Table of contentsTable of contentsTable of contents .........................................................................
Table of contents           2.10.1.5 Procedure ..............................................................................
Table of contents      3.2.2    Reference MOSFET (nMOS) .....................................................................
Introduction1 IntroductionThe biomedical analysis techniques require the development of smart sensorswith the following pr...
IntroductionThere are three basic electrochemical cell processes that are useful intransducers for sensor applications:   ...
IntroductionBecause the ISFETs were only for measuring pH it was not able to detectdissolved oxygen in the electrolyte flu...
Materials and methods2 Materials and methodsIn this chapter the used materials for the characterization of the sensor chip...
Materials and methods2.1.1 Purpose of useTo examine the sensor chips optically for visual manufacturing errors before theb...
Materials and methods2.2 Used PC softwareORIGIN PRO 8:        It is a professional data analysis and graphing software for...
Materials and methodsMS POWERPOINT 2007:          To make a presentation of this work with figures and animations.ADOBE IL...
Materials and methods2.3 Phosphate-Buffered Saline (PBS)PBS solution is used widely in biochemistry and biological researc...
Materials and methodsMORE FREE IONSTo make solutions with more dissolved free ions than 150mM of NaCl, we add8.8g to one l...
Materials and methodsThe motion of chloride ions at Ag/AgCl wire causes current, which can be                            e...
Materials and methods2.4.3 Producing assemblyElectrolysis by electrochemical oxidation of the silver wire in 0.1mMhydrochl...
Materials and methodsSo the whole reaction can be summed to:                                        2 Ag + HCl à 2 AgCl + ...
Materials and methods                Electrolysis current for producing Ag/AgCl. Graph 2-2This period can be also known fr...
Materials and methods2.5 Incubator                               The used incubator. Picture 2-3The used incubator is Kelv...
Materials and methodsThe incubator can be also used as faraday cage.2.5.2 Available settingsThe incubator can heat up to 3...
Materials and methods2.6 Regulated DC power supply unit                        The used Power supply [CONR08]. Picture 2-4...
Materials and methods2.6.2 Available settingsThe power supply has two outputs. The first output has a range of 0V to 3V at...
Materials and methods2.7 Voltalab® 80/10                       Measurement unit PGZ402 [RADI68]. Picture 2-52.7.1 Purpose ...
Materials and methods                     GUI interface of the VoltaMaster 4. Picture 2-6Some technical data of PGZ402 [RA...
Materials and methodsvoltage-current V-I curve is absolutely linear and there are no visible jumpsbetween the measurement ...
Materials and methodsPOT. CYCLIC VOLTAMMETRY         Cyclic voltammetry sweep the potential at a given rate and measure th...
Materials and methodsPULSE - CHRONO AMEPEROMETRY        The current flowing from REF to WORK is measured while the potenti...
Materials and methods2.8 Sensor chipsIn this assay, we have two kinds of chips to probe. Both chips have the same kindof s...
Materials and methods2.8.1 cMOS              1mm                         The cMOS chip and its sensors. Picture 2-12The cM...
Materials and methods                   Pins assignment (not true to size). Picture 2-13   PIN                  Chip cMOS ...
Materials and methods    10                                                                  Source    11                 ...
Materials and methods    57                                                       Auxiliary electrode    56               ...
Materials and methodsThe nMOS chips have the following objects:    a.   Temperature sensor: Using a temperature diode (TD)...
Materials and methods                 Pins assignment (not true to size)[WIES05]. Picture 2-16         PIN                ...
Materials and methods        16                      O2/CV-FET 1                                NME        18             ...
Materials and methods2.9 Pin box                        Picture of the used pin box. Picture 2-172.9.1 Purpose of useThe p...
Materials and methods2.9.2 Available connectors   PIN              Chip cMOS                  Chip nMOS                   ...
Materials and methods    27                                                           Reference electrode    28           ...
Materials and methods    57                                                                Auxiliary electrode    56      ...
Materials and methods2.10 Non-Semiconductor sensorsNon-Semiconductor sensors are the ones which are on the surface of the ...
Materials and methodsREGION I (ZERO CURRENT REGION):        The voltage U is not enough to reduce molecules at the work el...
Materials and methods                                          =−                                Diffusion flux. Equation ...
Materials and methodsREGION IV (DISSOCIATION REGION):        Over potential dissociates water molecules. This is visible b...
Materials and methods                            2            +       → 2                Chemical reaction to bind dissolv...
Materials and methodsThe nMOS chip has only one Clark sensor, where the cMOS has 5 Clark sensors.The single sensor of the ...
Materials and methods2.10.1.4        Measurement settings and parameters             are to be chosen, in this case 10 / ....
Materials and methods2.10.1.5        Procedure    1. Making several cycles at higher scan rate using the setting explained...
Materials and methods2.10.2         IDES Sensor (Impedimetric)2.10.2.1       IdeaAn electrochemical half cell consists of ...
Materials and methods        The measurements done with two-wire setup include not only the        impedance of the electr...
Materials and methods2.10.2.2          Equipment and itemsVOLTALAB 80:         Pot. Fixed Freq. EIS (Capacitance):        ...
Materials and methods       1mm                          IDES sensor on the cMOS chip. Picture 2-27The nMOS chip has a pol...
Materials and methods2.10.2.4     Measurement settings and parametersTo measure the impedance, a voltage of 30mV with a fr...
Materials and methodsUsing Ohm’s law the impedance can be easily calculated and plotted in real andcomplex components.    ...
Materials and methods2.11 Semiconductor sensorsSemiconductor sensors are in contrast to the non-semiconductor sensors have...
Materials and methods                                            =                        Diode law in respect to voltage....
Materials and methods2.11.1.2       Equipment and itemsINCUBATOR:         For a constant and adjustable environment temper...
Materials and methods1mm                                        30µm                      Temperature diode on the nMOS ch...
Materials and methodsthe small amount of the fluid (7µl), which has a smaller heat capacity than thesensor chip. So, the f...
Materials and methods2.11.2          Reference MISFET (nMOS)2.11.2.1        Idea                                MISFET [HE...
Materials and methodsBy applying a potential at the gate port, an electrical field is created, whichcreates within the emb...
Materials and methodsLINEAR/OHMIC REGION OR TRIODE MODE:        This operation mode is when the gate-source voltage UGS bi...
Materials and methodsSERSOR CHIPS           Chip            No. of sensors                           Gate area          nM...
Materials and methods2.11.2.3        Measurement assembly                   Schematic design of the measuring system. Pict...
Materials and methods2.11.3          ISFET Sensors for pH-Measurement2.11.3.1        IdeaThe pH of a solution is dependent...
Materials and methods2.11.3.2        Equipment and itemsVOLTALAB 80:        Voltammetry - Pot. Cyclic Voltammetry:        ...
Materials and methodsSENSOR CHIPS                                                                 Gate                    ...
