Chemical Quantities in Blood. 11. Biochemistry-1 Electrochemical Measurements of Biochemical Quantities. Hematocrit

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1 11. Biochemistry-1 Electrochemical Measurements of Biochemical Quantities Chemical Quantities in Blood Living cells are chemical factories where the input is metabolic food and the output is waste products. The functional status of an organ system can be evaluated by measuring the chemical input and output analytes of the cells. Biochemical Quantities Electrochemistry phmeasurement pco Measurement po Measurement po pco ph Ht or HCT or packed cell volume (PCV) or erythrocyte volume fraction (EVF) is the volume percentage (%) of RBCs in blood Hematocrit Hemoglobin To carry O from the lung to the tissues To release O to burn nutrients to provide energy to a protein molecule that binds to oxygen power the functions of the organism To collect the resultant CO to bring it back to the respiratory organs to be dispensed from the organism Hemoglobin forms an unstable, reversible bond with oxygen. 45% for men 40% for women Oxyhemoglobin Dissociation Curve ODC tells how blood carries and releases oxygen, and relates SO and po Partial Pressures gas mixture behaves in exactly the same fashion as a pure gas + = Oxyhemoglobin bright red Deoxyhemoglobin purple blue Partial pressure of oxygen po (mmhg) total pressure of a mixture of gases is Dalton's Law the sum of their individual pressures Biomedical Information Technology Lab 1

2 Significance of Blood Gases The fast and accurate measurements of the blood levels of the partial pressure of oxygen po, the partial pressure of carbon dioxide pco, and the concentration of hydrogen ions ph are vital in the diagnosis and treatment of many pathological conditions. pco mmhg po mmhg Electrochemical Sensors Convert chemical quantity into electrical potential or current by using electrochemical principles Two s + electrolyte solution + instrument Electrolyte is dissolved in water ions Ions move in electric field in the solution ionic current Ionic current electronic current Electrode interface between electrolyte and electric conductor Concentration of a substance to be measured is reflected in the potential between the s (potentiometric sensor), or current through the s (amperometric sensor) Basic Electrochemical System Ionic current is measured by a pair of which forms an interface between the electrolyte and the electric conductor to converts an ionic current into an electronic one. The concentration of the substance to be measured is reflected in the potential between the s, or current through the s. Ion-Selective Electrode (ISE) Potentiometric Sensor The object quantity is reflected in the potential difference When the electrolyte is dissolved in water, it dissociates into ions. The ion moves when an electric field is developed in the solution and causes ionic current. only a specific ion is permeable The change in ionic concentration is reflected upon the potential difference across the membrane. The change in potential difference is detected by two s placed in the solutions at both sides of the membrane. mperometric Sensor D, α: diffusion coefficient and the solubility of oxygen in the membrane p: partial pressure of the oxygen d, : Membrane thickness and area F: Faraday's constant 96, (1) C/mol, magnitude of electric charge per mole of electrons If oxygen is supplied by diffusion through a membrane, the current is 4FDp I d If a chemical reaction involves charge transfer between surface and substrate of the, the reaction will cause electric current Reference Electrode The potential is assumed to be 0, and used as a standard in determining the potentials of other s. cathode Pt O +H O+4e - 4OH - anode (reference) Reaction rate is governed by the concentration of the substrate and the potential between the and the solution Hydrogen a platinized platinum immersed in an acid solution g/gcl pure silver with a porous layer of silver chloride on its surface Calomel mercurous chloride (Hg Cl ) and pure mercury Biomedical Information Technology Lab

3 Energy Level and Transition If a potential difference between the metal and the electrolyte exists, the redox reaction corresponds to a move of a charge between two states at different potential levels, and thus it accompanies a gain or loss of electrical potential energy. Boltzman's distribution in thermal equilibrium energy difference transition probabilities between two states k: Boltzmann constant e(e E 0 ) e: electron charge Nernst Equation t thermal equilibrium, the transition rates on both directions between two energy levels are equal so that: n n exp kt Substituting ε equation and replacing populations of atoms in both states by respective activity a and a + ee E0 a a exp kt Thus, E becomes kt a E E 0 ln e a membrane is permeable to ion +, but impermeable to other ions, and is placed between solutions 1 and. Membrane Potential C : concentration of C+ in solution C 1 C: concentration of + 1 in solution 1 RT E E1 constant ln K a K Ba KCa... B C F K : selectivity coefficient, a +: activity of + If the potentials of solution 1 and are E 1 and E, respectively, then ion + gains an energy, c = e(e E 1 ), when it is moved from solution 1 to solution. In thermal equilibrium Electrolyte Solution Dissociation and association occur as B B When both direction reactions are balanced in equilibrium, the degree can be described as B a ab k K or B ab where [ + ], [B - ] and [B] are molar concentrations of +, B - and B, respectively a, a B and a B are activities of +, B - and B, respectively Both k and K are called equilibrium constants Ion Selective Electrodes The characteristics of ISEs depend on the electrical property of the ion-selective membrane. The response time of the ISE depends on the structure, size, and geometry of the, and the electrical property of the membrane material (sec-min). Ion-selective Field-effect Transistor (ISFET) The insulating layer (SiO ) on the n-channel is covered by the ion-selective membrane, and is exposed to the solution instead of the gate. fabricated on a p-type silicon substrate similar to MOSFET low impedance small size faster response (a) glass internal resistance 10 9 Ω (b) pressed-pellet membrane below 100kΩ (c) liquid ion-exchange membrane below 30 MΩ By placing a reference in the solution, a bias voltage V GS is applied onto the insulating layer on the conductive channel similar to applying bias voltage to an FET. The potential developed at the membrane by the existing ion in the solution is measured by the change in current through the channel. Biomedical Information Technology Lab 3

