Instrumentation, Lab Automation and Informatics – Part 1 PDF

Summary

This document provides an overview of instrumentation, lab automation, and informatics concepts. It details different spectroscopic techniques and their applications, emphasizing practical aspects like instruments, measurement principles, and data analysis.

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Instrumentation, Lab Automation and Informatics – Part 1 Chapter 4 Preamble PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details needed for the Unit exam Each student is responsible for READING t...

Instrumentation, Lab Automation and Informatics – Part 1 Chapter 4 Preamble PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details needed for the Unit exam Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES Unit Objectives are your study guide (not this PowerPoint) Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! Absorption Spectroscopy (1 ) Absorption spectroscopy is a spectroscopic technique that is used for measuring the absorption of radiation as it interacts with the sample. The radiation could be a function of either frequency or wavelength. A interrelated term is absorption spectrum which refers to the frequencies of light transmitted with dark bands when the electrons absorb energy in the ground state to reach higher energy states Absorption spectroscopy is related to the absorption spectrum because the sample used interacts with electromagnetic radiation (EMR) in the form of photons from the radiating field. Absorption Spectroscopy (2) The intensity of the absorption differs depending on the frequency and this variation is the absorption spectrum. This interaction of EMR in the form of photons with matter provides the principal means of measuring analytes in biological fluids. As much as the analytical systems have changed, the “black box” or measuring device within the system has fundamentally remained the same. Absorption Spectroscopy (3) Scattering of radiation Transmission of radiation in matter can be viewed as a momentary retention of the radiant energy by atoms, ions or molecules followed by reemission of the radiation in all directions as the particles return to their original electronic state. Rayleigh scatter Light scatter by molecules or aggregates of particles with dimensions significantly smaller than the wavelength of the radiation Example is the blue color of the sky. Tyndall effect Occurs with particles of colloidal dimensions Can be seen with the naked eye Raman scatter Involves absorption of photons producing vibrational excitation Always varies from the excitation energy by a constant energy difference Absorption Spectroscopy (3) Practical Aspects of Light Spectrophotometric techniques use either ultraviolet or visible light. Lambert Law Beer-Lambert law Graphs Percent transmittance versus concentration Absorbance versus concentration Absorption Spectroscopy (4) Practical Aspects of Light Lambert’s law States that for parallel monochromatic radiation that passes through an absorber material of constant concentration, the radian power decreases logarithmically as the light path increases arithmetically. Lambert proved that for monochromatic radiation that passes through an absorber of constant concentration, there is a logarithmic decrease in the radiant power as the light path increases arithmetically. A plot of percent transmittance versus cuvettes 1 through 6 for a series of solutions of identical concentrations and cuvette path length. This illustrates the nonlinearity of the relationship between percent transmittance and concentration. Absorption Spectroscopy (5) Practical Aspects of Light Beer-Lambert Law Based on previous work by Lambert, Beer discovered that for monochromatic radiation, absorbance is directly proportional to the light path, b, through the medium and the concentration, c, of the absorbing species. The work culminated in the Beer-Lambert law, or simply Beer’s law. The concentration of an analyte in solution can be determined by several different methods based on the Beer-Lambert law. Instrumentation (1) Typical photometer or spectrophotometer contains five significant components in either a single- or double-beam configuration. 