NORYATI BINTI MOHAMED SAPARI (HE110189)

BIOSENSOR (MEU 10403)

Assignment 1
Questions:-

1. Select a study on immobilization techniques for 3 types of biological components of enzyme

immobilization, peptide immobilization and antibody immobilization. Explain the study and the immobilization technique used. 2. Explain what is SAM (Self Assembled Monolayer)? Give and explain an example study. Remark: To be answer minimum in 1 page and maximum in 5 pages. Answers:-

1. Antibody Immobilization has been chosen for the study of biological components
immobilization for question 1 through a study taken from SPIE Digital Library website with the title of Micro Amperometric Immunosensor by Antibody Immobilizing with Electropolymerized Protein A as attached in Attachment 1. Introduction: In order to make a viable biosensor, the biological component has to be properly attached to the working electrode of a transducer. This process is known as immobilization of those particular biological materials. There are 5 common methods used for biological components immobilization, as follows:i. Adsorption

ii.

the process by which the biological materials being adsorbed on the surfaces of a substance (noble metals, charcoal, clay, cellulose, glass) through either physical adsorption (physisorption) or chemical adsorption (chemisorptions). physisorption is usually weak bonding and occurs via the formation of Van der Waals bonds, occasionally with hydrogen bonds or charge-transfer process. chemisorption is much stronger and involves the formation of covalent bonds. the most generally used model equation to describe adsorption is Langmuir Adsorption Isotherm. weakness of this method is adsorbed biological components is very susceptible to environments changes such as changes in pH, temperature, ionic strength and the substrate itself. this method is satisfactory for short-term investigations only.

Microencapsulation

iii.

an inert membrane (cellulose acetate dialysis membrane, polycarbonate, collagen, PTFE, synthetic polymer, nafion and polyurethane) is used to trap the biological components on the working electrode of the transducer. advantages are limit contamination, biodegradation, high degree of specificity, good stability to environments changes.

Entrapment

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NORYATI BINTI MOHAMED SAPARI (HE110189)

the biological components is mixed with polymer monomer solution which is then polymerized to a polymeric gel and trapping the biological components within the gel matrix. the most common polymer used is polyacrylamide and it is prepared by copolymerization of acrylamide with N,N-methylenebisacrylamide. polymerization can effected by UV radiation in the presence of vitaman B1 as photosensitiser. Other materials used are starch gels, nylon, silastic gels and conducting polymer such as polypyrroles which are particularly useful with working electrode of the transducer. disadvantage is large barrier are created, inhibiting the diffusion of the substrate, which slow the reaction thus increase the response time of the sensor. also lost of biological materials activity through the pores in the gel matrix but this problem can be overcome by cross-linking with e.g., glutaraldehyde.

iv.

Cross-linking the biological components is chemically bonded to solid supports or to another supporting material such as gel. bifunctional reagents such as glutaraldehyde are used as a binding agent to bind the biological materials to the solid supports. disadvantanges of this method it caused limitation diffusion of the substrate, damage to the biological components and poor mechanical strength. advantage, it is a useful method to stabilise adsorbed biological components.

v.

Covalent bonding

some functional groups of biological materials which are unused or are not essential for the catalytic activities (from nucleophilic groups in the amino acids of the biological components) can be covalently bonded to the support matrix such as support matrix from carboxyl group reacted with a carbodiimide. constrains was the reaction must work at low temperature, low ionic strength and in neutral pH environment. advantage is the biological components will not be lost or released from the biosensor during use.

Overall, the lifetime of the biosensor is expended with a proper immobilization method used. Typical lifetime of the same biosensor with different method of immobilization used are indicated as follows:a. b. c. d. Adsorption 1 day. Membrane entrapment/microencapsulation 1 week. Physical entrapment (cross-linking) 3 to 4 weeks. Covalent entrapment (covalent bonding) 4 to 14 months.

References (for both questions):1. Biosensors An Inroduction, 1997 (Brian Eggins, John Wiley & Sons) 2. Wikipedia, the free encyclopedia (http://en.wikipedia.org) 3. IEEE Journals through UTHM Library 4. ScienceDirect Journals through UTHM Library 5. www.thefreedictionary.com

6. 7. 8.

