Electrical study of the changes in fluorescence properties of Rhodamine B, for the design of future detection systems in microfluidic devices based on fluorescence

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(1)ELECTRICAL STUDY OF THE CHANGES IN FLUORESCENCE PROPERTIES OF RHODAMINE B, FOR THE DESIGN OF FUTURE DETECTION SYSTEMS IN MICROFLUIDIC DEVICES BASED ON FLUORESCENCE.. EDGAR ALBERTO UNIGARRO CALPA. UNIVERSIDAD DE LOS ANDES FACULTAD DE INGENIERÍA MAESTRIA EN INGENIERÍA ELECTRÓNICA Y DE COMPUTADORES. BOGOTÁ 2013. 1.

(2) ELECTRICAL STUDY OF THE CHANGES IN FLUORESCENCE PROPERTIES OF RHODAMINE B, FOR THE DESIGN OF FUTURE DETECTION SYSTEMS IN MICROFLUIDIC DEVICES BASED ON FLUORESCENCE.. EDGAR ALBERTO UNIGARRO CALPA. Trabajo de Grado presentado como requisito para obtener el título de Magíster en Ingeniería Electrónica y de Computadores. ASESOR: PhD. JOHANN FACCELO OSMA CRUZ COASESOR: PhD. FREDDY SEGURA QUIJANO. UNIVERSIDAD DE LOS ANDES FACULTAD DE INGENIERÍA MAESTRIA EN INGENIERÍA ELECTRÓNICA Y DE COMPUTADORES. BOGOTÁ 2013. 2.

(3) TABLE OF CONTENTS. 1.0 Introduction ............................................................................................................................................4 2.0 Materials and Methods .........................................................................................................................5 2.1 Reagents and Materials ...................................................................................................................5 2.2 Physical Vapor Deposition of Metals .............................................................................................6 2.3 Fluorescent Measurements .............................................................................................................6 2.4 Electrochemical Measurements .....................................................................................................7 2.5 Metallic Electrodes Modification .....................................................................................................7 2.6 Selective Wet Chemical Etching of Metals ...................................................................................8 2.7 Microfluidic System Fabrication ......................................................................................................9 2.8 In-channel Modification of Electrode..............................................................................................9 2.9 Electrical Tests ..................................................................................................................................9 3.0 Results ..................................................................................................................................................10 3.1 Electrochemical measurements ...................................................................................................10 3.2 Fluorescent measurements ...........................................................................................................10 3.3 Metallic Electrodes Modification ...................................................................................................12 3.4 Selective Wet Chemical Etching of Metals .................................................................................16 3.5 Microfluidic System Fabrication ....................................................................................................18 3.6 In-channel Modification of Electrode............................................................................................20 3.7 Electrical Tests ................................................................................................................................21 4.0 Conclusions .........................................................................................................................................24 5.0 Discussions ..........................................................................................................................................25 Acknowledgements ...................................................................................................................................26 References .................................................................................................................................................26. 3.

(4) 1.0 Introduction1 Fluorescence has been an extended research topic since the XVIII century. Some fluorescence most attractive properties are: high sensitivity, strong selectivity, fast response time and local observation. These allow the detection on a single molecule level. (Valeur B. 2002, Thompson R. 2005) Fluorescent molecules can be used through other interactions for the measurement of pH, temperature, concentration of reactants and dynamics of matter or living systems at a molecular or supramolecular level (Valeur B. 2002). The design of fluorescent sensors is of major importance because of the high demand in analytical chemistry, clinical biochemistry, medicine, environment research, etc (Thompson R. 2005). Numerous chemical and biochemical analytes can be detected by fluorescence: cations, anions, neutral molecules and gases. Most of the studies on fluorescent sensors are made on aqueous media in facilities with highly controlled environmental characteristics. Microfluidic devices have a micro-environment that can be easily controlled for the implementation of fluorescents sensors; also, their portability allows the possibility to carry out tests in situ (Tabeling P. 2006). Sensors fabrication on microfluidic devices requires a special attention, because most of the modification processes on the sensor substrate have to be done once the microfluidic channel is sealed to prevent damage. (Nguyen N & Wereley S. 2002). 1. Centro de Microelectronica Universidad de Los Andes - CMUA. 4.

