Advisor : R u -Li Lin Advisee :Shih-Min Chen

1. Detection of allergies using a silver nanoparticle modified nanostructured biosensor Advisor : Ru-Li Lin Advisee :Shih-Min Chen Southern Taiwan University of Science and Technology,Department of Mechanical Engineering, Tainan, TAIWAN Date ﹕2013/4/1

2. Outline • Paper Survey • Introduction • Experimental setup • Results and discussion • Conclusions • References

3. Introduction • The symptoms of the allergic response are determined by the types and concentrations of antigens in the ambient environment. • There are reportedly 21 allergen components in dust mite allergens.The group 2 allergen, Der p2, has been reported to be the key allergen. • It has been found that IgE, which is a useful index for allergy detection, is present in the serum of 80% of asthma patients, and can chemically bind with the carboxylterminal sequences of the Der p2 allergen. • Therefore, the Der p2 allergen can be used for the detection of the IgE concentration in a patient’s serum.

4. Experimental setup • Preparation of an AAO barrier-layer • Sputtering of Au thin film • Annealing • Packaging • Deposition of GNPs • Deposition of SNPs

5. Experimental setup-Schematic illustration of the proposed nanostructured biosensor

6. Experimental setup- Electrophoretic : The space uniform electric field, the phenomenon of dispersed particles in the fluid moving. Affect its deposition parameters generally include: the distance between the two electrodes (d), the drive voltage (V), electrophoresis time (t) and the concentration of the solution

7. Experimental setup-Sample preparation Immobilization of IgE.

8. Experimental setup-Sample preparation • The surface of the nanostructured sensor was cleansed bydrenching it in ethanol,acetoneand deionized (DI) water in turn,followedby an ultrasonic wave shaking for 5 min. • 30 μL of a 5 mM 11-MUA (11-mercaptoundecanoic acid) solutionwas then dispensed onthe sensor to create a self-assembled11-MUA monolayer as an anchor membrane. • Coating the sensor chip with a 30 μ L mixture of NHS and EDC (1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide) (molar ratio,1:2) for 50 min. The sensor was rinsed twice with a PBS buffersolution (pH 7.4).

9. The sensor chip was coated with a 25 μL (50 μg/mL) dust miteallergen Der p2 solution and then incubated for 30 min. The sensorwas again washed twice with a PBS buffer solution. A 20 μ L1% BSA solution was used as the blocking layer to the bindingsites of those Der p2 which were not tied to the 11-MUA layer.Thesample was then washed twice with a PBS buffer solution. • Coated with 25μL of a patient’s serum that had been dilutedusing a PBS solution at a ratio of 1:5. The sensor was incubatedfor 30 min before being washed twice in a PBS buffer solution. • Coating a 25μL diluted solution of the affinity purified goat antihumanIgEantibody. The diluted solution was prepared using aPBS solution at a ratio of 1:2000. The sensor was incubated for30 min and then rinsed twice with a PBS buffer solution.

10. Thirteen patient serum samples which had already beenexamined by ImmunoCAP (CAP; Pharmacia Diagnostics, Uppsala,Sweden) were provided by the Taichung Veterans GeneralHospital,Taichung, Taiwan.

11. Results and discussion - Device fabrication results (a) 3D electrode (b) flat electrode with a sodium citrate solution as the stabilizer.

12. Results and discussion - Conductivity enhancement analysis results Nyquist plots for the bare electrodes subjected to different applied voltages for SNP deposition

13. Results and discussion - Blood serum detection results Blood serum detection results

14. Results and discussion - Linear regression: △Ret and Immuno CAP Linear regression between △Ret and Immuno CAP detected value (1+≤scale≤4+)

15. Results and discussion - Measurement of the detection limit of the proposed device △ Ret as a function of the logarithmic concentration of IgE.

16. Conclusions • In this study, a nanostructured biosensor with uniformly deposited GNPs as the sensing electrode was implemented for the detection of a patient’s allergy level. • To enhance the charge transfer efficiency of the biosensor, SNPs were deposited on the GNP layer. • The blood serum detection results confirm that the proposed nanostructured biosensor can detect allergy diseases with small sample consumption, short sample preparation time, and little detection time.

17. References [1]Yi-Fen Liu , Jaw-Ji Tsai , Yu-Ting Chin, En-Chih Liao , Chia-Che Wu , and Gou-Jen Wang, Sensors and Actuators , "Detection of allergies using a silver nanoparticle modified nanostructured biosensor , " Sensors and Actuators B , vol 171– 172 , pp. 1095– 1100 ,2012 [2]Electrophoretic: http://brc.se.fju.edu.tw/protein/purify/sdspage.htm