Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
  • G01N21/272—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration for following a reaction, e.g. for determining photometrically a reaction rate (photometric cinetic analysis)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
• • G—PHYSICS
  • G05—CONTROLLING; REGULATING
  • G05D—SYSTEMS FOR CONTROLLING OR REGULATING NON-ELECTRIC VARIABLES
  • G05D21/00—Control of chemical or physico-chemical variables, e.g. pH value
  • G05D21/02—Control of chemical or physico-chemical variables, e.g. pH value characterised by the use of electric means

Definitions

• the present invention is in the area of apparatus and methods for automatically combining chemicals to form reactions, and relates more specifically to mixing assays of enzymes, substrate solutions and buffer, and measuring characteristics of the ensuing chemical reactions.
• An important use is for the determination of kinetic constants for enzyme catalyzed reactions.
• Biochemists in about the last quarter century have discovered the generalities and many of the details of the complex nature of the chemical reactions that take place in living organisms, as for example in the cells of human beings. Central to that chemistry is the synthesis of a large variety of proteins from the twenty known biological amino acids. In the complex process by which the genetic code on deoxyribonucleic acid (DNA) is copied and proteins are finally assembled according to the code, chemical reactions are catalyzed by other proteins called enzymes.
• DNA deoxyribonucleic acid
• An enzyme works as a catalyst by attaching to substrate molecules that are elements in the chemical reaction catalyzed, and bringing the elements into the close proximity required for reaction to occur. There are energy considerations in the process that take place to favor the direction of a particular reaction as well.
• the elements to which an enzyme attaches are called substrates of the enzyme, and every enzyme is specific to one or more substrates. The rate of an enzyme catalyzed process depends on the affinity of the enzyme for the substrate, the anount of the enzyme present, and the steady-state activity of a single enzyme molecule. With a very high substrate
• the specificity is expressed by the ratio of an enzyme's affinity for one substrate as opposed to it's affinity for another.
• Michaelis -Menten theory which assumes that an enzyme first combines with its substrate to form an enzyme- substrate complex, which then breaks down in a second step to form free enzyme and product. Based on this model the Michaelis - Menten equation was developed as the rate equation for reactions catalyzed by enzymes having a single substrate.
• v is the reaction velocity, which is the change of substrate (or product) concentration per unit time, measured at concentration [S] of free substrate.
• vmax is the maximum velocity the substrate can achieve.
• km is the Michaelis
• km and vmax can only be determined by a relatively large number of measurements at varying substrate concentrations.
• the procedure is to monitor optical density of the reactants and product mixture for a short time, and plot the optical density
• reaction rates are known to be sensitive to temperature changes.
• solutions to be mixed may not always be easily miscible, and may vary widely in viscosity, so efforts to obtain homogeneity can result in time delays as well. Also, such solutions do not always mix intimately in spite of time delaying efforts, so measured results are often innacurate.
• Robotic systems have been proposed and built for performing some of the manual operations.
• the time, accuracy, and mixing problems, however, have not been corrected.
• the robotic systems up to the present time have been severely limited in the range of
• the apparatus includes a supply system for providing an accurately metered plurality of solutions containing chemical reactants which when combined have a defined kinetic constant associated with their reaction.
• a mixing chamber system is connected to the supply system for mixing the plurality of solutions from the supply system.
• a temperature control system controls temperature of the mixing chamber system and the fluid delivery system.
• a reaction chamber is provided where a substantial portion of the reaction between the reactants occurs, the reaction chamber being connected to the mixing chamber system.
• a detection system is used for measuring a physical parameter that is a function of concentration of at least one of the reactants and reaction products in the reaction chamber.
• a computer system coupled to the supply system, to the mixing chamber system, to the temperature control system, and to the detection system, is used for automatically controlling the supply system to provide a plurality of sets of simultaneous metered volumes of the solutions to the mixing chamber system, each set corresponding to a predefined ratio of the reactants, for automatically controlling the temperature of the mixing chamber system, and for automatically causing the detection system to make measurements at a plurality of times for each set of the simultaneous metered volumes to
• the computer system is then used to analyze the data to determine kinetic constants and other
• Particular features of the apparatus that are significant include a new and unique mixing chamber system that uses a floating magnetic head inside the mixing chamber which is magnetically driven from outside the chamber so that the inside of the chamber can be contaminant free. Further, the design is significant in providing an extremely small mixing volume which can still achieve very high mixing efficiency.