4 ph Measurement The acid-base status of the blood is assessed by measuring the hydrogen ion concentration [H + ](mol/l) The ion concentration is described by ph=-log 10 [H + ] In a neutral solution, ph=7.0 The normal range of ph=7.40±0.03 ph quantity of hydrogen ions rate of excretion of CO respiratory acidosis + production of fixed acid diabetic ketoacidosis + abnormal losses of bicarbonate (principal hydrogen ion buffer in the blood) ph quantity of hydrogen ions rate of excretion of CO respiratory alkalosis + abnormal losses of acid prolonged vomiting=metabolic alkalosis ph is measured by utilizing a glass that generates an electric potential when solutions of differing ph are placed on the two sides of its membrane. ph Glass Capillary Electrode The sample is introduced into the capillary. The potential built up across the glass membrane is measured between a reference in the reference buffer solution outside the capillary and another reference connected to the sample via a salt bridge. pco Measurement HO HCO CO HCO3 H 3 CO a pco H HCO3 H HCO3 k' H CO a= mmol/liter/mmhg k 3 [H CO 3 ] is proportional to [CO ] CO H HCO a pco 3 pco Electrode CO diffuses across the membrane to establish the same concentration in both chambers. If there is a net movement of CO into (or out of) the chamber containing the buffer, [H + ] increases (or decreases), and the ph meter detects this change. Because the relationship between ph and -log(pco ) is proportional, it is necessary to calibrate before deriving pco from the measured ph. Once establishing a calibration curve of pco vs ph, the specimen's pco can be obtained by the measured ph value from this curve. CO -permeable membrane ph sensitive glass H loghco3 log a log pco log k 0 loghco log a log pco log k log p H 3 ph has a linear dependence on the negative of log(pco ) H CO 3 buffer solution Reference po Measurement Reduction reaction at cathode O HO 4e HO 4e 4OH 4KCl 4KOH 4Cl Oxidation reaction at anode 4g 4Cl 4gCl 4e The presence of O and the resulting chemical reaction can be thought of as producing in the circuit a variable source of current the value of which is directly proportional to the po level. 4OH The cathode (measurement ) is constructed of glass-coated Pt, and the anode (reference ) is made of g/gcl The reaction consumes O, which is a function of the area of the Pt exposed to the reaction solution and the permeability of the semipermeable membrane to O. po Electrode Twice calibration is done by using two gases of known O concentration. Firstly, the gas containing no O is provided. The po meter output is set to zero after equilibrium of O content is achieved (90s). Secondly, the gas with known concentration is used to determine the second point on the po --current calibration curve. O -permeable membrane H O glass-coated Pt reference Biomedical Information Technology Lab 4

5 Clark Polarographic Electrode In order to keep an electrolytic connection between s, a thin layer of electrolyte between the membrane and the surface is maintained (5-10 um). Connectors Insulator Leland Clark (1956), Clark oxygen Polarogram of Clark Electrode polarizing voltage of 600 to 800 mv is supplied for the reduction-oxidation reactions to occur. The value of po is determined by using the right chart that indicates the current through the s is proportional to po. Cathode (u, Pt, g) measurement node (g, g/gcl) reference HO H O e Electrolyte O -permeable membrane (polypropylene, polyethylene, 0 um) Cathode bias voltage, V (a) current vs. voltage for a po (b) current vs. po Galvanic-cell po Electrode O measurement in gas phase anode has higher ionization tendency to form a battery cell, and hence does not need the external voltage supply required for the cathode reaction. cathode Zirconia po Electrode Zirconia ceramic tube Measured gas Standard gas or air inside anode zirconia cylinder wall polyethylene membrane (80um) outside Micro porous platinum Limiting-current po Electrode O ion with a negative charge is transported to the positive side through the negative porous layer. Finally po under the porous layer becomes zero, and the current drain ceases as long as no O is supplied to the under the layer. If O diffuses through the porous layer, current flows in proportion to the diffused O flux. Consequently, it provides an output current proportional to po in the surrounding space. Zirconia electrolyte is sandwiched by two deposited thin platinum films as s, over an alumina porous substrate and a platinum heater. Configuration of Limitingcurrent po Sensor O is adsorbed and becomes negatively charged on the sintered metal oxide grains so that an energy barrier appears at the contact of the grains. The barrier height is sensitive to the gas pressure at the surface. The resistance varies inversely proportional to the square root of po Biomedical Information Technology Lab 5