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips
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Endversion1 skriptum characterization of miscellaneous multi parametrical silicon based biosensor chips

  1. 1. Table of contentsTable of contentsTable of contents .................................................................................................................. 11 Introduction .................................................................................................................. 42 Materials and methods.............................................................................................. 7 2.1 Microscopes..................................................................................................................... 7 2.1.1 Purpose of use ............................................................................................................................................ 8 2.1.2 Used equipment and items ................................................................................................................... 8 2.1.3 Available settings...................................................................................................................................... 8 2.2 Used PC software ........................................................................................................... 9 2.3 Phosphate-Buffered Saline (PBS) .......................................................................... 11 2.4 Ag/AgCl reference electrode ................................................................................... 12 2.4.1 Purpose of use ......................................................................................................................................... 12 2.4.2 Used equipment and items for production ................................................................................ 13 2.4.3 Producing assembly ............................................................................................................................. 14 2.4.4 Production procedure ......................................................................................................................... 15 2.5 Incubator ........................................................................................................................ 17 2.5.1 Purpose of use ......................................................................................................................................... 17 2.5.2 Available settings................................................................................................................................... 18 2.6 Regulated DC power supply unit ........................................................................... 19 2.6.1 Purpose of use ......................................................................................................................................... 19 2.6.2 Available settings................................................................................................................................... 20 2.7 Voltalab® 80/10 .......................................................................................................... 21 2.7.1 Purpose of use ......................................................................................................................................... 21 2.7.2 Available settings................................................................................................................................... 21 2.8 Sensor chips .................................................................................................................. 26 2.8.1 cMOS ............................................................................................................................................................ 27 2.8.2 nMOS ........................................................................................................................................................... 30 2.9 Pin box ............................................................................................................................. 34 2.9.1 Purpose of use ......................................................................................................................................... 34 2.9.2 Available connectors ............................................................................................................................ 35 2.10 Non-Semiconductor sensors ................................................................................... 38 2.10.1 Clark sensor (Amperometry) ........................................................................................................... 38 2.10.1.1 Idea ................................................................................................................................................... 38 2.10.1.2 Equipment and items ............................................................................................................... 41 2.10.1.3 Measurement assembly .......................................................................................................... 43 2.10.1.4 Measurement settings and parameters ........................................................................... 44Characterization of miscellaneous multi parametrical silicon based biosensor chips -1-
  2. 2. Table of contents 2.10.1.5 Procedure ...................................................................................................................................... 45 2.10.2 IDES Sensor (Impedimetric) ............................................................................................................. 46 2.10.2.1 Idea ................................................................................................................................................... 46 2.10.2.2 Equipment and items ............................................................................................................... 48 2.10.2.3 Measurement assembly .......................................................................................................... 49 2.10.2.4 Measurement settings and parameters ........................................................................... 50 2.10.2.5 Procedure ...................................................................................................................................... 51 2.11 Semiconductor sensors ............................................................................................. 52 2.11.1 Temperature Diode (Potentiometry) ........................................................................................... 52 2.11.1.1 Idea ................................................................................................................................................... 52 2.11.1.2 Equipment and items ............................................................................................................... 54 2.11.1.3 Measurement assembly .......................................................................................................... 55 2.11.1.4 Measurement settings and parameters ........................................................................... 56 2.11.1.5 Procedure ...................................................................................................................................... 56 2.11.2 Reference MISFET (nMOS) ................................................................................................................ 57 2.11.2.1 Idea ................................................................................................................................................... 57 2.11.2.2 Equipment and items ............................................................................................................... 59 2.11.2.3 Measurement assembly .......................................................................................................... 61 2.11.2.4 Measurement settings and parameters ........................................................................... 61 2.11.2.5 Procedure ...................................................................................................................................... 61 2.11.3 ISFET Sensors for pH-Measurement ............................................................................................. 62 2.11.3.1 Idea ................................................................................................................................................... 62 2.11.3.2 Equipment and items ............................................................................................................... 63 2.11.3.3 Measurement assembly .......................................................................................................... 65 2.11.3.4 Measurement settings and parameters ........................................................................... 66 2.11.3.5 Procedure ...................................................................................................................................... 66 2.11.4 O2-FET Sensors for DO-Measurement .......................................................................................... 67 2.11.4.1 Idea ................................................................................................................................................... 67 2.11.4.2 Equipment and items ............................................................................................................... 69 2.11.4.3 Measurement assembly .......................................................................................................... 72 2.11.4.4 Measurement settings and parameters ........................................................................... 72 2.11.4.5 Procedure ...................................................................................................................................... 