immobilization steps and biosensor characterization were specified by cyclic voltammetry and electrochemical impedance spectroscopy. The detection time and range of Ocn biosensor were determined as 45 min and 10–60 pg µL−1 Ocn concentration, respectively. The Ocn biosensor was successfully applied in artificial serum samples spiked with Ocn.