Question

Write down the details (can be in the form of a diagram) for the immobilization of bovine serum albumin onto a glass surface. The immobilization strategies should meet the following requirements:

• Involve affinity and covalent linkages

• Involve succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) as heterobifunctional linker

Answer 1

IMMOBILIZATION OF BOVINE SERUM ALBUMIN ONTO A GLASS SURFACE:-

INTRODUCTION

Biomolecule (proteins, drugs, etc.) immobilization onto the surface of glass surface is one of the main and efficient methods which are used in biotechnology. Immobilized molecules have several advantages compared to free molecules. Specifically, they have a wide practical application not only in the nanocomposite field, as drug carriers, in drug screening, in membrane bioreactors,and in biosensor synthesis but also in wide branches of modern medical diagnostics, genomics, proteomics and affinity chromatography. Due to the significant properties of immobilized molecules, such as a high kinetic and thermody-namic stability and resistance to various environmental factors, they are suitable and valuable for new applications in industry and medicine.

Affinity:-

pH of 5.0, a temperature of 25°C, and an immersion time of 4 h. Our results also show that small changes in temperature or pH that can affect the coatings, since these changes will affect the orientation adopted by the BSA protein on the glass surface, which tends to reduce its free energy, but that due to these changes, it will affect its attractive interactions, hydrogen bonds, and entropy due to the release of counterions and solvent molecules.

Covalent Linkages:-

The method for efficient protein encapsulation is a cross-linkage of the protein to the surface via a linker by means of covalent bonding during a chemical reaction. Covalent bonds are frequently used in the immobilization mechanism for microfluidic assays. The immobilization surface is activated via reactive reagents. The activated surface reacts with amino acid residues on the protein exterior and forms an irreversible linkage. One tends to rely on covalent immobilization when high, stable protein coverage is required. Bifunctional spacer molecules are a common approach to form an irreversible bond between proteins and the immobilization surface. In such an approach, one end of a spacer molecule is covalently linked to an activated surface and then a protein is covalently linked to the other end of the spacer. Alternatively, another spacer or protein capture agent (e.g., streptavidin) is cross-linked on the other end. Unreacted active functional groups are blocked or deactivated. A covalent bond can be formed on the active sites of the proteins, resulting in reduced activity. This method allows creation of immunoassay protein chips. Covalent immobilization was used to attach albumin onto porous polyethylene membranes using strongly attached polydopamine as a spacer. A protein (bovine serum albumin, BSA)-coated polymer was also used as a matrix for enzyme immobilization. This protein-coated polymer provides a novel matrix for covalent immobilization of enzymes. Also, cross-linkage allows the synthesis of various materials for immunosorbent assays.

SMCC AS HETEROBIFUNCTIONAL LINKER:-

Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is a heterobifunctional amine-to-sulfhydryl crosslinker, which contains two reactive groups at opposite ends: N-hydroxysuccinimide-ester and maleimide, reactive with amines and thiols respectively. SMCC is often used in bioconjugation to link proteins with other functional entities (fluorescent dyes, tracers, nanoparticles, cytotoxic agent. For example, a targeted anticancer agent – trastuzumab emtansine (antibody-drug conjugate containing an antibody trastuzumab chemically linked to a highly potent drug DM-1) – is prepared using SMCC reagent.

Peptides were conjugated to aminated microwell surfaces through the specific reaction between C-terminal cysteines and the maleimide-activated surfaces, 10 mM SMCC was prepared in 16PBS buffer, pH 7.4. Next, 30 mLof SMCC was added into each aminated microwell and incubated for one hour at room temperature. The microwell plate was then briefly washed with pure water three times. Then, 30 mLof a 300 mM peptide solution, prepared in 16PBS pH 7.4 plus 1 mM TCEP, was added to the appropriate SMCC-activated microwells. The reaction was incubated for 4 hours at room temperature, in the dark. After the conjugation reaction was complete, the microwells were washed for 5 minutes in 16TBST, three times, followed by three washes in water. To immobilize the enzyme on peptide-modified surfaces, 30 mL of 25 nM biotin-labeled b-G always incubated in the peptide-modified microwells for two hours in 10 mM phosphate buffer, pH 7.3 with 100 mM MgCl2 and 0.05% Tween 20 (v/v%), at room temperature. The microwells were washed for 5 minutes in 16TBST, three times, followed by three washes in phosphate buffer. At this point, the b-Gal-bound microwells were ready for testing. Covalent attachment of b-Gal to NHS (N-Hydroxysuccinimide)-activated surfaces was performed using BS3 homogeneous amine-reactive cross-linker, as recommended by the manufacturer. First, 30 mL of 2 mg/mL BS3 prepared in 16PBS, pH 7.4 was incubated.