(5) In the present work the implementation of a device with fluorescent electrodes inside a microchannel was studied. Electrochemical techniques were used to modify the metal microelectrodes with rhodamine B. The rhodamine B molecule was one of the first fluorescent dyes to be used as laser dye. Its absoption and emission spectra are quite narrow and the Stokes shift is small. It emits fluorescence in the range 500-700nm. Rhodamine B has a quantum yield of 0.54 in ethanol (Kubin R. & Fletcher A., 1983) and it is sensitive to temperature (Karstens, T. and Kobs K., 1980) and presents radiochemiluminiscence (Jiang Q., et al, 2006). Approaches to the use of rhodamine B as a sensor have been done for pH (Gao F. et al., 2005) and for temperature (Low P. et al, 2007). Other type of fluorescent sensor using rhodamine B was presented by. (Spehar-Deleze A. et al, 2006), where hot. electron electrochemiluminiscence process was used to generate photons depending on the concentration of certain DNA sequences. The aim of this study was to measure the influence of electrical signals on the fluorescent molecules inside a microchannel. The results obtained here can be used in future designs of fluorescent tests inside microfluidic devices and in the integration with electrical sensors or actuators.. 2.0 Materials and Methods 2.1 Reagents and Materials Rhodamine B (RhB) was purchased from Panreac (Spain). Developer (Microposit MF319) and positive photoresist (Microposit SC 1827) were purchased from Shipley. 5.

(6) (USA). Gold wires (Au, 99.99%), Aluminum wires (Al, 99.99%), Copper pellets (Cu, 99.99%) and Chromium pieces (Cr, 99.95%) were purchased form Kurt J. Lesker (USA). Stripper photoresist (JT Baker, USA), potassium chloride pentahydrated (KCl•5H 2O, JT Baker, USA) and ammonium sulfate were purchased from Sigma-Aldrich (USA). Glass slides of 76.2 mm in length, 25.4 mm in width and 1 mm thick were purchased at the local market. All other compounds used were analytical degree. 2.2 Physical Vapor Deposition of Metals Edwards auto 306 (UK) evaporator was used to deposit single and multi-layers of gold, chromium, aluminum and copper metals on glass slides. Deposition pressure was kept below 1E-5 mbar, the average currents in the process were 3.4 A, 3.8 A, 3.0 A and 3.2 A for gold, chromium, copper and aluminum respectively, the evaporation was made on Tungsten dimple boats (Lesker,USA). Multi-layer deposition process was done in a single run of the Edwards system. Aluminum and copper electrodes were fabricated with a thickness of 200 nm and 300 nm respectively. Besides, chromium-copper (Cr/Cu) and chromium-gold (Cr/Au) electrodes were fabricated evaporating a layer of 20 nm of Cr before a 200 nm layer of Cu and 100nm of Au respectively. The aluminum-copper (Al/Cu) electrodes had a deposition layer of 400 nm of Al and a layer of 200 nm of Cu. 2.3 Fluorescent Measurements An optical microscope Olympus CX21 (USA) was modified to allow fluorescent measures using optical filters, light-emitting-diodes (LED) and a support for couplingdigitals-cameras at the ocular (Segura S, 2011). A Sony Cyber-shot (USA) digital. 6.

(7) camera was used as sensor to capture the fluorescent images. LED light was used as an excitation source for the fluorescent samples, and Cokin (France) camera filters was used to filter the excitation source light from the light emitted by the sample. A Cokin A.003 red filter, and four green LEDs of 525 nm were used to measure rhodamine B fluorescence. Image analysis and quantification on fluorescence were made with open source software ImageJ (USA), image sequences were taken at the clean room facilities in a dark room. In every test, the camera was programmed to the same capture parameters using ISO3200 and Focal Aperture F2.7. The microscope, camera, filter and LEDs were kept at the same position for comparative measurements. 2.4 Electrochemical Measurements Cyclic voltammetry (CV) was controlled by potensiostat/galvanostat Autolab PGSTAT128N (Metrohm, USA). Data was acquired by the software Nova version 1.6. A conventional three-electrode system was used to carry out the electrochemical measurements at room temperature inside a Faraday cage. CV was performed using a 0.05M KCl•5H2O solution with 0.96 mM of RhB. The measurements were performed within a range of 0.2 to 1.2 V vs. Ag/AgCl/KCL3M at a 100 mV/s scan rate. 2.5 Metallic Electrodes Modification Rhodamine B electrochemical immobilization analyses were tested on electrodes of aluminum, copper and gold. A 30 seconds step signal at the oxidation potential measured from the cyclic voltammetry was applied to the electrodes with a source power HP6276B (Hewlett Packard, USA).. 7.