Elsevier Journals on Biosensor & Bioelectronics (www.elsevier.com) Youtube (TutorTom10 on basic on atoms) Youtube (Campbellteaching on basic of cell biology)

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NORYATI BINTI MOHAMED SAPARI (HE110189)

Summarization of immobilization technique used in the chosen study: From the study chosen, it can be summarizing as below (refer Attachment 1):a. The objectives of the study are:i. To fabricate a micro plane amperometric immunosensor for detection of human immunoglobulin G (HIgG) on silicon wafer based on MEMS (Micro-ElectroMechanical Systems) technology which is small in size, less in reagent consumption, easy integrated with IC and highly sensitive to quantitatively assay antigen and antibody. ii. To investigate the effectiveness of electrochemical polymerization immobilization technique as a new method for the orientation-controlled immobilization of antibody by using pre-coupling with staphylococcal protein A (SPA) and polypyrrole (PPy) as the foundation in co-electropolymerize biomolecule and polymer at the working electrode surface (only 1mm 2) for the antibody attachment. Immobilized antibody used in this study is goat antiimmunoglobulin G (goat anti-HIgG). b. The antibody immobilization process in the study details as below:i. First the micro amperometric immunosensor were fabricated on silicon wafer by using standard silicon micromachining process with outcome of the design is a much smaller microsensor compare to the size of conventional amperometric immunosensor and this microsensor only needs a few microlitre of reagents for detection process. ii. Then the polypyrrole (PPy) conductive polymer was electropolymerized onto the sensitive area of the working electrode on the pre-treatment fabricated micro amperometric immunosensor. This PPy will become a transition layer for staphylococcal protein A (SPA) immobilization. iii. Then the staphylococcal protein A (SPA) will run a co-electropolymerized with PPy for SPA attachment on PPy on the working electrode. These steps were taken for minimising SPA denaturation caused by direct contacting with the working electrode surface. These actions also increase more active adhesion sites for SPA for effectiveness of antibody attachment onto SPA. iv. Then the antibody was attached to the working electrode that already coated with co-electropolymerized PPy and SPA by a single drop of 1l antibody with 250g/ml of concentration to form the functional groups binding with the SPA. The Fc fragment of the antibody will specifically bind with the SPA for completing the antibody immobilization attachment process on the micro amperometric immunosensor working electrode. v. Finally, 1l of bovine serum albumin (BSA) was dropped on the same working electrode and incubated at 4C for 2 hours to prevent non-specific adsorption onto the working electrode and afterwards rinsed with phosphate buffered saline (PBS). vi. The completed fabricated micro immunosensor with antibody immobilization attachment were stored at 4C.

c. The completed fabricated micro amperometric immunosensor in this study were tested

with immunoglobulin G (HIgG) through sandwich assay procedure with enzyme labelled antibody (horseradish peroxidase HRP conjugated goat anti-HIgG) for catalytic reaction to take place and fulfil its design purpose.

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NORYATI BINTI MOHAMED SAPARI (HE110189)

2. Self Assembled Monolayer (SAM) Introduction: Self assembled monolayer (SAM) is a formation induced by the strong chemisorptions (chemical adsorption - is a sub-class of adsorption, driven by a chemical reaction occurring at the exposed surface) between the substrate and head group of selected organic molecule provides one of the most elegant approaches towards making ultra-thin organic films of controlled thickness (Ulman, 1991; Dubois and Nuzzo, 1992; Bain and Whitesides, 1989a). Self assembled related to molecules which can be defined as the process by which molecules adopt a defined arrangement without guidance or management from an outside source. SAM is related to intermolecular self assembly from the 2 types of molecular self assembly. The other one more type of molecular self assembly is intramolecular self assembly which is commonly called folding. The monolayer films produced by self-assembly allows tremendous flexibility with respect to several application depending upon their terminal functionality (functional group) either hydrophilic or hydrophobic control or by varying the chain (tail) length which give distance control. For example, SAM of long chain of alkanethiols (the most commonly used molecules for SAMs formation) produced highly packed and ordered surface, which can provide a membrane like micro-environment which useful for immobilising biological components. The high selectivity characteristic of biological components such as antigen-antibody bonding when integrated with an electrochemical, optical or piezoelectric transduction mode of analyte recognition with SAM as their strong immobilization foundation offers great promise to exploit them as efficient and accurate biosensors. Figure 1 below shows basic representation of SAM structure.