73 2.11.5 CV-FET (an extended O2-FET Sensor) .......................................................................................... 74 2.11.5.1 Idea ................................................................................................................................................... 74 2.11.5.2 Measurement settings and parameters ........................................................................... 75 2.11.5.3 Procedure ...................................................................................................................................... 753 Results and Discussion ........................................................................................... 77 3.1 Non-Semiconductor sensors ................................................................................... 77 3.1.1 Clark sensor ............................................................................................................................................. 77 3.1.1.1 cMOS chips .................................................................................................................................... 78 3.1.1.2 nMOS chips ................................................................................................................................... 79 3.1.2 IDES Sensor .............................................................................................................................................. 80 3.1.2.1 cMOS chips .................................................................................................................................... 80 3.1.2.2 nMOS chips ................................................................................................................................... 80 3.2 Semiconductor sensors ............................................................................................. 82 3.2.1 Temperature Diode .............................................................................................................................. 82 3.2.1.1 cMOS chips .................................................................................................................................... 82 3.2.1.2 nMOS chips ................................................................................................................................... 83-2- Characterization of miscellaneous multi parametrical silicon based biosensorchips
  3. 3. Table of contents 3.2.2 Reference MOSFET (nMOS) .............................................................................................................. 85 3.2.3 ISFET Sensor ............................................................................................................................................ 86 3.2.3.1 cMOS chips .................................................................................................................................... 86 3.2.3.2 nMOS chips ................................................................................................................................... 87 3.2.4 O2-FET Sensor ......................................................................................................................................... 90 3.2.4.1 cMOS chips .................................................................................................................................... 90 3.2.4.2 nMOS chips ................................................................................................................................... 91 3.2.5 CV-FET Sensor (nMOS) ....................................................................................................................... 934 Problems and Solutions ......................................................................................... 99 4.1 Contacting errors ........................................................................................................ 99 4.2 Loosing of the passivation layer ......................................................................... 100 4.3 Noise.............................................................................................................................. 103 4.4 Signal drops while measuring ............................................................................. 104 4.5 Digital rounding errors .......................................................................................... 104 4.6 Unclean sensor surface .......................................................................................... 1055 Conclusions and outlook ...................................................................................... 1066 Acknowledgments .................................................................................................. 1097 Indexes....................................................................................................................... 110 7.1 Index of pictures ....................................................................................................... 110 7.2 Index of graphs.......................................................................................................... 111 7.3 Index of equations.................................................................................................... 112 7.4 Index of tables ........................................................................................................... 1128 List of abbreviations and symbols .................................................................... 1149 Bibliography ............................................................................................................ 11910 Appendix ............................................................................................................... 123Characterization of miscellaneous multi parametrical silicon based biosensor chips -3-
  4. 4. Introduction1 IntroductionThe biomedical analysis techniques require the development of smart sensorswith the following properties: mass fabrication, low cost, low power and ease ofuse. In this goal, various sensors have been developed to cover the needs of thebiomedical researches. In these researches, biological cell cultures are analyzedunder different conditions. The biochemical activities of these cultures changesome parameters of the environment which they live in. This environment can beenclosed and protected from any outer effects, so any changes by the livingbiological cells can be detected using various detecting methods. One of thesemethods is the electrochemistry, which is the detecting of electrical signalscaused by chemical reaction.An electrochemical cell is a chemically and electrically isolated environment.Therefore the isolated environment, which the biological cells live in, can behandled as an electrochemical cell. Electrochemical cell. Picture 1-1-4- Characterization of miscellaneous multi parametrical silicon based biosensor chips
  5. 5. IntroductionThere are three basic electrochemical cell processes that are useful intransducers for sensor applications: 1. Potentiometry, the measurement of a cell potential at zero current. 2. Voltammetry and analogue amperometry, in which an oxidizing potential is applied between the cell electrodes and the cell current is measured. 3. Conductometry, where the conductance and resistance of the cell is measured by an alternating current bridge method.Semiconductor sensors have the advantage that they have smaller dimensionsthen other materials and several sensor types can be easily integrated in onechip. Electronic miniature circuits and structures e.g. memory or amplifier canproduced in the same wafer with the sensor at the same time. On the other hand,only mass produced semiconductor sensors are economically producible.Alternatively, researches are also done using thin film technology to producesensors on glass or ceramic. This is cheaper and easier.Because the rapid development the semiconductor production and the highquality at small dimensions, the silicon sensors are not to disregard. Thereforethe Lehrstuhl für medizinische Elekronik – the Chair for medical electronics- atTechnische Universität München has developed silicon sensor chips to monitorthe activity of living cell.The most important parameters to measure are oxygen concentration and pHvalue under monitoring temperature and adhesion. Parameter Silicon Thin film [MICH06] technology technology Temperature pn diode Pt1000 Dissolved Clark Sensor Clark Sensor oxygen O2-FET pH ISFET Metal oxide Used sensors on silicon and thin film technologies. Table 1-1For the pH measurement, the ion-sensitive field effect transistor (ISFET) wasused. It provides all the requested advantages and its potentiometric principle iswell adapted to the detection of ions for pH value. Thus, many researches toincrease the pH sensitivity were done for the development of ISFETs.Characterization of miscellaneous multi parametrical silicon based biosensor chips -5-
  6. 6. IntroductionBecause the ISFETs were only for measuring pH it was not able to detectdissolved oxygen in the electrolyte fluid without disturbing it with othersubstances to cause a chemical reaction resulting in change of pH value. It wasnot possible to limit this chemical reaction to be locally, so the same fluid can beused again. A solution for this problem was to use electrochemical half reactions,which can be controlled very locally and without the need to add othersubstances. The electrochemical half reactions can be produced by applying apotential at an electrode, which is small enough to keep the reaction locally. Theproduced ions are only in the surrounding area but in the same time they areenough to produce an electrical potential to be detected by the ISFET sensor.For this an O2-FET was developed and evaluated successfully. The work idea forO2-FET was also to be generalized to measure other dissolved materials thanoxygen. This requires the improvement of the O2-FET measurement proceduresfrom a pulse operating mode to a cyclovoltammetrical scan mode, so themeasured values are significant to concentration of substances we want todetect.In addition to O2-FET, a Clark type sensor -which is also on the same chip-, can beused for measuring dissolved oxygen and confirm the results of the O2-FET.The main work points in this assay are: 1. Examine the sensor chips of visible production errors. 2. Investigating available measurement methods. 3. Theoretical explanation of the measuring methods. 4. Construction of measurement system for each sensor. 5. Procedure of measurements. 6. Discussion of the measured data. 7. Determination of malfunction and failure sources. 8. Development and improvement the measurement procedures.-6- Characterization of miscellaneous multi parametrical silicon based biosensor chips