(8) Absorption test was made by immersion of the samples on a 0.05M of KCl•5H2O with 0.96 mM of RhB solution, times within 1 and 3 hours with 30 minutes intervals were analyzed. The samples were washed with deionized water and dried with nitrogen gas after every single run. 2.6 Selective Wet Chemical Etching of Metals Consecutive etching processes were performed in the microelectrodes fabrication. Each metalic layer was removed with a selective slow etch rate solution. The isotropic wet etching was made by substrate immersion on the etchant solution, at room temperature without agitation. The proccess time depends on the etch rate of each etchant solution. The solution for etching aluminum was a mixture of HF/H2O in a volumetric ratio 1:10 containing 0.19 M of NH4SO4 with an average etch rate of 5 nm/s. chromium was attacked with a solution of HF 40% with an etch rate of 0.3 nm/s. Copper was removed using a solution of FeCl3 52% (w/v) with an etch rate of 35nm/s (Unigarro, et. Al. 2011). The electrodes patterns were transferred onto the thin metal film by traditional optical lithography methods: the positive photoresist was spin-coated and patterned using a maskless UV light exposure system (Model SF-100, Intelligent Micro Patterning, USA); then, the soluble photoresist was removed by the developer. A multiple exposure program was used for the electrodes fabrication, with exposure windows of 700 x 700 pixels. Each pixel correspond to a square of 5 μm or 15 μm, both technologies were used for the metallic microelectrodes fabrication.. 8.

(9) 2.7 Microfluidic System Fabrication A copper layer (> 100 nm) was deposited onto a glass slide by thermal evaporation, and the fluidic microsystem pattern was transferred to the substrate by optical lithography. Then, the microsystem copper pattern, obtained after the development, was directly used as wet etching mask. The exposed region of glass was attacked with HF 40% and the remained Cu mask was removed using FeCl3 52% (w/v). (Velez, 2011) Microsystem inlet and outlet were made using a commercial moto-tool. The holes were drilled, immersed under water, at 20,000 rpm using a diamond coated tip with 2 mm of diameter. The microsystem and electrodes slides were assembled using UV light curable glue. Previous to coating the microsystem with a glue layer, the channel was protected using a red color dye solution to prevent channel clogging. 2.8 In-channel Modification of Electrode Rhodamine B was selectively deposited on the working microelectrode inside the sealed fluidic microsystem by electrochemical techniques. The microchannel was filled up with a solution of 0.05M of KCl•5H2O with 0.96 mM of RhB. Rhodamine B was electrodeposited by applying 1 V steep signal between the microelectrodes and a platinum wire electrode for 30 s using a source power HP6276B (Hewlett Packard, USA). 2.9 Electrical Tests Voltage and current measures were made with a multimeter Fluke 179 (USA). Electrical clamps with copper foil were used to avoid scratches on the microelectrodes. 9.

(10) surfaces. Signal wave generator BK precision 4011A (USA) and source voltage Protek DF1731SB (USA) were used on the electrical tests.. 3.0 Results In order to measure the changes on fluorescence caused by electrical signals a high conductivity substrate for the signal transmission was needed. Aluminum, copper and gold were selected as metal substrates for this study. For minimal external interference the distance between the electrodes and the fluorophores was reduced by electrochemical deposition techniques on the metallic microelectrodes. Also, the rhodamine B immobilization in the metallic electrodes is necessary to use the electrodes as a sensor embedded in a microfluidic device. 3.1 Electrochemical measurements Cyclic voltammetry was used to determinate the parameters for rhodamine B electrochemical immobilization study on metallic substrates. The results obtained from CV using a graphite electrode are shown in Figure 1. The anodic direction of CV presents an anodic peak at Epa = 0.97 V, this peak is caused by the molecule oxidation. The cathodic direction has a peak caused by rhodamine B reduction in Epc = 0.89 V (Figure 1). These results are consistent with the work presented for (Austin, et al, 1986). 3.2 Fluorescent measurements A Cokin A.003 red filter with a cutoff frequency greater than 560 nm, and four green LEDs with a light emission center on 525 nm were selected based on rhodamine B. 10.