Figure 1: Basic Representation of SAM structure

From Figure 1 above the substrate can be taken from any noble metal (rhodium, silver, iridium, platinum, gold, ruthenium, palladium, osmium, mercury and rhenium) due to strong affinity of sulphur from the head group of alkanethiols molecules to these types of metals. One important characteristic that SAM provides is the head group shows a specific, reversible affinity for substrate which gives advantage for biological components which they will degrade back into individual molecules that can be broken down by the body if used as SAM formation only by their kinds. An example study related to SAM application with biosensor has been chosen from MDPI website (http://www.mdpi.net/sensors) with the title of Electrical Characterization of a Thiol SAM on Gold as a First Step for the Fabrication of Immunosensors based on a Quartz Crystal Microbalance as attached in Attachment 2. Summarizations of chosen study are as follows:

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i.

Objective of the study:

a. To develop a biosensor based on quartz crystal microbalance (QCM) technique for

antigen (rabbit IgG) detection with functionalized crystal surface by self assembled monolayer (SAM) technique. b. To characterize insulating properties and estimate coverage area of the self assembled monolayer (SAM) adsorbed (chemisorptions) on quartz surface (gold) by using cyclic volatmmetry and impedance spectroscopy technique respectively. c. To characterize vibration molecule frequencies of the SAM on quartz surface by using infrared spectroscopy for proven occurrences of high formation of covalent gold-sulphur bonding. d. To integrate antibody immobilization via cross-linker coupling with Nhydroxysuccimide (NHS) and 1-(3-(dimethylamino)propyl)-3-ethylcarbodimine hydrochloride (EDC) with SAM as a strong foundation for the antibody (rabbit antiIgG) immobilization technique.

ii.

The SAM formation process in the study details as below:

a. First the crystal surface of the QCM was pre-treatment by cleaning it with piranha
solution and rinsed with deionised water. Then the crystal needs to be dried over a stream of N2 gas. Finally, an UV-cleaning process for 5 minutes was used to remove all unwanted hydrocarbons adsorbates. b. The SAM formation was carried out by soaking the completed pre-treatment crystal in a 1mM mercaptoundecanoic acid solution (dissolved in ethanol) for overnight. Mercaptoundecanoic acid solution is the SAM molecules chose for this study. c. Then, in the next day the crystal with SAM formation was rinsed with ethanol for soft cleaning purposes. d. The formation of the SAM on the crystal surface and all required characteristics of the SAM formation were determined by using cyclic voltammetry, impedance spectroscopy and infrared spectroscopy.

iii.

The antibody immobilization process in the study details as below:

a. First the crystal with completed SAM formation was need to be attached with

cross-linker antibody immobilization coupling agent of 0.5 M NHS and 0.2 M EDC solution which has been prepared in millipore water. b. The solution was drop onto the crystal by using micropipette (100l) and then inserting into the flow-injection pump for the solution to make contact with the crystal for 45 minutes. After that the crystal were rinsed with phosphate buffer saline (PBS) for cleaning and maintaining a constant pH in the physiological range. c. Next, a solution of 5g/ml of antibodies (rabbits anti-IgG) in acetic/acetate buffer (pH=5) was applied to the crystal by using micropipette (100l) for 1 hour for binding process of functional groups of cross-linker NHS/EDC to be taken place with the Fc region/fragment of the antibodies. After that the crystal were rinsed with 0.05% Tween 20 (polysorbate 20) and with PBS solution. d. The non-binding or non-covered surface of cross linker coupling agent with the antibodies was blocked by using a solution of 0.1% bovine serum albumin (BSA) applied to the crystal by using micropipette (100l) for 30 minutes. Then the crystal rinsed with PBS. iv. Antigen detection process in the study details as below: 2 different antigens (rabbit IgG) concentration prepared in PBS were applied via injected pump onto the QCM for testing the immunosensor functionality for antigenantibody binding occurrences fulfilling this study intended purposes.

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