  7. 7. Materials and methods2 Materials and methodsIn this chapter the used materials for the characterization of the sensor chips arepresented. Recommended working steps and available setting of the usedequipment are also described.2.1 Microscopes The used microscopes with digital cameras. Picture 2-1Characterization of miscellaneous multi parametrical silicon based biosensor chips -7-
  8. 8. Materials and methods2.1.1 Purpose of useTo examine the sensor chips optically for visual manufacturing errors before thebeginning of the evaluating.Comparing the pictures of the sensors before and after measuring will give lot ofinformation about its aging process and it is opportunity to specify commonerrors of the chips.2.1.2 Used equipment and itemsDIGITAL CAMERAS: Nikon E4300: Was used to take the pictures using the first microscope with the high magnification factor. Nikon E5400: It was connected to the second microscope.CARD READER: To transfer the photos taken by the camera from the memory card, where the cameras save the photo files, to a PC using the USB port.2.1.3 Available settingsThe pictures were taken with the digital cameras. The digital camera wasconnected to the microscope by an optical adapter with lens. Additional theoptical zoom of the camera is also used. An accurate zoom factor thereforecannot be given.The first microscope has a bigger zoom factor and it can only magnify theindividual sensors on the chip. The second microscope cannot magnify as goodas the first one, but it used for taking pictures of the whole chip surface.-8- Characterization of miscellaneous multi parametrical silicon based biosensor chips
  9. 9. Materials and methods2.2 Used PC softwareORIGIN PRO 8: It is a professional data analysis and graphing software for engineers. It can handle huge amount of data more efficient than other programs. Its multi-sheet workbooks, publication-quality graphics, and standardized analysis tools provide a tightly integrated workspace to import data, create and annotate graphs, explore and analyze data, and publish work.VOLAMASTER 4 V7.08: It is software with an easy configurable measurement sequence editor for the Voltalab measuring unit. It gives the possibility to monitor the detected response signal in real time and record these values in data tables. The program VoltaMaster 4 has also the ability to show the captured data in graphs, apply filters, and change parameters to highlight information.MS WORD 2007: A good known word processing software. The version 2007 uses a new file format called docx. Word 2000-2003 users on Windows systems can install a free add-on called the "Microsoft Office Compatibility Pack" to be able to open, edit, and save the new Word 2007 files. Alternatively, Word 2007 can save to the old doc format of Word 97-2003 and edit it, but then is not possible to use the “Equation Editor” any more.MS PAINT: A simple graphics painting program that has been included with almost all versions of MS Windows. The used Windows version is Vista, which has more undo levels and better crop functions. The main improvement is to add zoom slider, which increased the work speed with small objects. The program can edit and save in the most known non layer graphic file formats.Characterization of miscellaneous multi parametrical silicon based biosensor chips -9-
  10. 10. Materials and methodsMS POWERPOINT 2007: To make a presentation of this work with figures and animations.ADOBE ILLUSTRATOR CS3: Used to design some figures in vector graphics format.MS EXCEL XP/2007: To plot the raw data of the acquired measurements in graphs and diagrams.MATHTYPE 6.0: A plug-in for MS Office package as an alternative to the Equation Editor which comes with MS Office.ADOBE ACROBAT PROFESSIONAL 8: To make a PDF version of this electronic document for the publication. Files in PDF format are platform independent and contain the fonts used in the document.MS VISIO 2007: Used to design some figures in vector graphics format, it contains also a graphic library to use in making data flow diagrams and work plans.- 10 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  11. 11. Materials and methods2.3 Phosphate-Buffered Saline (PBS)PBS solution is used widely in biochemistry and biological research. That’sbecause its osmolarity and ion concentration usually match those of the humanbody, and because it maintains a constant pH value. = ℎ Molarity Equation. Equation 2-1 Components Mole Weight Concentration Molarity [MICH06] (g/mol) (g/l) (mM)KH2PO4 136 0.20 1.47NaCl 58.5 8.00 138Na2HPO4 * 2H2O 178 1.44 8.1KCl 74.6 0.20 2.68 PBS buffer composition. Table 2-1The PBS solution used has a pH value of about 7.15.BONDING DISSOLVED OXYGENIn addition, to bond from air dissolved oxygen molecules in the PBS it is enoughto add 10g sodium sulfite Na2SO3 to 1l PBS. For an accurate measurement thissolution must be used fresh. The resulted PBS has a pH value of about 8.10. Substance Mole Weight Concentration Molarity [GEST08] (g/mol) (g/l) (mM)Na2SO3 126 10.00 79.4 Used sodium sulfite concentration for bonding dissolved oxygen. Table 2-2Characterization of miscellaneous multi parametrical silicon based biosensor chips - 11 -
  12. 12. Materials and methodsMORE FREE IONSTo make solutions with more dissolved free ions than 150mM of NaCl, we add8.8g to one liter PBS to double the molarity to 300mM. To make severalconcentrations it is easier to dilute a higher concentrated solution with PBS. Forconcentrations below molarity of a usual PBS we add deionised water. Substance Mole Weight Concentration Molarity [MICH06] (g/mol) (g/l) (mM)NaCl 58.5 16.80 288 Concentration of the NaCl to double the amount of the free ions. Table 2-32.4 Ag/AgCl reference electrodeReference electrode is an electrode which has a stable and known potential. Thestability of the electrode potential is reached by employing a redox system withconstant concentrations.2.4.1 Purpose of useReference electrodes are used to keep the electrolyte at a constant potential,without causing electrical current to flow within the electrolyte. The referenceelectrode is difficult to build on the silicon chip by using integrated circuittechnology. That is because a reference electrode uses an electro chemicalreaction to move ions from an electrode into solution.A silver/silver chloride wire is used as reference electrode due these features: - Stable standard potential of 0.2V [MACA78]. - Non-toxic components. - Simple construction. - Inexpensive to manufacture.- 12 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  13. 13. Materials and methodsThe motion of chloride ions at Ag/AgCl wire causes current, which can be e- + AgCl ↔ Ag + Cl-explained as [FARM98]: Reference electrode current. Equation 2-2The corresponding Nernst equation for this reaction is: = − ln [ ] The voltage of reference electrode. Equation 2-3To avoid current to flow through the electrode and then to the electrolyte, a 3MKCl solution is used.2.4.2 Used equipment and items for productionVOLTALAB:(PULSE-CHRONO POTENTIOMETRY) The current that will flow though the electrolyte is set to constant value. The corresponding voltage is also recorded.SILVER AG WIRE: Cut in handy 4cm peaces wire.PLATINUM PT WIRE: One peace 4cm wire.HYDROCHLORIC ACID HCL SOLUTION: With a molarity of 0.1M.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 13 -
  14. 14. Materials and methods2.4.3 Producing assemblyElectrolysis by electrochemical oxidation of the silver wire in 0.1mMhydrochloric acid HCl solution: - Ag as anode at the plus pole (Work-Prot) of the voltage source Voltalab. - Pt as cathode at the minus pole (Ref-Port) of Voltalab. Wiring schema for the production of Ag/AgCl electrode. Picture 2-2While producing an AgCl on the Ag wire the following chemical reactionshappen: On the Ag-Anode side: 2Ag + 2 HCl à 2 AgCl + 2 H+ + 2 e- (AgCl is darker than Ag) Half reaction the Ag side. Equation 2-4 On the Pt-Cathode side: 2 H + + 2 e- à H 2 (H2 bubbles rise on Pt) Half reaction the Pt side. Equation 2-5- 14 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  15. 15. Materials and methodsSo the whole reaction can be summed to: 2 Ag + HCl à 2 AgCl + H2 The whole chemical reaction for producing Ag/AgCl electrode. Equation 2-62.4.4 Production procedure 1. A constant current of 4mA to flow through the electrodes is applied 2. Becoming the silver wire darker and rising hydrogen gas on the platinum wire is an indicator for building silver chloride. 0 50 100 Time [s] 150 200 250 -0,7 -0,8 -0,9 -1 -1,1 Voltage [V] -1,2 -1,3 -1,4 -1,5 -1,6 -1,7 The measured electrolysis voltage at 4mA for producing Ag/AgCl. Graph 2-1 3. After few minutes (4 minutes) the hydrogen bubbles will stop to develop on the platinum side, this means the silver chloride is already reached its maximal thickness on the silver wire.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 15 -
  16. 16. Materials and methods Electrolysis current for producing Ag/AgCl. Graph 2-2This period can be also known from the electrolysis current curve below, wherethe current a 1mA doesn’t change anymore, if we applied a constant voltageinstead of current.- 16 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  17. 17. Materials and methods2.5 Incubator The used incubator. Picture 2-3The used incubator is Kelvitron t6030 from Heraeus Instruments. It has avolume of 30l and offers enough space to set the sensors and its pin box, withouthaving an unneeded free volume to heat. The more volume there is to heat themore time is needed to reach the target temperature.2.5.1 Purpose of useTo make and keep a constant tempered environment for temperature dependentmeasurements.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 17 -