• Another feature of the apparatus is use of an accurately driven syringe system, which in combination with an automated sample delivery system, and a versatile software control system, provides completely automatic operation for analyzing kinetic
• Fig. 1 is a block diagram presenting important elements of the first preferred embodiment of the invention.
• Fig. 2 is a perspective view of an arrangement of components of the first preferred embodiment.
• Fig. 3 is a perspective view of an autosampler unit showing an automatic sample changer.
• Fig. 4 is an elevation view, partially in section, showing a motor driven syringe unit.
• Fig. 5 is a schematic of an arrangement of valves and syringe units in the first preferred embodiment
• Fig. 6 is a broken section view showing the construction and operation of a mixer in the first referred embodiment.
• Fig. 7 (A) is a section view of a mixer module including the mixer of Fig. 6.
• Fig. 7 (B) is a section view of a connection between a fluid line and the mixer of Fig. 6 and Fig. 7 (A).
• Fig. 8 is a face view of a display and keyboard module for operator control.
• Fig. 9 illustrates a syringe control menu for control of syringe movements.
• Fig. 10 (A) is a block diagram of software
• Fig. 10 (B) is the Main Menu of the Kinetic
• Fig. 10 (C) is Screen 1 of a Measure Parameter Menu.
• Fig. 10 (D) is Screen 2 of the Measure Parameter Menu.
• Fig. 10 (E) is a setup menu for the Kinetic
• Fig. 10 (F) is a first screen of a Measure Menu.
• Fig. 10 (G) is the second screen of the Measure Menu.
• Fig. 10 (H) is a Setup Menu
• Fig. 10 (I) is a Debug Menu.
• Fig. 11 shows a printout of a typical setup for determination of Mikaelis constant for a particular exemplary enzyme using the apparatus of the invention.
• Fig. 12 shows a printout of measurement data using the setup of Fig. 11.
• Fig. 13 shows a plot of the derivative of the absorption as a function of time for different
• Fig. 14 is a printout showing the different turnover numbers for different wavelengths of the detector system for the exemplary enzyme.
• Fig. 15 is a plot of the turnover number versus substrate concentration for the exemplary enzyme.
• Fig. 16 is a plot of turnover number for several determinations illustrating the reproducibility of the apparatus of the invention.
• Fig. 1 is a block diagram showing equipment comprising an automated assay system, hereinafter called the "Assayomate.” Shown there is a first preferred embodiment of the present invention,
• An autosampler module 11 is a temperature-controlled reservoir region for presentation of stock and sample solutions such as buffer, substrate, and enzyme which the computer-automated system may use to develop assays of widely varying concentrations for measurement to determine kinetic constants.
• Stock solutions like buffer solutions and water, that may be used in a wide variety of assay preparations, may be kept in the autosampler in relatively large quantities in
• inhibitors, and accelerators for example, are placed in probes in a rack that may be moved on a stage relative to a pipette that is robotically manipulated.
• the stage is a rotating, indexing carrousel.
• Fluid transfer lines represented as path 13 connect the reservoirs and the pipette in the
• the valve module is for switching sample and stock solutions to and from the autosampler and other parts of the apparatus.
• An array of motor-driven syringes 17 is connected to the autosampler through valves in the valve module.
• the syringes are for drawing solutions from stock solutions and probes in the autosampler, and for feeding the drawn solutions in precise amounts at precise rates for assay preparation.
• the driven syringes deliver solutions via valve module 15 to a unique motor-driven mixer 19 which is designed to insure a complete and rapid mixing of solutions, even with wide viscosity variations.
• Solutions delivered by means of the syringes go to the mixer through fluid-transfer lines between the valve block and the mixer, represented by lines 14a, 14b, and 14c. There is one line for each syringe, so there may be more than the three representative lines shown.
• the fluid transfer lines between the valve block and the mixer are routed side-by-side and within a larger tubing through which temperature-controlled water is circulated.
• the mixture enters a cuvette 21 as a result of the forward drive of the syringes through the mixer, and action is stopped. After mixing and stop-flow,
• a spectrophotometer is used to measure change in optical density relative to time, which is a measure of change in concentration of reactants and products from which reaction velocity values may be determined.