1. A stable source of radiant energy 2. A device that isolates a specific region of the electromagnetic spectrum 3. A sample holder 4. A photo detector 5. A read- out device Instrumentation (2) Radiant-energy sources Provide polychromatic light Must generate sufficient radiant energy or power to measure the analyte of interest Two types Continuum Emits radiation that changes in intensity very slowly as a function of wavelength Line Emit a limited number of discrete lines or bands of radiation Instrumentation (3) Wavelength selectors Monochromators Spectroscope modified for selective transmission of a narrow band of the spectrum. Quality of a monochromator is described by: Nominal wavelength Wavelength in nanometers at peak light transmittance Effective bandwidth Range of wavelengths at a point halfway between the baseline and the peak. Bandpass Total range of wavelengths transmitted Filters Absorption filters and interference filters Instrumentation (4) Wavelength selectors Prism Monochromators Grating Monochromators Instrumentation (5) Wavelength selectors Cuvettes Cuvettes or cells hold samples and reagents that are made of material transparent to radiation in the spectral region of interest. Flow-cell or flow-through cuvette Instrumentation (6) Wavelength selectors Radiation transducers Transducer is a device that converts one form of energy to another. Photovoltaic or Barrier Layer Cell Basic phototransducer used for detecting and measuring radiation in the visible region Vacuum Phototubes Semicylindrical cathode and a wire anode (i.e., the positive electrode) sealed inside an evacuated transparent envelope. Instrumentation (7) Wavelength selectors Radiation transducers Photomultiplier Tubes PMTs are commonly used when radiant power is very low, which is characteristic of very low analyte concentrations. Output signal from the PMT is amplified up to approximately one million times. Silicon Diode Transducers More sensitive than vacuum phototubes but less sensitive than PMTs. Consist of a reverse bias p-n junction formed on a silicon chip. The p-n junction is constructed by the fusing p-type and n- type semiconductive materials. Instrumentation (8) Wavelength selectors Radiation transducers Multichannel Photon Transducers Consists of an array of small photoelectric-sensitive elements arranged either linearly or in a two-dimensional pattern on a single semiconductor chip. Photodiode Arrays Several hundred photodiodes set side-by-side on a single integrated circuit (IC), or “chip” In comparison to the PMT, the PDA has a lower dynamic range and higher noise. Charge-Transfer Devices (CTD) is a generic term that describes a detection system in which a photon, striking the IC semiconductor material, releases electrons from their bound state into a mobile state. Charge-coupled devices (CCDs) Charge-injection devices (CIDs) Instrumentation (9) Signal Processors and Readout Processing of an electrical signal received from a transducer is accomplished by a device that: 1. Amplifies the electronic signal 2. Rectifies alternating current (ac) to direct current (dc) or the reverse 3. Alters the phase of the signal 4. Filters it to remove unwanted components Quality Assurance in Spectroscopy (1 ) Several photometric parameters that must be monitored periodically to ensure optimal performance. Wavelength accuracy Bandwidth Photometric accuracy Linearity Stray light Accuracy suggests the closeness of a measurement to its true value. Quality Assurance in Spectroscopy (2) Assessment of photometric accuracy is performed using glass filters or solutions that have known absorbance values for a specific wavelength. Linearity of a spectrometer can be determined with these tools. Stray light Can have a significant impact on any measurement of absorbance by a solution Can be evaluated by using special cutoff filters Quality Assurance in Spectroscopy (3) Types of Photometric Instruments Spectroscope Optical instrument used for visual identification of atomic emission lines Colorimeter User compares the observed color of the unknown sample to a standard or a series of colored standards of known concentrations Photometer Consists of a light source, monochromatic filter and photoelectric transducer, signal processor, and readout. Quality Assurance in Spectroscopy (4 ) Types of Photometric Instruments Spectrometer An instrument that provides information about the intensity of radiation as a function of wavelength or frequency Spectrophotometers Spectrometers equipped with one or more exit slits and photoelectron transducers that permit determination of the ratio of the power of two beams as a function of wavelength Single-beam instrument represents the simplest type of spectrometer. Quality Assurance in Spectroscopy (5) Types of Photometric Instruments Double-beam instrument splits or chops the monochro- matic beam of radiation into two components. Two fundamental instrument designs for double-beam spectrophotometers: Double beam in space Double beam in time Reflectometry (1 of 2) Reflectometer A filter photometer that measures the quantity of light reflected by a liquid sample that has been dispensed onto a nonpolished surface. Two types of reflectance: Specular reflectance Occurs on a polished surface (e.g., a mirror), where the angle of incidence of the radiant energy is equal to the angle of reflection Diffuse reflectance Occurs on nonpolished surfaces Occurs within the layers and depends on the properties and characteristics of the layers themselves Reflectometry (2 of 2) Reflectometers Components of a reflectometer are very similar to a photometer Tungsten-quartz halide lamp serves as a source of polychromatic radiation Light passes through a slit and is directed onto the surface of a urine dipstick “pad” or dry slide. A wavelength selector, such as a stationary filter or filter wheel for multiple analytes, is used to isolate the wavelength of interest. A. polychromatic light source, Solid-state photodiodes are typically used to B. monochromator, detect the reflected radiant energy. C. slit, D. diffuse reflective surface, E. Lens, F. photodetector, G. readout device. Atomic Absorption Spectroscopy AAS is used for quantitative analysis of metals such as calcium, lead, copper, and lithium. Time-consuming to perform, is labor intensive, and requires meticulous laboratory techniques. Measures the amount of EMR absorbed by elements in their ground state Amount of absorbed radiation is directly proportional to the concentration of the metal in solution. (Go ) Molecular Luminescence Spectroscopy (1) Fluorometry Fluorescent spectroscopy is widely used because of its inherent high sensitivity and high specificity. High specificity results from dependence on two spectra and the possibility of measuring the lifetimes of the fluorescent state. Excitation and emission spectra Compounds that are excited at the same wavelength but emit at different wavelengths are readily differentiated. Molecular Luminescence Spectroscopy (2) Fluorometry Principles of fluorometry Luminescence Based on an energy exchange process that occurs when valence shell electrons absorb EMR, become excited, and return to an energy level lower than their original level Fluorescence Light emission from a singlet-excited state Phosphorescence Light emission from an excited triplet state Molecular Luminescence Spectroscopy (3) Fluorometry Instrumentation Conventional design of fluorometers places the detector at a 90° angle to the polychromatic light source. Sources Intensity Wavelength distribution of emitted radiation Stability Filters and Monochromators Transducers PMTs are the most common transducers found in fluorescent instruments. Newer fluorometers on the market today use diode-array and CTDs. Cuvettes or Cells Used for fluorescent measurement may be rectangular or cylindrical. May be made of glass or quartz Molecular Luminescence Spectroscopy (4) Fluorometry Applications of Fluorescent Spectroscopy Fluorescence polarization immunoassay Measurement of fluorescent-labeled bound fraction is determined in the presence of fluorescent-labeled free fraction Referred to as a homogenous immunoassay Time-resolved fluorescent immunoassay Front-surface fluorometry Chemiluminescence Differs from fluorescence and phosphorescence Light is produced from a chemical or electrochemical reaction and not from electromagnetic energy stimulation of electrons, resulting in emission of photons. Light Scatter Techniques Nephelometry Measurement of the light scattered by particles in solution Typical nephelometer consists of a light source, a collimator, a monochromator, a sample cuvette, a stray light trap, and a photo- detector. Turbidimetry Measurement of the reduction in light trans- mission caused by particle formation. Light transmitted in the forward direction is detected. The amount of light scattered by a suspension of particles depends on the specimen concentration and particle size. Refractometry The principle of refractometry is based on the refraction of light as it passes through a medium such as glass or water. When light passes from one medium into another, the light beam changes its direction at the boundary surface if its speed in the second medium is different from that in the first. Critical angle The angle created by the bending of the light Refractivity The ability of a substance to bend light Osmometry (1 of 2) The measurement of the osmolality of an aqueous solution such as serum, plasma, or urine As the osmolality of a solution increases: 1. Osmotic pressure increases 2. Boiling point is elevated 3. Freezing point is depressed 4. Vapor pressure is depressed Osmometry (2 of 2) Freezing-Point Osmometer Consists of a sample chamber containing a stirrer and a thermistor connected to a readout device Sample is rapidly supercooled to several degrees below its freezing point. Then agitated with the stirrer to initiate freezing. Rate at which