IMMOBILIZATIONS OF ANTI-JEV ANTIBODIES IN HUMAN SERUM FOR DIRECT IMMUNOSENSORS Tran Quang Huy 1,2 , Phan Thi Nga 1 , Nguyen Thi Hong Hanh 1 , Mai Anh Tuan 2 1) National Institute of Hygiene and Epidemiology (NIHE), N°1 – Yersin Str., Hanoi 2) International Training Institute for Materials Science (ITIMS), Hanoi University of Technology (HUT), N°1 Dai Co Viet Road, Hanoi Email: huytq@nihe.org.vn Da Nang - 2009 HỘI NGHỊ VẬT LÝ CHẤT RẮN VÀ KHOA HỌC VẬT LIỆU TOÀN QUỐC LẦN THỨ 6 (SPMS-2009) Đà Nẵng 8-10/11/2009 CONTENTS • Introduction Introduction • Immobilizations of Antibodies Immobilizations of Antibodies • Results and Discussion Results and Discussion • Conclusion Conclusion INTRODUCTION BIOSENSORS Mechanism: based on the specific bio-chemical reactions: + Enzyme – substrate (enzyme sensor) + DNA hybridization (DNA sensors) + Antigen – antibody (Immunosensor) A perspective of traditional methods and biosensors IMMUNOSENSORS Based on the specific reaction of antigen - antibody Virus: envelop (poteins or antigens) and capsid (containing genetic material: DNA or RNA) Antibody: immunoglubulin molecule to recorgnize and block strange agents (antigens of viruses, bacteria, etc) Signal THE PRINCIPLE OF IMMUNOSENSORS FOR JEV DETECTION Si Pt/Au Micro – Electrodes Antibody Virus sample Bio-compatible materials PROBLEMS 1- Transducer (fabrication technology) 2 – Immobilization of sensitive biological elements (antibody) IMMOBILIZATIONS IMMOBILIZATIONS - Molecules may be immobilized either passively through:  Hydrophobic  Ionic interactions  Covalently by attachment to activated surface groups. - The immobilization process should occur selectively in the presence of common functional groups, including amines, thiols, carboxylic acids, and alcohols.  Amino-NH2,  Carboxy-COOH,  Aldehyde-CHO,  Thiol-SH,  Hydroxyl-OH [...]... C, D] JEV detection using immunosensors [SR 830 Lock -In Amplifier (ITIMS)] The detection signal of the sensor using APTES – 10% glutaraldehyde for immobilization of anti- JEV antibodies contained human serum is 2- to 3- fold higher than other methods The response time of JEV antigen detection was about 5 minutes and got stable condition within 20 minutes after immersion of the immunosensor in JEV antigen... working electrodes area for 30 min - Immobilization with 1 mg/ml anti- JEV antibodies contained human serum for 1 hour - Blocked with 2% BSA solution in PBS for 30 minutes Washing with phosphate buffered saline (PBS) at pH 7.0 and air dried Covalent immobilisation of antibody using glutaraldehyde 10% RESULTS AND DISCUSSION Biosensors after anti - JEV serum immobilization Sensors are immobilized with antibodies. .. Up-down sensors CLEANING PROCEDURES sensors 1 2 3-aminopropyl-triethoxy-silance (APTES) Experimetal immobilizations of anti JEV serum 1 APTES – Antibody - Immobilization with 1 mg/ml anti- JEV antibodies contained human serum for 1 hour - Blocked with 2% BSA solution in PBS for 30 minutes - Washing with phosphate buffered saline (PBS) at pH 7.0 and air dried 2 APTES – Glutaraldehyde – Antibody - The silanized... glutaraldehyde solution for 30 min - Immobilization with 1 mg/ml anti- JEV antibodies contained human serum for 1 hour -Blocked with 2% BSA solution in PBS for 30 minutes - Washing with phosphate buffered saline (PBS) at pH 7.0 and air dried 3 APTES – Glutaraldehyde – Protein A - Antibody - The silanized sensor with 10% (v/v) aqueous glutaraldehyde solution for 30 min - 5µl protein A solution (1mg protein A/1ml PBS,... glutaraldehyde (G.A) and then immobilized with anti- JEV antibodies contained serum (1mg/ml in PBS pH 7.0) (D) - The sensor coated with APTES, added 10% glutaraldehyde (G.A), then protein A (1mg/ml in PBS) and immobilized with anti- JEV antibodies contained serum (1mg/ml in PBS pH 7.0) FTIR spectra [Nicolet 6700 FT-IR machine (HUT)] FTIR spectra showing the stretching modes of chemical bonds on the sensor surface... was 60-times diluted in comparison with the initial solution used for MAC ELISA test CONCLUSION  Potential development of direct immunosensors using anti JEV serum as probes for rapid JEV detection in outbreaks  The signal obtained by using 3-aminopropyl-triethoxy-silance (APTES) and 10% glutaraldehyde to immobilize the antibodies was 2 to 3-fold as higher as those obtained using different attachment... methods  The response time of JEV antigen detection occurred was 5 minutes and stable within 20 minutes after immersion of the immunosensor in the diluted solution of JEV antigen, 60 times in comparison with the initial solution used for MAC ELISA test Development of Biosensors/Biochips at ITIMS Four biosensors can be integrated on the chip BIOSENSOR GROUP AT ITIMS THANK YOU FOR YOUR ATTENTION! ...Preparation of Surface for Antibody Immobilization: • Modification of the electrodes surface to create functional groups • Modification of antibodies for covalent attachment to the surface • Cleaning the surface of electrode • Modification of the surface to create fuctional groups by: + Polymer Coating  Glow Discharge  Dipping  Spin Coating  Electrochemical deposition + Langmuir... modified with different chemical methods for antibody immobilization The region of 3600–3200 cm−1 is characteristic of free amino functional groups of the surface treated with APTES only (Fig A) The vibration band for asymmetric Si-O-Si stretching mode can be seen in the range of 1100 – 1050 cm-1, and C- N stretching mode in 1300 – 1000 cm-1 However, the stretching modes are shifted slightly by different... immobilization Sensors are immobilized with antibodies Immunosensors are kept in tubes at 40C Morphology of the sensor surfaces treated with different immobilization methods [FE-SEM S-4800 (NIHE)] (A) - The cleaned sensor surface (B) - The sensor surface coated with APTES only and directly immobilized with anti- JEV antibodies contained serum (1mg/ml in PBS pH 7.0) (C) - The sensor coated with APTES, added . solution for 30 min - Immobilization with 1 mg/ml anti- JEV antibodies contained human serum for 1 hour. -Blocked with 2% BSA solution in PBS for 30 minutes. - Washing with phosphate buffered saline. area for 30 min - Immobilization with 1 mg/ml anti- JEV antibodies contained human serum for 1 hour. - Blocked with 2% BSA solution in PBS for 30 minutes. Washing with phosphate buffered saline. FT-IR machine (HUT)] JEV detection using immunosensors The detection signal of the sensor using APTES – 10% glutaraldehyde for immobilization of anti- JEV antibodies contained human serum is 2-