• a shutter 23 opens and exposes a transparent opening through the cuvette. Light from a source 25 passes through the cuvette and the mixture in the cuvette. Then it passes through a prism 27 and
• a diode array 29 impinges on a diode array 29.
• the changing signal from the diode array is delivered to a spectrophotometer control unit 31.
• other analytical instruments may be used, such as, for example, an electrochemical detector, a
• a microprocessor-based control unit 35 is
• the computer in the first preferred embodiment also has an
• microprocessor control unit 35 has a printer/plotter and a disk drive for storage.
• An RS-232 communication interface allows the computer to be connected to a larger host computer (not shown) for accomplishing tasks such as further data manipulation and display.
• the microprocessor unit has a printer-plotter and disk drive for storage.
• Computer 37 is connected to microprocessor module 35 by means of a communication bus through link 41 and module 35 is connected to the photometer control unit by means of link 43.
• the communication bus is an IEC bus.
• microprocessor unit operates the syringe module through link 49, the valve module through link 45, the mixer apparatus through link 47, and the autosampler through link 51.
• links 45, 47, 49, and 51 comprise more than a single electrical line, and some lines are for power while others are for control signals, such as microswitch closures, for example.
• autosampler 11, syringe module 17, valve module 15, and control unit 35 are mounted in a single cabinet, and the associated keyboard and display unit 39 are mounted in a face of the cabinet.
• the mixer and cuvette are a separate unit connected to the main cabinet by a tubing through which dedicated fluid lines are directed and through which temperature-controlled water is circulated to maintain sample fluids at a specific temperature prior to reaction.
• the water control unit is not shown in Fig. 1.
• the apparatus of the invention is used to study biochemical reactions, such as the determination of kinetic constants in enzyme catalyzed reactions, which cannot be measured directly for the reasons described above in the Background of the Invention.
• the method has to employ typically a large number of measurements of a dependent variable while varying an independent variable.
• the syringes are sized so that a single ingestion of the necessary solutions from the reservoirs and probes in the autosampler is enough to mix and inject all of the assays necessary to determine the kinetic constants for a particular enzyme and substrate. This is important for two reasons: First, a refill during the determination of a kinetic constant may introduce a systematic error due to enzyme degradation. Second, if no refill is needed, the autosampler unit is free to perform other functions while the determination is still in process.
• the keyboard unit associated with control module 35 can be used to input specific commands in a manual mode and to initiate programmed subroutines, as well as to initiate automatic determinations.
• programmed procedures there are procedures for wash, for loading stock materials, and for injection of the materials to form assays of varying concentrations to generate data for determination of kinetic constants for a specific enzyme.
• the amounts of enzvme, substrate and other solutions for measurements of a data point are determined by the software and appropriate signals are generated through the microprocessor unit to cause the specified mix to take place by selected drive of the several syringes.
• the materials are urged to and through the mixer, and the mixer is driven to ensure an homogeneous mix of the materials.
• the mixture enters the cuvette very rapidly, and measurements are made with the analytical instrument dedicated to the
• the shutter is opened to allow the photometric sequence to take place, during which data from the diode array is sent to the
• Each data point may be
• the speed of the mix and full preparation for a single data point is within 0.3 second, as opposed to typically 5 seconds or more for manual methods. Hence, fast reactions can be characterized. Also, sequencing is rapidly accomplished, so determinations that
• Fig. 2 provides an overview of the apparatus of the first preferred embodiment in order to better explain the organization of the units comprising the system shown in Fig.1, and to illustrate other
• Autosampler 11 is a part of main cabinet 53, and microprocessor unit 35 with keyboard and display unit 39 is mounted in the same cabinet.
• the syringe array and valve block are also mounted in cabinet 53, although not seen in the view of Fig. 2.
• a mixer module 20 which includes mixer 19 is separate and connected to cabinet 53 by tubing 55 through which the various fluid lines pass. Control and power lines are arranged alongside tubing 55, and temperature-controlled water passes through the tubing as well.
• the length of connecting tubing and power and control lines to the mixer module are such that the module may be mounted in an analytical instrument, such as a commercially available spectrophotometer devices, such as a Hewlett-Packard Model 8451 diode-array spectrophotometer, which is used for data collection in the first preferred embodiment for determination of kinetic constants.