(11) emission and absorption spectrum (Figure 2). Figure 3 shows the modification diagram made on the Olympus cx21 microscopy. The microscope 10x lens was used for the fluorescent measures. 5. Current [µA]. 4. Epa. 3 2 1 0 -1. Epc. -2 0. 0,2. 0,4. 0,6. 0,8. 1. 1,2. 1,4. Voltage [V] vs Ag|AgCl|KClsat. Normalized Emision [AU]. Figure 1. Cyclic voltammograms of 0.96mM rhodamine B on 0,05M KCl·5H2O made with a graphite electrode. The measurements were done within a range of 0.2 to 1.2 V at a scan rate of 100 mV/s. Anodic direction (black arrow).. 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0. 230. 430 630 Wavelenght [nm]. Figure 2. Normalized emission ( ) an absorption ( (Prahl S., 2012). ) rhodamine B spectrum in ethanol.. 11.

(12) LEDs were aligned to the lens focal point; optical filter was placed between the microscopy ocular and the. An image taken with the system is shown in Figure 10.. Figure 3. A) (1) Digital camera, (2) optical filter, (3) coupling camera system, (4) microscope ocular. B) LEDs pointed to the 10x lens focal point and a microfluidic system.. Image J software allows the image intensity quantification of a selected area in an images sequence. In order to perform an analysis the images were arranged and aligned for software analysis. The plugin “intensity vs time plot” allows to measure the intensity and to export the data for further analysis. The maximum value measured with the fluorescent system of a rhodamine B saturated solution was 110.45, and the intensity of a glass slide without any fluorescent or metallic structure was 6.72. The value obtained on the glass slide test is caused by the optical filter transmission response; however it’s possible to make quantification with the proposed system.. 3.3 Metallic Electrodes Modification Aluminum, gold and copper microelectrodes with square shape and 300 μm side were fabricated using soft lithography methods and isotropic wet etching.. 12.

(13) Intensity, arbitrary units. 28,38. 6,69. Al. 8,80. 7,79. 8,15. 7,27. Al + RhB. Au. Au + RhB. Cu. Cu + RhB. Sample Figure 4. Electrodes Intensity before and after electrochemical immobilization. A 30 s step signal of 1 V was applied between the sample and a platinum electrode, immersed in a solution of 0.96mM rhodamine B on 0,05M KCl·5H2O. Electrochemical immobilization was probed on metallic microelectrodes, Figure 4, shows the fluorescence intensity before and after the process. The electrodes were immersed in a solution of 0.96mM rhodamine B on 0,05M KCl•5H2O. A step signal of 1.0 V was applied between the working electrode and a reference platinum electrode during 30 s. Power supply positive terminal was connected to the metallic microelectrode and the ground reference was connected to the platinum electrode. After the process electrodes were cleaned with deionized water and nitrogen gas. Only copper remains with rhodamine B after the cleaning process. Gold and aluminum electrodes show a little increment on the fluorescent intensity (Figure 4). Rhodamine B absorption on aluminum, gold and copper microelectrodes was measured on intervals of 30 min during 3 hours, the electrodes were immersed in a solution of 0.96mM rhodamine B on 0,05M KCl•5H2O.. 13.