  18. 18. Materials and methodsThe incubator can be also used as faraday cage.2.5.2 Available settingsThe incubator can heat up to 300°C. Therefore, it is not possible to have atemperature below environment temperature in the room. Although, it acceptssettings below room temperature, but this practically cannot be realized. Coolingdown takes several hours. So, when measuring at many temperatures, it is easierand faster to begin with the lowest temperature. Damped oscillations of the incubator. Graph 2-3Heating up the air in the incubator to a constant target temperature needsrelatively long time compared e.g. to a fan oven. This is because the oscillation ofthe heating process of the incubator, which uses pulsed operating of the heatingelements without circulating the air. The bigger the difference between targetand start temperatures is, the bigger is the oscillation amplitude and time to get aconstant target temperature.- 18 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  19. 19. Materials and methods2.6 Regulated DC power supply unit The used Power supply [CONR08]. Picture 2-4Laboratory power supply VLP-1303 PRO delivers constant potential differencebetween its input minus port and output plus port. The potential difference canbe adjusted manually and displayed with its corresponding current flowingthrough the ports.The voltmeter is used to control the adjusted voltage. The display of the powersupply has not enough digits to display the applied voltage exactly. The displaycan have here a rounding error up to 100%, because the missing second andthird digit after the radix point, which can be 99, a voltage of 0.099V can beshown inaccurate on the units display as “00.0V”.2.6.1 Purpose of useThe voltage supplied by this unit is used to raise the potential of the gate abovethe source potential on the reference MOSFET of nMOS chips. This potentialbuilds the electrons channel between source and drain. Through this channel cancurrent flow. The width of this channel is controlled by the applied voltage atgate using this power supply. This voltage must be very constant; otherwise thesmall changes of this voltage can affect the transistor current very much, so thecharacterization cannot be done as desired.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 19 -
  20. 20. Materials and methods2.6.2 Available settingsThe power supply has two outputs. The first output has a range of 0V to 3V at amaximal current of 3A. The second output has a range of 3V to 6V at maximalcurrent of 2A.The unit -beside the supplying of a constant voltage- can also limit the currentflow through the first output. To do that; turn the control AMPERE clockwiseuntil the red LED for current limiting (CC or OL) referring to the output goes offand the green LED for voltage limiting (CV) lights up. Then the VOLT control canbe used to adjust the desired output voltage.It is not possible to limit current at the second output, that’s why it has only onecontrol to adjust. By using the pushbutton, the voltage of the second output canbe displayed. Simply, hold the button down as long as is wished to see the valueson the display.- 20 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  21. 21. Materials and methods2.7 Voltalab® 80/10 Measurement unit PGZ402 [RADI68]. Picture 2-52.7.1 Purpose of useVoltaLab 80 and its basic version VoltaLab 10 are simple and easy to configurepotentiostats PGZ402/100 and electrochemical software VoltaMaster 4combinations, for recording, analyzing and evaluating of electronic andelectrochemical elements. The VoltaLab unit is connected to a PC via the RS232interface port.2.7.2 Available settingsVoltalab has the software GUI VoltaMaster 4. VoltaMaster 4 v7.08 is an easyconfigurable measurement sequence editor. It gives the possibility to monitorthe detected response signal in real time and record these values in data tables. Ithas a huge amount of possible configuration settings to measure and evaluatecircuits connected to the system. Voltammetry, amperometry and coulometryare only some examples of the methods, which Voltalab can be used for.The program VoltaMaster 4 has also the ability to show the captured data ingraphs, apply filters, and change parameters to highlight information.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 21 -
  22. 22. Materials and methods GUI interface of the VoltaMaster 4. Picture 2-6Some technical data of PGZ402 [RADI68]:Specifications Working rangeMaximum compliance voltage ±30VMaximum current output ±1AMaximum polarisation voltage ±15VA/D converter 16bitMeasurement period 500msMax. scan rate 20V/sMax. frequency 100kHzMin. frequency 1mHzDynamic Impedance Driven up to 100mV/sStatic manual & Static auto up to 1V/sFeedback manual & Feedback auto up to 20V/s Specifications cable of the PGZ402. Table 2-4The next graph shows an example measurement at a 10MΩ resistor. For thismeasurement one side of the resistor is connected to the WORK-input of thePGZ402 and the other side is connected to the REF- and the AUX-input. The- 22 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  23. 23. Materials and methodsvoltage-current V-I curve is absolutely linear and there are no visible jumpsbetween the measurement ranges. [WIES03] Measurement of a 10MW resistor with the PGZ402 unit. Graph 2-4OPEN CIRCUIT POTENTIAL: The Open Circuit Potential corresponds to the WORK potential measured versus the REF potential. As the name of the measurement method implies the circuit is open so there is no current to flow and measure. A measuring sequence of 30 seconds is enough to calibrate to a drift threshold near zero. Available settings for Open Circuit Potential measuring method. Picture 2-7Characterization of miscellaneous multi parametrical silicon based biosensor chips - 23 -
  24. 24. Materials and methodsPOT. CYCLIC VOLTAMMETRY Cyclic voltammetry sweep the potential at a given rate and measure the current. The curve obtained is known as a "voltammogram", where voltage to current values are plotted. A ranging for current measurement is available depending on the scan rate. Available settings for Pot. Cyclic Voltammetry measuring method. Picture 2-8PULSE - CHRONO POTENTIOMETRY The WORK potential is measured versus the REF potential while the current is maintained at a pre-set value. Available settings for Chrono Potentiometry measuring method. Picture 2-9- 24 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  25. 25. Materials and methodsPULSE - CHRONO AMEPEROMETRY The current flowing from REF to WORK is measured while the potential between them maintained at a pre-set value. Available settings for Chrono Ameperometry measuring method. Picture 2-10IMPEDANCE - POT. FIXED FREQ. EIS (CAPACITANCE) The WORK potential versus REF is imposed and the electrochemical impedance is recorded at one fixed frequency with an AC signal. A real time plot displays Zimaginary and Zreal versus potential. Available settings for Pot. Fixed Freq. EIS (Capacitance) measuring method. Picture 2-11Characterization of miscellaneous multi parametrical silicon based biosensor chips - 25 -
  26. 26. Materials and methods2.8 Sensor chipsIn this assay, we have two kinds of chips to probe. Both chips have the same kindof sensors, which are temperature, Clark, IDES, ISFET and O2-FET sensors.The first produced chip lot was manufactured at Micronas AG. We refer to this lotwith the name cMOS. The second was produced at the Lehrstuhl für MedizinischeElektronik and we name it nMOS. Although both chips are in cMOS technologyand in nMOS channel structure, we select this notation from its developmenthistory.At the early stages, sensors were made on glass chips, and then came out thesilicon cMOS compatible production technology, and with the next design, it hasbeen more specifically so it is called nMOS referring to the n channel structure ona p-substrate. It is not to mix up with the cMOS and nMOS pair, where it refers todigital circuit design.The following short compression can be useful to know more about thecomponents on the both sensor chips: cMOS nMOS d=6mmChip reservoir A=28mm² V=7µL 68 contactsChip board A=24x24mm²Die area A=12.5x14.5mm² A=7.5x7.5mm²TD 1CLARK d=35µm(Work electrode) A=960µm²IDES A=~3mm² A=10.2mm² 3x (+4x O2-FETs) 4x (+2x O2-FETs)ISFET AGate=100x3µm² AGate=100x10µm² 4x 2xCV/O2-FET ANME=2096µm² ANME=2600µm²REF-FET not available 1x Fast compare between cMOS and nMOS chips. Table 2-5- 26 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  27. 27. Materials and methods2.8.1 cMOS 1mm The cMOS chip and its sensors. Picture 2-12The cMOS chips have the following objects: a. Temperature sensor: Using a temperature diode (TD). b. Adhesion sensor: One IDES with a contact area of about 3mm². c. Electrode: Metal electrode made of palladium. d. pH value sensors: 7 ISFET sensors including the sensors of 4 O2-FETs. e. Dissolved oxygen sensors: 5 Clark type sensors and 4 O2-FET sensors.The used sensor chips for this project have the names u01, u02 and u03. All arefrom the same batch and were examined under microscope for visual noticeableproduction errors on the chip surface before beginning of the measurements.The examination under microscope is repeated casually to prevent anymeasurements may interpreted mistakenly and falsify the results.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 27 -
  28. 28. Materials and methods Pins assignment (not true to size). Picture 2-13 PIN Chip cMOS Connector 1 ISFET A Drain 2 O2/CV- -FET A Source 3 Drain ISFET B 4 Source 5 Cathode Temperature diode 6 Anode 7 Drain ISFET C 8 Source 9 ISFET D Drain- 28 - Characterization of miscellaneous multi parametrical silicon based bios biosensor chips