this heat of fusion is released from the ice being rapidly formed reaches equilibrium with the rate of heat removed. Known the freezing point of the solution. Diagram of a uniform freezing point depression curve illustrating the entire process which begins at number 1, fast cool, and ends with a direct readout in units of mOsmol/kg. Electrochemistry (1) Potentiometry Electrical potentials are produced at the interface between metal and ions of that metal in a solution. To measure the electrode potential, a constant-voltage source is needed as the reference potential. Reference electrode Electrode with a constant voltage Indicator electrode The measuring electrode Electrochemistry (2) Potentiometry Nernst equation Measured cell potential is related to the molar concentration by the Nernst equation Useful for predicting the ECell = cell potential electrochemical cell E0 = Cell potential under standard conditions potential given the R = Universal gas constant (8.314 J/(mol*K)) T = Temperature concentrations of oxidized n = Number of electrons transferred in the reaction and reduced species for a F = Faraday constant (96485 C/mol) given electrode system Q = Reaction Quotient Electrochemistry (3) Potentiometry Reference selective electrodes In most electroanalytical applications, it is desirable the half-cell potential of one electrode be known, constant, and completely insensitive to the composition of the solution under study. Important attributes include: 1. Potential is reversible and obeys the Nernst equation 2. Electrode exhibits a potential that is constant with time 3. Electrode returns to its original potential after being subjected to small currents 4. Electrode exhibits little hysteresis or lag with temperature cycling Electrochemistry (4) Commonly used ion-selective electrodes in Potentiometry clinical laboratories. Ion-selective electrodes Ion-selective electrode Key membrane component ISE is a membrane-based Sodium Silicate in glass electrochemical transducer capable of Dodecylmethyl-14-crown-4 responding to a specific ion. Lithium (ether) ISEs measure ion activities, specifically Calcium di-(n-decyl)phosphate in Calcium free ion concentration. di-(n-octylphenyl)-phosphate ISEs provide several advantages over Magnesium N’N’’N’’’-imin-6,1-hexandiyl-tris “wet chemistry” and photometric (N-heptyl-N-methyl-malonamide) techniques. Hydrogen Neutral carrier tridodecylamine Chloride Solvent polymeric membranes Potassium Valinomycin Electrochemistry (5) Potentiometry pH electrodes Consists of a small bulb located at the tip of the electrode made of layers of hydrated and nonhydrated glass. Inside the electrode is a chloride ion buffer solution. PCO2 electrodes A pH electrode contained within a plastic “jacket” Plastic jacket is filled with a sodium bicarbonate buffer and has a gas-permeable membrane across its opening. Coulometry An analytical method that involves measuring the quantity of electricity (in coulombs) needed to convert the analyte quantitatively to a different oxidation state. A coulomb is the quantity of electricity or charge that is transported in one second by a constant current of one ampere. Laboratory application includes the measurement of chloride ions in serum, plasma, CSF, and sweat samples. Amperometry (1) Measurement of the current flow produced by an oxidation-reduction reaction Measurement of chloride in samples involves the use of two electrochemical methods: Coulometry Amperometry Chloride titrator Includes a pair of silver electrodes that serve as the indicator electrodes. When all of the chloride in the sample has been consumed, silver ions appear in excess Amperometry (2) PO2 gas-sensing electrode Clark PO2 electrode consists of a gas-permeable membrane, usually polypropylene, that allows dissolved oxygen to pass through. Other Techniques (1) Voltammetry Comprises a group of electroanalytical methods in which information about the analyte is derived from the measurement of current as a function of an applied potential obtained under conditions that promote polarization of an indicator, or working, electrode Based on the measurement of a current that develops in an electrochemical cell under conditions of complete concentration polarization. A minimal consumption of analyte takes places Anodic stripping voltammetry Measurement of lead in whole blood samples can be performed in the clinical laboratories Technique consists of three major steps: 1. Reduction of lead and deposition of the lead onto the electrode. 2. “Resting period” in which stirring is halted but the potential remains on the electrode. 