(14) Intensity, arbitrary units. 40 35 30 25 20 15 10 5 0 0. 50. 100 Time [min]. 150. 200. Figure 5. Electrodes Intensity during the absorption processes. Electrodes were immersed in a solution of 0.96mM rhodamine B on 0,05M KCl·5H2O during 3 hours and measures were taken on 30min intervals. ( ) Au electrodes ( ) Al electrodes ( ) Cu electrodes.. After the process electrodes were cleaned with desinizated water and nitrogen gas. Figure 5, shows the electrodes fluorescent level. Copper electrodes increase their fluorescent response with time, after 2 hours the Cu electrode was saturated. Al and Au electrodes were not modified by the absorption process. The absorption process can be controlled with the step signal level applied to the copper electrodes. Figure 6 shows the samples increase on the fluorescent level with the voltage increment used for the electrochemical deposition. Voltages greater than 2 V remove the copper from the substrate, that’s the reason for the abrupt decrease in the fluorescent level at that point. The immobilization process stability was measured applying voltages on the modified copper electrodes submerged into deionized water; the voltages were near E pa and Epc. Negative voltages were applied trying to remove the copper of the glass substrate. 14.

(15) (Figure 7). Under deionized water the absorption process is stable in the reduction and. Intensity, arbitrary units. oxidation voltages measured in the cyclic voltammetry. 45 40 35 30 25 20 15 10 5 0 0. 0,5. 1. 1,5 Voltage [V]. 2. 2,5. Intensity, arbitrary units. Figure 6. Rhodamine B intensity on copper microelectrodes at different electrochemical deposition voltages.. 36 31 26 21. 16 11 6 1 -2,5. -1,5. -0,5 Voltage [V]. 0,5. 1,5. Figure 7. Immobilization stability for voltages near Epa and Epc Parameters are the same that on Figure 4.. 15.

(16) 3.4 Selective Wet Chemical Etching of Metals Techniques for metals selective wet etching were developed. Using those techniques it was possible to fabricate electrodes with several metallic layers by continuous steps of soft lithography and etching. A. B. C. Figure 8. Mask used for multi-layered metallic microelectrodes. A) Continuous electrode mask, B) Interdigitated electrode mask, C) Protective mask.. Figure 10. A) Copper microelectrodes fabricated on Aluminum substrate 25 x images with Aligner microscope (German). B) Image before the rhodamine B immobilization Olympus CX21 with modification for fluorescence C) Image after rhodamine B immobilization Olympus CX21 with modification for fluorescence. 16.

(17) Chromium and copper continuous electrodes were fabricated by doing a photolithography process with the mask (Figure 8.A), in the exposed area both metals were etched, copper etching was made with a solution of FeCl3 52% (w/v) by immersion for 2 seconds, and Chromium etching was made with a solution of HF 40% for 40 seconds. The process maximum resolution was around 60 μm, due to the chromium etchant which also attacks the glass substrate. A second process of lithography using the mask of Figure 8.C and wet etching was done in order to obtain a copper electrode above the chromium layer. The electrodes fabricated are shown in Figure 9.B.. Figure 9. A) Al/Cu interdigitaded electrodes, B) Cr/Cu continuous electrodes with RhB.. Al/Cu interdigitated electrodes were fabricated by following a similar process to the described above, after a photolithography process using mask of Figure 8.B unprotected areas of both metals were etched. Copper was removed by a 2 seconds etching process on FeCl3. Aluminum was etched in a solution of a mixture of HF/H2O in a volumetric ratio 1:10 containing 0.19 M of (NH4)2SO4 for 30 seconds. The maximum resolution of this process was 15 μm. A second photolithography process for the fabrication of a copper electrode above the aluminum layer was done using mask of Figure 8.C, the remaining copper was etched with a FeCl3 solution.. 17.

(18) Selectivity on the etching processes were probed by the fabrication of those electrodes, chromium and aluminum etchant solutions showed high selectivity. However copper etchant solution also etches aluminum. Repetitive etching processes on the aluminum caused an increase in its porosity. The final copper etching process performed after the fabrication steps of multiple layer metal electrodes had a maximum resolution of 5 μm as shown in Figure 10.A. The electrochemical immobilization was selective over the different metallic structures. Fluorescent images in Figure 10.C show selective immobilizations of rhodamine B on copper structures. Rhodamine B was not immobilized neither in aluminum nor in chromium. 3.5 Microfluidic System Fabrication The microfluidic device (figure 11) contains two layers; the bottom layer had the microelectrodes used for the electrical tests and immobilization of rhodamine B and the upper layer contains the microfluidic channel with the inlet and outlet (Figure 12). The design length was 13.35 mm, width 360 μm and depth 50 μm. A 3,5 mm. Figure 12. Microfluidic channel.. The microfluidic channel profiles were measured with Dektak 3 Profilometer (USA), Figure 13 shows the measures taken at the beginning, middle and end of the channel. B. The measured depth was 51020 nm, and the measured width was 358.33 μm.. 18.