  29. 29. Materials and methods 10 Source 11 Substrate x1 Sub x1 15 Source ISFET E 16 NME O2/CV-FET 1 18 Drain ISFET F 17 Drain O2/CV-FET F 20 Working electrode 22 Clark sensor Auxiliary electrode 24 Reference electrode ISFET F 23 NME O2/CV-FET F 25 Working electrode 26 Clark sensor 2 Auxiliary electrode 27 Reference electrode 28 Anode 29 Anode 2 IDES 31 Cathode 32 Cathode 2 ISFET F 30 Source O2/CV-FET F 33 Auxiliary electrode 34 Clark sensor 3 Working electrode 35 Reference electrode 36 Substrate x2 37 Reference electrode 38 Substrate x3 Sub x3 50 Working electrode 51 Clark sensor 4 Reference electrode 53 Auxiliary electrode 52 Substrate x4 54 Working electrode Clark sensor 5 55 Reference electrodeCharacterization of miscellaneous multi parametrical silicon based biosensor chips - 29 -
  30. 30. Materials and methods 57 Auxiliary electrode 56 NME ISFET G 58 Source O2/CV-FET G 59 Drain ISFET A 60 NME O2/CV-FET A Pins assignment of the pin box. Table 2-6Pin numbers within yellow colored cells means that numbered pin, whichbelongs to a sensor, does not exist on the pin box output. (See “Pin box” chapter2.9 on page 34)2.8.2 nMOS 1mm The nMOS chip and its sensors. Picture 2-14- 30 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  31. 31. Materials and methodsThe nMOS chips have the following objects: a. Temperature sensor: Using a temperature diode (TD). b. Adhesion sensor: One big IDES with a contact area of about 10mm². c. pH value sensors: 6 ISFET sensors including the sensors of 2 O2-FETs. d. Dissolved oxygen sensors: A single Clark type sensor and 2 O2-FET sensors.The used sensor chips for this project have the names f5, f8, i5 and c10. All arefrom the same batch and were examined under microscope for visual noticeableproduction errors on the chip surface before beginning with the measurements.The letter in the name of the sensor chip corresponds to the horizontal placingthe sensor chip on the wafer, and the number after it is for the vertical place. The sensor chips on the nMOS 4 inch wafer. Picture 2-15The examination under microscope is repeated casually to prevent anymeasurements may interpreted mistakenly and falsify the results.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 31 -
  32. 32. Materials and methods Pins assignment (not true to size)[WIES05]. Picture 2-16 PIN Chip nMOS Connector 1 Drain ISFET A 2 Source 3 Drain ISFET B 4 Source 5 Cathode Temperature diode 6 Anode 7 Drain ISFET C 8 Source 9 Drain ISFET D 10 Source 11 Substrate x1 Sub x1 15 ISFET E Source- 32 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  33. 33. Materials and methods 16 O2/CV-FET 1 NME 18 Drain 20 Working electrode 22 Clark sensor Auxiliary electrode 24 Reference electrode 28 Anode 29 Anode 2 IDES 31 Cathode 32 Cathode 2 63 Drain ISFET E 64 NME O2/CV-FET 2 65 Source 66 Drain 67 REF-MISFET Gate 68 Source Pins assignment of the cMOS chips. Table 2-7ISFET E has no contact pin for its source contact on the pin box output. Thereforeit is colored in the table with yellow.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 33 -
  34. 34. Materials and methods2.9 Pin box Picture of the used pin box. Picture 2-172.9.1 Purpose of useThe pin box is an adaptor, which converts the contact pins from the base of thesensor chip board using a PLCC68 socket to BNC connector type. The BNC is anisolated connector type used widely by most of measuring units in labs. The casehas ports for 48 lines including a connector for the grounding of the aluminumcase.Although the PLCC68 socket has 68 contacts, which is more than the availableoutputs connector on the pin box, there is no need to have all the 68 pins of thesocket to have BNC outputs. That’s because the sensors on the chip need only amaximum of 46 lines to operate.- 34 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  35. 35. Materials and methods2.9.2 Available connectors PIN Chip cMOS Chip nMOS Connector 1 ISFET A Drain ISFET A 2 O2/CV-FET A Source 3 Drain ISFET B ISFET B 4 Source 5 Cathode Temperature diode Temperature diode 6 Anode 7 Drain ISFET C ISFET C 8 Source 9 Drain ISFET D ISFET D 10 Source 11 Substrate x1 Substrate x1 Sub x1 13 14 15 Source ISFET E ISFET E 16 NME O2/CV-FET 1 O2/CV-FET 1 18 Drain ISFET F 17 Drain O2/CV-FET F 19 20 Working electrode 22 Clark sensor Clark sensor Auxiliary electrode 24 Reference electrode 21 ISFET F 23 NME O2/CV-FET F 25 Working electrode Clark sensor 2 26 Auxiliary electrodeCharacterization of miscellaneous multi parametrical silicon based biosensor chips - 35 -
  36. 36. Materials and methods 27 Reference electrode 28 Anode 29 Anode 2 IDES IDES 31 Cathode 32 Cathode 2 ISFET F 30 Source O2/CV-FET F 33 Auxiliary electrode 34 Clark sensor 3 Working electrode 35 Reference electrode 36 Substrate x2 37 Reference electrode 38 Substrate x3 Sub x3 39 40 41 42 43 44 45 46 47 48 49 50 Working electrode 51 Clark sensor 4 Reference electrode 53 Auxiliary electrode 52 Substrate x4 54 Working electrode Clark sensor 5 55 Reference electrode- 36 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  37. 37. Materials and methods 57 Auxiliary electrode 56 NME ISFET G 58 Source O2/CV-FET G 59 Drain ISFET A 60 NME O2/CV-FET A 61 62 63 Drain ISFET E 64 NME O2/CV-FET 2 65 Source 66 Drain 67 REF-MISFET Gate 68 Sourcegrounding Pins assignment of the nMOS chips. Table 2-8Pin numbers within yellow colored cells means that numbered pin does not existon the pin box output. Empty yellow cells are pins which does not havecorresponding sensor on the chip.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 37 -
  38. 38. Materials and methods2.10 Non-Semiconductor sensorsNon-Semiconductor sensors are the ones which are on the surface of the chipand have no contact with the silicon semiconductor layer. Clark and IDES sensorsare produced by silicon technology using metallization and oxidation, but theyare isolated with an oxide layer from the silicon.2.10.1 Clark sensor (Amperometry)2.10.1.1 IdeaVoltammogram is applying a voltage ramp to an electrolyte to determine avoltage region where voltage is essentially independent of current.A typical voltammogram of aqueous solutions e.g. PBS in range of 0 to -1.4V hasseveral regions. These regions vary according to dissolves substances in thesolution. The regions of a solution, which is with oxygen dissolved, can beillustrated and explained as fallowing. [BRIS06] Typical voltammogram of Clark sensor. Graph 2-5- 38 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  39. 39. Materials and methodsREGION I (ZERO CURRENT REGION): The voltage U is not enough to reduce molecules at the work electrode. The current there is almost zero.REGION II (INTERMEDIATE REGION): The ability of the oxygen molecules to pass the electrochemical double layer (inner and outer Helmholz plane) to the work electrode limits the current. Cause of diffuse current of dissolved oxygen [ISRA07]. Picture 2-18REGION III (PLATEAU REGION): Transport of oxygen molecules to the work electrode is causing a electrolyte solution. ∝ diffusion current, which is relative to the concentration of oxygen in the . This is limited to current. The width of the region is dependent on the diffusion of the oxygen molecules. This can be explained with Ficks first law, which is used in steady-state diffusion, i.e., when the concentration within the diffusion volume does not change with respect to time.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 39 -
  40. 40. Materials and methods =− Diffusion flux. Equation 2-7 Where: D is the diffusion coefficient or diffusivity, is the concentration of oxygen in the solution, x is the position. And the electrical current caused by diffusion is = Diffusions current. Equation 2-8 Where: n is the number of free transported electrons. F is the Faraday constant. A is area of the cross section. x is the position. is the diffusions flux. In addition, using Laplace transformation we get[BARD00]: √ ∗ ( )= √ Diffusion Current respect to time t. Equation 2-9 For current after a long time and a temperature of 25°C, it can be simplify to: =4 Oxygen concentration current. Equation 2-10 Where r is radius of the work electrode.[MUGG02]- 40 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  41. 41. Materials and methodsREGION IV (DISSOCIATION REGION): Over potential dissociates water molecules. This is visible by the hydrogen formation in gas form. Solutions without dissolved oxygen have almost this region only.2.10.1.2 Equipment and itemsVOLTALAB 80: Voltammetry - Pot. Cyclic Voltammetry: To get a curve we use a potential ramp as input parameter and read the current response of the Clark sensor, in the range of zero to -1.4V. To avoid current flowing through the reference electrode, we use an auxiliary electrode.PIN BOX ASSIGNMENT: Sensor Auxiliary Working Reference No. electrode electrode electrode 4 22 20 24 Pins assignment of the Clark sensor. Table 2-9 Sensor number 4 on cMOS chips has the same contact pin numbers as the single sensor on nMOS chips.SOLUTIONS: - PBS: Phosphate buffered solution with pH value of 6.5 with from air dissolves oxygen. The oxygen saturation in PBS has a concentration of 7.8811mg/l or 0.25mM. - Calibration solution: Na2SO3 (M=126g/mol) added as 1g to 100ml PBS, enough to bind the oxygen molecules in the PBS solution.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 41 -
  42. 42. Materials and methods 2 + → 2 Chemical reaction to bind dissolved oxygen. Equation 2-11SERSOR CHIPS 1 2 Reference elektrode 3 4 5 Working electrode Auxiliary electrode1mm 250µm Clark sensor on the cMOS chip. Picture 2-19 Auxiliary electrode Working electrode reference 1mm 250µm electrode Clark sensor on the nMOS chip. Picture 2-20Working electrode is circle shaped and has diameter of 35µm on both chips. Theauxiliary and reference electrodes are surrounding the working electrode in ringform. The reference electrode is as big as about one third surface area of theauxiliary electrode. On the cMOS chips, this ring is directly surrounding theelectrode. On the other side, the ring of the nMOS chip has a distance of about250µm from the working electrode.- 42 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  43. 43. Materials and methodsThe nMOS chip has only one Clark sensor, where the cMOS has 5 Clark sensors.The single sensor of the nMOS has the same contacts of the sensor number 4 onthe cMOS chips.2.10.1.3 Measurement assembly Schematic design of the measuring system. Picture 2-21 Measurement assembly. Picture 2-22Characterization of miscellaneous multi parametrical silicon based biosensor chips - 43 -