3. Lead is stripped from the electrode back into the solution by oxidation to the ionic form. Other Techniques (2) Conductometry Electrolytic conductivity is a measure of the ability of a solution to carry an electric current. The reciprocal of resistance, 1/R is called the conductance, given the symbol G, and is expressed in reciprocal ohms, or mhos. Resistivity The electrical resistance in ohms measured between opposite faces of a 1.00-centimeter cube of an aqueous solution at a specific temperature. Measurement is accomplished by using a resistivity meter. Impedance Electrical impedance measurement is based on the change in electrical resistance across an aperture when a particle in a conductive liquid passes through this aperture. Used primarily in the hematology laboratory to enumerate leukocytes, erythrocytes, and platelets Separation Techniques (1) Electrophoresis and Densitometry Electrophoresis Separation of charged compounds, typically proteins, applied to a solid or semisolid support and immersed in a liquid medium pH at which an amino acid exists mainly as the zwitterion is called the isoelectric point and the pH at that point is called the pI. Conductivity of a solution increases with its total ionic concentration. Separation Techniques (2) Electrophoresis and Densitometry Densitometry Basically an absorbance measurement Densitometer Measures the absorbance of the stained compounds on a support medium or electrophoretic strip. Light source, monochromator, and movable carriage to move the electrophoretic strip between the monochromator and photodiode and a photodetector. Separation Techniques (3) Electrophoresis and Densitometry Capillary Electrophoresis Typical CE system consists of a fused silica capillary, two electrolyte buffer reservoirs, a high-voltage power supply, and a detector linked to a data acquisition unit. Electroosmosis Motion of a liquid when a voltage is applied between the ends of an insulating tube that contains that liquid Isoelectric Focusing IEF techniques are similar to electrophoresis except that the separating molecules migrate through a p H gradient. pH gradient is created by adding acid to the anodic area of the electrolyte cell and adding base to the cathode area. Has been useful in measuring serum isoenzymes of acid phosphatase, creatine kinase, and alkaline phosphatase. Separation Techniques (4) Chromatography Retention time (RT) Time it takes a compound to elute off the column once it has been injected Can be used to determine a compound’s identity Resolution (RS) Measure of the ability of a column to separate two or more analytes in a sample Several factors have a significant impact on the ability of the chromatographic system to separate compounds: Column retention factor (k’) Column efficiency (N) Column selectivity (α) Factors Controlling the Resolution of Multicomponent Mixtures of Analytes (k’) Strength of solvent: polarity Strength of packing material: surface area or amount of stationary phase Temperature N Flow rate: linear velocity Column length Average particle size of packing material Viscosity of solvent Mass of injection α Chemistry of solvent: functional groups Chemistry of packing material: functional groups Chemistry of samples: presence of hydrogen bonds or derivatization Separation Techniques (7 of 13) Thin-Layer Chromatography Used in many laboratories as an initial screening technique for the detection of drugs of abuse in urine (DAU) Stationary phase is manufactured as a thin layer or coating of adsorbent that is bonded to a solid support such as glass or plastic. Solid adsorbent may consist of a basic silica material or a more complex adsorbent. Most compounds have a characteristic identifier known as Rf Separation Techniques (9 of 13) Gas Chromatography (GC) The basic design of a GC consists of the following components: Carrier gas supply Sample injection device and GC inlet Column Detector Data system Separation Techniques (8 of 13) Gas Chromatography (GC) Uses a “carrier” gas to move compounds through a stationary phase located within a column Widely used technique for decades because of: High resolution Low detection limits Accuracy Short analytical times Detectors used in a gas chromatography must provide optimum performance characteristics: High sensitivity Good stability and reproducibility Linear response over a wide concentration range Expanded temperature range from room temperature to between 300 and 400°C Short response times Separation Techniques (11 of 13) Liquid Chromatography (High-Performance Liquid Chromatography) Techniques use lower temperatures for separation, thereby achieving better separation of thermolabile compounds. Uses small, rigid supports and special mechanical pumps producing high pressure to pass the mobile phase through the column Five commonly used separation techniques: Adsorption Partition Ion exchange Affinity Size exclusion Separation Techniques (12 of 13) Liquid Chromatography (High-Performance Liquid Chromatography) HPLC Instrumentation Typical system consists of: Liquid mobile phase Sample injector (manual or automatic) Mechanical pump Column Detector Data recorder HPLC Instrumentation Two methods for delivery of mobile phase(s): Isocratic Uses one mobile phase Gradient Involves the use of two or more mobile phases that are automatically programmed to pump for a specific interval of time Mass Spectroscopy (1) Used to identify unknown compounds, determine concentrations of known substances, and study the molecular structure and chemical composition of organic and inorganic material Involves the following steps: 1. Atomization 2. Conversion of a substantial fraction of the atoms formed in step 1 to a stream of ions 3. Separation of the ions formed in the second step on the basis of their mass-to-charge ratio (m/z) m is the mass of the ion in atomic mass units and z is its charge 4. Counting the number of ions of each type or measuring current produced when the ions formed from the samples strike a transducer. Mass Spectroscopy (2) Mass-to-Charge Ratio Measurement commonly used in MS Obtained by dividing the atomic or molecular mass of an ion by the number of charges that the ion bears Often shortened to the more convenient term mass Mass Spectroscopy (3) Three components of mass spectrometers are: Ion source 1. Electrospray ionization (ESI) 2. Matrix-assisted laser desorption ionization (MALDI) 3. Surface-enhanced laser desorption ionization (SELDI) Mass analyzer Ion detector Mass Spectroscopy (4) Mass Analyzers Five types ESI-QqQ (triple quadrupole), ESI-QIT (quadrupole ion trap), MALDI-ToF-MS (time of flight), ESI-QqToF (quadrupole time of flight) ESI-FTMS (Fourier transform) Mass Spectroscopy (5) Mass Analyzers Time of Flight Consists of a metal flight tube m/z ratios of the ions are determined by accurately and precisely measuring the time it takes ions to travel from the MALDI or SELDI sources to the detector. Quadrupole Ion Trap Fourier Transform Ion Cyclotron Mass Spectrometer MALDI-ToF Diagram Scintillation Counters (1) Scintillations are flashes of light that occur when gamma rays or charged particles interact with matter. Chemicals used to convert their energy into light energy are called scintillators. PMT detects light either directly or through an internal reflecting fiber optic. A scintillation counter is an instrument that detects scintillations using a PMT and counts the electrical impulses produced by the scintillations. Two types of scintillation methods exist: Crystal scintillation Liquid scintillation Scintillation Counters (2) Crystal Scintillation Counters Used to detect scintillations created by interaction of gamma particles from radioisotopes with matter Liquid Scintillation Counters Used to detect and count photons that are produced when beta radiation from radioisotopes interacts with matter Nuclear Magnetic Resonance Spectroscopy Nuclear magnetic resonance Occurs when the nuclei of certain atoms are immersed in a static magnetic field and exposed to a second oscillating magnetic field, the magnetic component of EMR Basic components of an NMR spectrometer consist of: Magnet used to separate the nuclear spin energy state Transmitter that supplies the radio frequencies (RFs) or irradiating energy Sample probe Computer Flow Cytometry (1) Flow cytometer Instrument that measures multiple cell parameters and other types of particles as they flow individually in front of a light source Detects and correlates the signals from multiple detectors at various angles Key Features of a Flow Cytometer Cells or particles Particle Any of the objects flowing through a flow cytometer Event Anything that has been interpreted by the instrument to be a single particle Illumination Laser light Serves as a source of illumination for most flow cytometers Provides intense light in a narrow beam Flow Cytometry (2) Key Features of a Flow Cytometer Fluidics In flow cytometry, the particles need to be suspended in a fluid. Decreases the probability that multiple cells will group together at the analysis point Detector Lenses are used to collect the light and focus the beam of radiation onto a photodiode. Only light that has been refracted or scattered as it strikes a will be diverted enough to strike the forward-positioned lens and the photodiode behind it. Data All the data about each of a group of cells is stored in data files in an array where each cell has the associated output from each detector. Software packages available will produce histograms and scattergrams of any set of parameter values for the cells