(19) Depth [ Å] x 10000. Distance [µm]. 0 -10. 0. 200. 400. 600. -20 -30 -40 -50. -60. Figure 13. Profilometer measures at the ( ) beginning, (---) middle and ( microchannel.. ) at the end of the. Figure 11. Microfluidic system assembling A) (1) AL/Cu interdigitated microelectrodes. B) Microfluidic device assembling process. (2) Microfluidic channel, (3) Microelectrodes, (4) Microfluidic system.. The microsystem and electrodes slides were assembled using UV light curable glue. 40 seconds before the assembly UV glue was put on the microchannel, and then both slides were aligned and hold together under a constant pressure for 30 seconds. Then, the channel was strong enough to be filled up with a red color dye solution to prevent. 19.

(20) channel clogging. Finally, the system was exposed to UV light for one minute. The result can be seen in Figure 13. The microsystem device dimensions are width 25.4 mm and length 26 mm.. 3.6 In-channel Modification of Electrode Rhodamine B immobilization on a sealed microchannel has the same parameters that the ones performed on the electrodes outside the microsystem. Figure 14 show the immobilization process on the interdigitated electrode inside a microchannel. Figure 14.C was taken after cleaning the microsystem with deionized water during 10 minutes. The immobilization of rhodamine B resists a water flow without losing fluorescence neither changing the electrodes form.. A 300 μm. B. 300 μm. C. 300 μm. Figure 14. In channel Modification of Interdigitated Electrode. A) Electrode on the microsystem. B) Under fluorescent microscopy channel with rhodamine B on the walls. C) Copper interdigitated microelectrode with rhodamine B.. 20.

(21) 3.7 Electrical Tests Continuous microelectrodes of Cr/Cu were used to study the effect of electrical current on the rhodamine B molecule. In a dried microfluidic system it was applied currents between 5 mA to 30 mA. Data were taken after 10 minutes of every current increase for the system stabilization. Figure 15, show that for 10 mA to 30 mA the intensity on the fluorescence level decreases. The continuous current applied to the Cr/Cu electrode causes the quenching on rhodamine B by thermal heating, this results are consistent with work of (Low P. et. Al., 2007).. Intensity, arbitrary units. 50 45 40 35 30 25 0. 5. 10. 15 Current (mA). 20. 25. 30. Figure 15. Current applied to Cr/Cu microelectrodes. Electrode resistance was 700 Ω. Data collected 10 min after every increase on current level.. Cu/Cr continuous electrode cooling process was registered after 5, 10, 50 and 100 min. No change was registered on the fluorescence intensity measured (Figure 16).. 21.

(22) Intensity, arbitrary units. 50 45 40 35 30 25 20 15 10 5 0 1. 10 Time (min). 100. Figure 16. Cr/Cu microelectrode thermal relaxation.. Variable current signals were applied to a Cr/Cu electrode inside a fluidic microsystem filled up with deionized water, square and sinusoidal waves were tested with a frequencies spam from 1Hz to 1MHz, the signal magnitude was 0.5 mA. No change was registered on fluorescence intensity levels emitted by rhodamine B (Figure 17).. Intensity, arbitrary units. 35 30 25 20 15 10 5 0 1. 100 10000 Frequency (Hz). 1000000. Figure 17. Variable current applied to Cr/Cu microelectrodes. Data collected 5 min after every change on frequency. ( ) Square wave ( ) Sine wave.. 22.