  44. 44. Materials and methods2.10.1.4 Measurement settings and parameters are to be chosen, in this case 10 / . - To reduce capacitive effects caused by polarization slower scan rates An example for a voltammogram voltage. Graph 2-6 −1.4 , so no need to scan more than this value. - By PBS the disassociation of the water within it begins already below is in around −10 . Therefore, the range of the measured current must be within ±1µ , otherwise the Voltalab unit -due the change to a - smaller accuracy range- will not be able anymore to detect small currents in nA range - The influence of the temperature is to ignore, due the small effect of the temperature on the diffusions constant, which is under 2%.[HITC78]. = - The diffusions constant D is an exponential function of temperature T: Diffusions current. Equation 2-12 Where: is the diffusions constant at a reference temperature, is the activation energy for diffusion, R is gas law constant.- 44 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  45. 45. Materials and methods2.10.1.5 Procedure 1. Making several cycles at higher scan rate using the setting explained in the previous chapter will deliver more accurate results. 2. Repeating the measurement again with the same parameters but this time using a PBS solution without oxygen dissolved in it. 3. Choose an operation point from the tableau region with significant difference between the measurement with and without oxygen.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 45 -
  46. 46. Materials and methods2.10.2 IDES Sensor (Impedimetric)2.10.2.1 IdeaAn electrochemical half cell consists of the resistance of the electrolyte solution,the capacity of the electrochemical double layer q.v. Clark sensor (Amperometry)and the resistance of the charge transfer. Using impedance measurement we cancalculate the imaginary component as like capacity and the real component asthe resistance.In order to determine impedance, complex Ohm’s law is used: ( ) = ̅ ( ) Complex Ohm’s law. Equation 2-13For impedance measurement, a two-wire electrical measurement assembly isused. However, when the impedance to be measured is relatively low, or theimpedance of the probe is relatively high, a 4-point probe measurement willyield more accurate result.TWO-WIRE MEASUREMENT METHOD: A known alternating voltage at a defined frequency is applied across the unknown impedance Z. This voltage source is alternating symmetric at zero volts and it should not generate a current. In other words, the voltage source must have a high resistance at chosen frequency. The current that flows through the probe is measured. The impedance can then easily determined by dividing the applied current by the measured current. An ideal circuit for measuring an impedance Z. Picture 2-23- 46 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  47. 47. Materials and methods The measurements done with two-wire setup include not only the impedance of the electrolyte but also the impedance of the leads and contacts. This may be a problem falsifying the results. When using an impedance meter to measure values above few ohms or picofarads, this added small impedance is usually not a problem. However, when measuring low impedances or when contact and lead resistance and capacity may be high, obtaining accurate results with a two-wire measurement may be problematical. Realistic circuit incl. interfering components. Picture 2-24FOUR-WIRE MEASUREMENT METHOD: A solution for the problem of two-wire measurements is using the four- wire measurement setup. Because a second set of probes are for sensing and since the current I0 though the electrolyte is negligible small, only the voltage drop across the device under test is measured. As a result, impedance measurement is more accurate. Four-wire impedance measurement circuit. Picture 2-25Characterization of miscellaneous multi parametrical silicon based biosensor chips - 47 -
  48. 48. Materials and methods2.10.2.2 Equipment and itemsVOLTALAB 80: Pot. Fixed Freq. EIS (Capacitance): To measure the impedance, an alternating sinus voltage is applied and the resulted current is measured.PIN BOX ASSIGNMENT: Sensor Anode Anode No. 2 Cathode Cathode No. 2 IDES 28 29 31 32 Pins assignment of the IDES sensor. Table 2-10SOLUTIONS: - De-ionized water. - PBS: Phosphate buffered saline solution. It has a molar concentration of about 150mM of NaCl. - PBS solutions with 75, 225, and 300mM of NaCl. -SERSOR CHIPS - nMOS chips have a visible sensor area of about A=8mm², while cMOS chips have about one third of it. 2mm IDES sensor on the nMOS chip. Picture 2-26- 48 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  49. 49. Materials and methods 1mm IDES sensor on the cMOS chip. Picture 2-27The nMOS chip has a polygon shaped IDES and it covers almost the half visualarea of the fluid contact surface. The IDES on the cMOS is much smaller andrectangular. On the both of the chips, the IDES sensor is placed centered and theother sensors types is surrounding it.2.10.2.3 Measurement assembly Schematic design of the measuring system. Picture 2-28The impedance measurement assembly is good enough to achieve clear resultsusing the two-wire method. The Voltalab and the isolated BNC cables haveinsignificant effect on the measured values, due its low electrical resistance andcapacity.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 49 -
  50. 50. Materials and methods2.10.2.4 Measurement settings and parametersTo measure the impedance, a voltage of 30mV with a frequency of 10kHz isapplied and the resulted current for 20 seconds is measured. AC signal for impedance acquisition. Graph 2-7The applied sinus voltage is alternating at zero with an enough frequency toavoid current flow. Influence of frequency on impedance[BRIS06]. Graph 2-8- 50 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  51. 51. Materials and methodsUsing Ohm’s law the impedance can be easily calculated and plotted in real andcomplex components. ̅= ̅ + ̅ = + = ̅ ( ) = ̅ ( ) =2 Real and complex component of impedance. Equation 2-142.10.2.5 Procedure 1. Making several cycles using the setting explained in the previous chapter with a PBS solution of 75mM NaCl. 2. Repeating the measurement again with the same parameters but this time using PBS solutions with steps of 75mM to 300mM. 3. The resulted measurements should be vary in real component.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 51 -
  52. 52. Materials and methods2.11 Semiconductor sensorsSemiconductor sensors are in contrast to the non-semiconductor sensors havestructures within the silicon semiconductor layer. Temperature diode, ISFET andCV/O2-FET all share the silicon layer with different doped regions.2.11.1 Temperature Diode (Potentiometry)Temperature change effects the properties of semiconductors, and this willfalsify the measurements. Therefore sensors falsified by temperature must beadjusted with a correction factor relatively to the temperature. When using livingcells the cell activity is temperature dependent.2.11.1.1 IdeaThe characteristic curve of a p-n diode shows a direct temperature dependency.This can be explained with the electronic band structure model. Operating such adiode with a current in forward bias and a voltage , gives us Schockley’sdiode law [MSZE98]: = ( − 1) Schockley’s diode law. Equation 2-15For ≫ = Schockley’s simplified diode law. Equation 2-16Where: is the thermal diode current, is the saturation current, is the voltage across the diode, is the thermal voltage.The diode equation in respect of voltage can be written as:- 52 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  53. 53. Materials and methods = Diode law in respect to voltage. Equation 2-17The thermal voltage UT is a known constant defined by: = Thermal voltage. Equation 2-18Where: q is the magnitude of charge on an electron (elementary charge), k is Boltzmann’s constant, T is the absolute temperature of the p-n junction in kelvins.The voltage change is −2.25 / in the range from −50° to +150°C. [STEP06].So is approximately 26 mV at room temperature of 300K. [MOHR00]. I-V characteristic curve of a diode and the influence of temperature. Graph 2-9Characterization of miscellaneous multi parametrical silicon based biosensor chips - 53 -
  54. 54. Materials and methods2.11.1.2 Equipment and itemsINCUBATOR: For a constant and adjustable environment temperature.VOLTALAB 80: Voltammetry - Pot. Cyclic Voltammetry: To get a diode curve we use a potential ramp as input parameter and read the current response of the diode, in the range of zero to 3V. Pulse - Chrono Potentiometry: At chosen fixed work current we measure the voltage as a function of the temperature change.PIN BOX ASSIGNMENT: Sensor cathode Anode TD 5 6 Pins assignment of the temperature diode. Table 2-11SERSOR CHIPS 1mm 15µm Temperature diode on the cMOS chip. Picture 2-29- 54 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  55. 55. Materials and methods1mm 30µm Temperature diode on the nMOS chip. Picture 2-30The diode on the nMOS chip has a remarkable bigger area than the pn diode ofthe cMOS. This will cause different behavior for the temperature dependency.The pn diode is isolated with the protection layer and therefore it has no directcontact to the electrolyte. This makes the temperature sensor electrolyteindependent, so there is no aging caused by contacting with fluids.2.11.1.3 Measurement assembly Schematic design of the measuring system. Picture 2-31For fast tests, fluids with different temperatures can be used instead of theincubator. But characterizing and long term measurements are not possible dueCharacterization of miscellaneous multi parametrical silicon based biosensor chips - 55 -