in the data file. Microscale Technologies (1) Lab-on-a-chip A total microanalysis system (μTAS) incorporating sample preparation, separation, detection, and quantification on a microchip surface. Micro machining Process of fabricating labs-on-a-chip and micromachines Optodes Optical sensors Used in clinical laboratory instrumentation designed to measure blood gases and electrolytes Microscale Technologies (4 of 13) Biosensors A device with a biologically sensitive coating comprising an antibody, receptor protein, or biocatalysts Comprises a biologically sensitive material (a biocatalyst) in contact with a suitable transducing system that converts the biochemical signal into an electrical signal Biocatalysts Enzymes Multienzyme systems Antibodies Membrane components Organelles Bacteria Mammalian or plant tissues. Biocatalysts are responsible for the sensitivity and specificity of the biosensors. Microscale Technologies (5 of 13) Types of biosensors used in laboratories and their respective transducer systems include: Electrochemical Potentiometric Amperometric Conductimetric Piezoelectric Applied using crystals of quartz coated with an adsorbent Calorimetric Biological component attached to a heat-sensing transducer or thermistor Reaction between these two components generates a specific amount of heat Optical Uses fiber-optic technology to measure the reflected fluorescence light from immobilized chemicals at the end of small fiber-optic probes Types of Biosensors Used in Clinical Laboratories Transducer Measurement Applications N a plus, K plus, C a plus plus, L i plus Ion-selective electrodes Potentiometric Na+ , K + ,Ca++ , Li+ P C O sub 2 Gas-sensing electrodes Potentiometric Enzyme electrodes Amperometric PCO2 Glucose Conductimeter Conductance Urea nitrogen Piezoelectric crystals, Mass change Immunosensor, volatile acoustic waves gases and vapors Thermistor Calorimetric Enzymes Luminescence Optical pH, enzyme substrates Microscale Technologies (7 of 13) Biosensor Technologies Enzyme-based biosensors with amperometric detection Development of electrodes to measure cholesterol, pyruvate, alanine, and creatinine Enzyme-based biosensors with potentiometric and conductometric detection Developed for the measurement of BUN, glucose, creatinine, and acetaminophen. Enzyme-based biosensors with optical detection Used to measure analytes such as glucose, cholesterol, and bilirubin Affinity biosensors Use binding proteins, antibodies, or oligonucleotides as an immobilized biological recognition element. Microscale Technologies (9 of 13) Biosensor Technologies Point of Care (POC) POC Testing “any test that is performed at the time at which the test result enables a decision to be made and an action taken that leads to an improved health outcome.” Devices should: Be portable, Have consumable reagent cartridges Generate results within minutes Require minimum operating steps Have the capability to perform tests on whole blood specimens Have flexible test menus Contain built-in/integrated calibration and quality control Require ambient temperature storage for reagents POCT devices are designed to provide both qualitative and quantitative measurements. Strips Sensors Table 4-3 Analytical Principles and Analytes Measured Using P OCT Devices Analytical Principle Analyte Reflectance Urine and blood chemistries (e.g., glucose) Lateral-flow immunoassay Infectious disease agents, cardiac markers, human chorionic gonadotropin Electrochemistry Glucose, pH, blood gases, electrolytes, metabolites (e.g., creatinine and urea nitrogen) Light scattering Coagulation Immunoturbidimetry HbA1c, urine albumin Spectrophotometry Blood chemistry Fluorescence pH, blood gases, electrolytes, metabolites Multiwavelength spectrophotometers Hemoglobin species, bilirubin Time-resolved fluorescence Cardiac markers, drugs, C-reactive protein Electrical impedance Complete blood count Microscale Technologies (12 of 13) Biosensor Technologies Nanotechnology Defined in part as the manipulation of living and nonliving matter at the level of the nanometer Quantum physics takes over from classic physics A nanometer is 10-9 meter in length. 10-12 puts us in the realm of the nucleus of an atom Laboratory applications are not described in the true sense of nanotechnology but rather as miniaturization Atom Technology A spectrum of new technologies that operate at the nanoscale and smaller. Transdisciplinary Borrows from physics, engineering, molecular biology, and chemistry Atomtech Postamble READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. USE THE UNIT OBJECTIVES AS A STUDY GUIDE All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives

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