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

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

Investice do rozvoje vzdělávání 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

Investice do rozvoje vzdělávání 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

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

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

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

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

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

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

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

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

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

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

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

Investice do rozvoje vzdělávání 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-modification Avidin-Biotin Testing of system selectivity Publication: Manuscripts for JACS and LOC Investice do rozvoje vzdělávání

Investice do rozvoje vzdělávání Acknowledgment Ultrasensitive Protein Detection Investice do rozvoje vzdělávání

Thank you for your attention Investice do rozvoje vzdělávání