  56. 56. Materials and methodsthe small amount of the fluid (7µl), which has a smaller heat capacity than thesensor chip. So, the fluid will get the temperature of the chip in a short time.2.11.1.4 Measurement settings and parametersA diode characteristic curve is U-I curve. That means we measure the current independence on the applied voltage. Instead of choosing voltage as an operationpoint and measuring its current, we set a current as operation point and measureit’s correspond voltage. That is because the voltage is easier and more accurateto measure using a simple electrical circuit than measuring a current.The supplied current can be easily generated with a voltage to current amplifiercircuit.2.11.1.5 Procedure 1. Make a fast test to determine the resulted current range within a voltage from zero to 3 volts. Our target is to get smallest current as an operation point. A higher current causes more internal heating of the diode, which is not only falsifying the real temperature of the sample, but it can also rise its temperature to unwanted values especially for living cells. 2. At room temperature, measuring the current for a given voltage ranging from zero to maximal 3 volts, and repeat it at higher temperatures. It’s not to forget, that in the course of the day, the room temperature can be vary according to the sunlight, operating of electrical equipment and the number of persons sharing the same room. All this produce extra heat in the room and may cause to bias the results. So using an incubator with a temperature a little above room temperature will give a more clear result without having temperature variations when measuring. 27°C seems to be easy to realize and keep constant by the incubator. The used incubator needs about an hour to heat up and to remain at a constant temperature, and another one after reaching the target temperature, to let the sensor chips and its terminal box also to reach this temperature. 3. Determining the best operation point, at lowest current with significant temperature influence. This can be done easy by reversing the voltage- current U-I curve to current-voltage I-U curve and selecting the biggest voltage range at the same current.- 56 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  57. 57. Materials and methods2.11.2 Reference MISFET (nMOS)2.11.2.1 Idea MISFET [HENN05]. Picture 2-32A MISFET is an active part. It works like a voltage controlled resistor. It has threeports (electrodes): Gate, Source and Drain.As basic material a low p doped silicon substrate is used. In this substrate twohigh n doped regions are embedded. These two regions make the drain andsource ports. Between these two regions there must be a p doped region so weget an npn structure. Though this npn flows for now no current, because it is likea np diode which is connected afterwards with a pn diode. When the first diodeallows flowing current through it, the second one will block it.Above the p doped region, which is between the n regions, is an isolation layerand then a metal layer. This construction builds the gate port.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 57 -
  58. 58. Materials and methodsBy applying a potential at the gate port, an electrical field is created, whichcreates within the embedded p region an n electrons channel. The size of thischannel is proportional to the gate potential. Source-drain current. Graph 2-10, Picture 2-33Usually source and drain pins are interchangeable, but the manufacturing maybe not made symmetric.The MISFET has three operation modes:CUT-OFF, SUB-THRESHOLD OR WEAK INVERSION M ODE: This operation mode is when the gate-source voltage UGS smaller than threshold voltage of the device Uth. The transistor is turned off. This means there is ideally no current flows through the transistor, because there is no conducting n-channel between source and drain. In reality, the Boltzmann distribution of electron energies is allowing some electrons at the source to enter the n channel and flow to the drain. This results in a sub-threshold leakage current.- 58 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  59. 59. Materials and methodsLINEAR/OHMIC REGION OR TRIODE MODE: This operation mode is when the gate-source voltage UGS bigger than the threshold voltage Uth and drain-source voltage is smaller than the difference between source-gate UGS and threshold Uth voltages. The transistor is turned on. This means, that the n channel between the drain and source has been created: This allows current to flow through the transistor. The MISFET operates in this mode like a controllable resistor. This can be done by the gate voltage. This current has also dependency on the gate’s width and length and the isolating layer electrical capacitySATURATION MODE OR ACTIVE MODE: This operation mode is when the gate-source voltage UGS is bigger than the threshold voltage Uth and drain-source voltage is bigger than the difference between source-gate UGS and threshold Uth voltages. The transistor is turned on. This means that the n channel between the drain and source has the maximal capacity, which allows current to flow through it. The drain current is now weakly dependent upon drain voltage and controlled primarily by the gate-source voltage.2.11.2.2 Equipment and itemsVOLTALAB 80: Voltammetry - Pot. Cyclic Voltammetry: To get the characteristic curve of the ISFET we use a potential ramp as input parameter and read the current response.VOLTAGE SOURCE: Applying several voltages on the gate port, to control the current between source and drain.PIN BOX ASSIGNMENT: Drain Gate Source REF- 63 64 65 MISFET Pins assignment of the cMOS chips. Table 2-12Characterization of miscellaneous multi parametrical silicon based biosensor chips - 59 -
  60. 60. Materials and methodsSERSOR CHIPS Chip No. of sensors Gate area nMOS 1 3x100µm² cMOS 0 n/a Pins assignment of the cMOS chips. Table 2-13The reference transistor is identical in contraction to the ISFET sensor, which isdescribed and evaluated in the next chapter. The characteristic curves of thereference are in the same range of the ISFET. So a malfunction of the reference isa good indicator for the malfunction ISFET, without using any fluids to test. 1mm 100 µm Reference MISFET on the nMOS chip. Picture 2-34Above is a picture of the die. The MISFET is located in the top right corner of it.The transistor can be seen only before the packaging. The package for theprotection of the bonding and the plastic fluid reservoir above it covers thetransistor completely.- 60 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  61. 61. Materials and methods2.11.2.3 Measurement assembly Schematic design of the measuring system. Picture 2-35No need for fluids to operate the reference transistor. Transistors havetemperature dependency, so operating the transistor for a long time may causeto heat and that will effect the measuremesnt. Using fluid can make the transistorheating being less, and that’s by taking some heat from the surface of the chip tothe fluid.2.11.2.4 Measurement settings and parameters For the characteristic curve of the reference MISFET, the used potential ramp of the UDS is in the range of 0V to 5V. The UGS is in 1V steps from 0V to 5V.2.11.2.5 Procedure 1. Measuring IDS while applying UDS in a ramp from 0 to 5V. The power supply is not yet connected the gate port. 2. Repeating the measurement of IDS while increasing USG in 1V steps from 0V to 5V.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 61 -
  62. 62. Materials and methods2.11.3 ISFET Sensors for pH-Measurement2.11.3.1 IdeaThe pH of a solution is dependent on the concentration of hydrogen ions orits correspondent hydroxide ions. The higher is the concentration ofhydroxide ions in a solution, the higher is its pH value. = −log [ ] = 14 − = 14 + log [ ] ∆ ( ) = − log [ ( )] = 14 + log [ ( )] pH value dependency on the concentration of . Equation 2-19ISFET has an ion sensitive layer. On this layer the gathering ions create apotential. This potential is the ISFET controlling potential of gate. The n-channelwithin the semiconductor of the ISFET is established and allows the current toflow though the transistor from source to drain. The higher is the gate vs. sourcepotential, the wider is the n-channel and higher is the current flow from sourceto drain. Effect of the hydroxide on the source drain current. Graph 2-11, Picture 2-36- 62 - Characterization of miscellaneous multi parametrical silicon based biosensor chips
  63. 63. Materials and methods2.11.3.2 Equipment and itemsVOLTALAB 80: Voltammetry - Pot. Cyclic Voltammetry: To get the characteristic curve of the ISFET we use a potential ramp as input parameter and read the current response. Pulse - Chrono Potentiometry: At chosen fixed work current we measure the voltage as a function of the pH change.PIN BOX ASSIGNMENT: Drain Source ISFET A 1 2 ISFET B 3 4 ISFET C 7 8 ISFET D 9 10 ISFET E 18 15 Pins assignment of the ISFET sensors. Table 2-14 ISFET E is also in the same time an O2-FET with a surrounding NME.SOLUTIONS: - PBS: Phosphate buffered saline solution with a pH value of 7.3 - A seconds PBS solution with a pH of 6.8.REFERENCE ELECTRODE: - Ag-AgCl electrode.Characterization of miscellaneous multi parametrical silicon based biosensor chips - 63 -
  64. 64. Materials and methodsSENSOR CHIPS Gate Drain Source 1mm 100µm ISFET sensor 4 on the cMOS chip. Picture 2-37 Gate Drain Source 1mm 100µm ISFET sensor on the nMOS chip. Picture 2-38The placing of the ISFET sensors on both chips is different. While the sensors oncMOS chip are evenly distributed on the chip surface, the ones of the nMOS chipare on the both sides of the IDES sensor, which is located in the middle of thechip.- 64 - Characterization of miscellaneous multi parametrical silicon based biosensor chips

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