6 Metal-oxide po Sensors Gold s are deposited on a small ceramic tube, and tin oxide layer is formed on it. Catalysts are added to the metal oxides so as to modify the sensitivity to a specific gas. By combining metal oxide and catalyst, different gasses such as CO, alcohols, H, O, H S, halogenated hydrocarbons, and NH 3 can be detected. (a) Taguchi gas sensor 50 um alumina substrate (b) Thick-film micro gas sensor The electric conductivity of a solution is almost proportional to the ionic concentration salt meter. affinity to specific analyte Impedimetric Electrode The impedance varies when the analyte binds on it. The impedance change in the recognition element is detected by measuring impedance between two s. Electrolyte solution (a) structure Resistance arises when charges move between the and solution, whereas capacitance arises when charges are bounded in the layer. Both components are used for detecting analyte. When the analyte causes a change in charge transfer, it can be detected by a change in resistivity, while if the analyte causes a change in dielectric property, or a change in permittivity, it can be detected by capacitance change. When capacitive detection is used, the surface is usually covered with an insulating layer so as to reduce charge transfer. Rw, Cw: working resistance and capacitance Rc, Cc: counter resistance and capacitance Rs: solution resistance Z: recognition element impedance (b) equivalent circuit Impedimetric Interdigitated Electrode When a small amount of analyte is bound on the surface, the resulting change in impedance is small. If the analyte is labeled by an enzyme so that the enzyme catalyzes a chemical reaction, one enzyme molecule can produce many product molecules as long as there exist substrate molecules in reasonable concentration. If the product molecule is insoluble and precipitates onto the surface, the resistance and capacitance will be increased. Such a catalyzed reaction can be regarded as a chemical amplification that increases the sensitivity of impedimetric detection significantly. Catheter Clark po Electrode intravascular measurement g cathode Membrane g anode mm Epo xy The electrolyte is initially deposited on the tip, and it is dip coated by PVC so as to form a membrane of about 5 um. When it is immersed in blood, water vapor diffuses through the membrane and forms a liquid electrolyte layer in min. Wire to anode Wire to cathode one hollow tube within another hollow tube Epoxy resin Sample hole Catheter is inserted into the radial or brachial artery, or into the central vessels via the femoral artery or vein to measure ions such as sodium, potassium, and calcium and catabolites such as glucose and urea. (a) side view Insulating support (b) top view Bilumen catheter Skin as a Membrane Normally the skin is not very permeable to gases. t higher temperatures the skin's ability to transport gases is improved. The heat dilates the blood vessels, blood flow increases and gas transport becomes facilitated. Skin surface Skin surface a nonliving layer of dehydrated cells that serves as a barrier. Its electrical resistance is rather high, which reduces the risk of hazardous electrical currents entering the body. a living layer consisting of proteins, lipids, and melanin-producing cells. The vasodilatation by skin heating and the associated increased perfusion is a prerequisite for tcpo measurements. Transcutaneous po (tcpo ) Electrode Cathode (Pt or u) node (g/gcl) Cross section of the human skin perfused by capillaries arranged in vertical loops The capillaries receive blood from arterioles and release blood to venules arterial-capillaryvenule system. rteriovenous anastomoses exist in local skin areas especially in the palms, feet, and face. They are muscular regions innervated by the sympathetic nervous system. KCl electrolyte tcpo assesses the oxygen tension in the skin under the and not in the systemic circulation, i.e. local level, not systemic level of po. To "arterialise" capillary blood flow, skin heating to is necessary. Biomedical Information Technology Lab 6

7 tcpo & tcpco Combined Electrode tcpo reflects the function of several physiological processes forming the links of O transport chain, e.g., gas exchange in the lung, blood circulation at various levels in the circulatory system, gas exchange at the cellular level, etc. tcpco varies inversely with alveolar ventilation and gives instant information about the effectiveness of ventilation. Reference anode Cathode (Pt or u) for po (g/gcl) Glass ph for pco Gas diffusion membrane permeable for both O and CO Biomedical Information Technology Lab 7