Připraveno ve spolupráci s projektem OPVK NANOTEAM

1. Připraveno ve spolupráci s projektem OPVK NANOTEAM • Budování výzkumných týmů a rozvoj univerzitního vzdělávání výzkumných odborníků pro mikro- a nanotechnologie (NANOTEAM) • Reg. č. CZ.1.07/2.3/.00/09.0224 Investice do rozvoje vzdělávání

2. Gold-amalgam microelectrodes array for ultrasensitive protein detection 28. 11. 2014 Radim Hrdý, Camila M. Campos, Pavel Neužil Investice do rozvoje vzdělávání

3. Outlineelectrodes Array for Ultrasensitive Protein Detection • Introduction • Electrochemical detection • Microelectrodes • Amalgam electrodes • Chip design • Chip design and fabrication • Bonding problem • Electrochemical characterization • Conclusion Investice do rozvoje vzdělávání

4. Electrochemical protein detectionlectrodes array for ultrasensitive protein detection • Actual trends • Selective and high sensitive detection • Multi and fast detection • Low cost • High reproducibility • Sensitive method and modified electrodes • Array of microelectrodes • System integrated on the chip • Mass production Investice do rozvoje vzdělávání

5. Electrochemical methodsArray for Ultrasensitive Protein Detection http://www.ni.com/

6. „Semi - Difference Cyclic Pulse Votametry“Array for Ultrasensitive Protein Detection Vin t Vout t http://www.ni.com/ Investice do rozvoje vzdělávání

7. Advantages of microelectrodesltrasensitive protein detection • Measurement inside the diffusion layer • Short distance between electrodes suppress the charging current • Steady-state is attained rapidly • Faster electrochemical reaction ,Andrew O. Simm, Analyst, 2005,130, 1303-1311

8. Mercury electrodes • Wide potential window • Possibility to detect proteins or µRNA • High affinity to –SH (thiol) group http://www.autolabj.com

9. Chip design and fabrication • 3D system of 9 x 4 electrodes array • 2x 200 nm gold layer • 1 um Parylen interlayer • All working electrodes are connected • Changing of interelectrode distance by electrochemical deposition Parylen gold deposited metal

10. Chip design and fabricationfor ultrasensitive protein detection • Problem of bottom gold layer over etching • Proof by SEM and EDX

11. Chip design and fabricationarray for ultrasensitive protein detection Reparation and gold deposition • 0,1M KAu(CN)2 + 0.1M H3BO4 • Platinum wire as counter electrode • Poor wettability of electrolyte • Using TWEEN 80 as surfactant Mercury deposition • Higher wettability • Short circuits of top electrodes

12. Bonding Problemsltrasensitive Protein Detection • Soldering • Differential preheating of glass chip • Poor wettability of solder • Application of high pressure • EPOTEK H31-D Conductive Glue • Elimination soldering problem • Manual deposition • New board design

13. Electrochemical characterization Ultrasensitive Protein Detection • Conditions • 0.1 M KCl and K3[Fe(CN)6] + K4[Fe(CN)6] 1:1 • Concentration 1 mM – 1uM • Scan rate 200 – 50 mHz= 640 - 160 mV.s-1 • Amplitude 800 mV Fe(CN)63- + e- → Fe(CN)6 4- Fe(CN)64--e- → Fe(CN)6 3- V V V V

14. Electrochemical characterization Ultrasensitive Protein Detection Gold deposition with TWEEN 80 Thickness calculated according to Faradays law – 0.5 µm V V

15. Electrochemical characterization Ultrasensitive Protein Detection Mercury deposition Thickest of Hg layer 0.1 µm 50 mHz V V V V

16. Summary Ultrasensitive Protein Detection • Microelectrode array was designed • New electrochemical system was developed • Amalgame electrodes were tested • Future plans • Repeated fabrication of chips • Set the LOD (limit of detection) • Surface of electrodes bio-modificationAvidin-Biotin • Testing of system selectivity • Publication: Manuscripts for JACS and LOC Investice do rozvoje vzdělávání

17. Acknowledgment Ultrasensitive Protein Detection Investice do rozvoje vzdělávání

18. Thank you for your attention Investice do rozvoje vzdělávání