(23) Variable current signal amplitude effects were also tested. Sinusoidal current signal with frequencies of 60 Hz, 1 KHz, 100 KHz, and 1 MHz with magnitudes of 1 mA 5 mA and 10 mA were measured. As a response on the increment on the current magnitude, the fluorescent level shows a diminution caused for the thermal heating on the microelectrode (Figure 18).. Intensity, arbitrary units. 36 35 34 33 32 31 30. 29 28 10. 100. 1000 10000 Frequency (Hz). 100000 1000000. Figure 18. Sinusoidal current applied to Cr/Cu microelectrodes. Data collected 5 min after every change on frequency. ( ) 1 mA ( ) 5 mA ( ) 10 mA.. Al/Cu interdigitated microelectrodes were used to study the effects of applying constant and variable voltages to rhodamine B. Variable voltages were applied with to an Al/Cu microelectrode inside a microchannel filled up with deionized water. Square and sinusoidal voltage waves with a magnitude of 5 V were tested in a spam of frequencies from 1 Hz to 1 MHz. Figure 19 shows that there was no change on the sample fluorescent intensity. Continuous voltages were applied to Al/Cu microelectrodes inside a microchannel with deionized water. Voltages higher than 5 V applied for more than 20 seconds. 23.

(24) remove the electrode from the substrate, because of that no other studies with continuous voltages were done.. Intensity, arbitrary units. 35 30 25 20 15. 10 5 0 1. 10. 100. 1000. 10000. 100000 1000000. Frequency (Hz). Figure 19. Variable voltage applied to Al/Cu microelectrodes. Data collected 5 min after every change on frequency. ( ) Square wave ( ) Sine wave.. 4.0 Conclusions Modifications made to the Olympus CX21 microscopy with the imageJ software allow the measure and quantification of fluorescent samples inside a fluidic microsystem. Metals selective etching techniques were developed and used for the fabrication of multilayered metal microelectrodes. Also, selective etching on copper showed a maximum resolution of 5 μm. Fluidic microsystems with metallic microelectrodes were fabricated, the method develop does not affect the metallic surfaces composition allowing further immobilization processes.. 24.

(25) The rhodamine B immobilization process was studied and methods for in-channel modification of copper electrodes were successfully developed on this work. Selective immobilization was probed, with a maximum resolution of 5 μm. Current and voltage signals were applied to the microelectrodes with rhodamine B, and it was possible to measure the effect caused by thermal heating on the rhodamine B molecule. Electrodes fluorescent response was not modified for the frequency and the wave form changes on the current signal applied. Effect of continuous voltage variations in the electrodes was impossible to measure because the damage caused for the electrolysis process when voltages above 5 V were applied. Variable voltages with a 5 V magnitude do not affect the intensity on the rhodamine B fluorescence.. 5.0 Discussions The results of this research probed the integration of metallic microstructures and fluidic microdevices, also the modification of those microstructures by electrochemical immobilization with a fluorophore. The knowledge of those fabrication techniques and the integration developed with each other are the first step in the design on sensors embedded on microsystems. All devices presented here were fabricated at the cleanroom facilities at los Andes University reaching the laboratory highest resolution of 5 μm.. 25.

(26) The results gathered from the electrical study show the possibility of using rhodamine B electrodes to sense the temperature inside a microchannel at specific places and geometries. The fluorescent microelectrodes are strong enough to support a water flow without suffering changes inside the microsystem, perhaps substances that cause rhodamine B degradation can be detected by the losses on the fluorescence intensity levels. Variable electric signals do not affect the fluorescence level of rhodamine B immobilized on copper electrodes. This allows the design of electrical examinations inside the microfluidic channel based on the knowledge that fluorescent decays are not caused by the electrical signal.. Acknowledgements The author would like to thank especially to his advisor Dr. Johann Osma, his coadvisor Dr. Fredy Segura, Universidad de los Andes Electric and Electronic Engineer Department, and CMUA (Centro de Microelectrónica de la Universidad de los Andes). Also thank for his participation in this project to Juan C. Gonzalez.. References Valeur B.; “Molecular Fluorescence- Principles and Applications” WHILEY-VCH, Germany, 2002. Thompson R.; “Fluorescence Sensors and Biosensors”, CRC press. Taylor and Francis,2005. Spehar-Deleze A., Suomi J., Jiang Q, Nico de Rooij, Koudelka-Hep M. and, Kulmala S.; “Heterogeneous oligonucleotide-hybridization assay based on hot electron-induced electrochemiluminescence of a rhodamine label at oxide-coated aluminum and silicon electrodes”, Electrochimica Acta 51, issue 25, 5438-5444, 2006.. 26.

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