• an analytical instrument such as a commercially available spectrophotometer devices, such as a Hewlett-Packard Model 8451 diode-array spectrophotometer, which is used for data collection in the first preferred embodiment for determination of kinetic constants.
• Computer 37 in the first preferred embodiment is an HP 85 computer and has a printer 61 and a plotter 63 attached.
• the HP 85 computer in some instances may be connected to a more powerful host computer, such as an HP 150, for more extensive data analysis, and there is an RS-232
• Peltier units for controlling temperature in the apparatus, and electronic units to control the Peltier units.
• the entire connected system is small enough to be arranged on a desktop.
• the main cabinet is more extensive, and encompasses a built-in parallel processing computer, the water temperature control unit, the spectrophotometer or other analytical instrument, and an internal printer-plotter and disk drive, as well as electronic controls for Peltier elements.
• This instrument may be remotely controlled via an RS-232 or IEEE-488 bus using software which runs on a Mac II or an IBM PS/2.
• Autosampler Module
• Fig. 3 shows a part of Autosampler Module 11 within cabinet 53 of Fig. 2.
• the Autosampler Module may be accessed through a door from outside the
• a typical temperature inside the Autosampler enclosure is 4 degrees
• Two stock reservoirs 65 and 67 rest on a shelf 69 within the cabinet.
• Reservoir 65 has a tubing 71 that passes from the reservoir thru a back wall of the unit, and reservoir 67 has a similar tubing 73.
• the tubings are flexible, small-bore fluid transfer tubes, and are connected at the other end to ports at the valve module.
• the stock reservoirs are for storage of stock solutions, such as substrate, inhibitor, or other chemical reagents. A solution can be changed or replaced if needed by lifting the tube from a reservoir and switching containers.
• the stock reservoirs are used for solutions that may be common to a large variety of experiments. Although only two such reservoir containers are shown in Fig. 3, a larger number may be placed in the Autosampler, each with a dedicated fluid tube. In the first preferred embodiment there are typically two to four stock reservoirs, for use as needed.
• an automated sample changer 75 for presenting different sample solutions automatically.
• An indexing stage 77 extending through shelf 69 is driven from below by a stepper motor 79 so that each of several probes, such as probe 81, supported by the stage, may be presented to a pipette 83.
• the pipette is carried by a slide carriage 85 in a guide frame 87, and the slide carriage is driven up and down by another motor drive below (not shown). The pipette is raised to clear a probe to allow the indexing stage to turn without interference, and lowered when a probe is in position.
• Pipette 83 is connected to a fluid transfer tube 89, and the other end of the tube connects to an opening at the valve module so that probes may be drawn into one or another syringe of the syringe module through the valve module.
• An upper limit switch 91 signals the uppermost position of the pipette
• a lower limit switch 93 signals the lowermost position of the pipette translation.
• stage 77 Only four probes are shown supported by stage 77 in Fig. 3, for purposes of illustration, but there are many more in the Assayomate. Typically, there are as many as 50 probes arranged in circular rows.
• the sample changer allows a wide variety of sample
• determining kinetic constants and also allows for empty containers to be provided to recieve processed samples and other effluent.
• the presence of empty containers also allows for mixing of sample solutions to different concentrations before introduction to the mixer in an analytical procedure. For example, if the initial concentration of a enzyme is too large, the enzyme can be diluted accurately using an empty container and using the syringes to control the dilution.
• An additional Peltier unit is incorporated into a part of the rotary stage, typically covering about 25% of the stage area for supporting containers, and is used to maintain certain sample solutions at a temperature other than the ambient selected temperature within the Autosampler enclosure.
• There is typically a wash station incorporated on the rotary stage which is a position supporting a relatively large container for water or other reagent to be used to wash syringes and flush lines when desired.
• Fig. 4 is a sectioned view of a single syringe unit 95 that is one of several units that comprise syringe array 17 in the first preferred embodiment.
• a Hamilton syringe 97 is operated by a motorized drive to draw solutions from probes and stock reservoirs in the autosampler module, through valves in valve block 15, and to inject solutions as required to mixer module 19 through the valve block after switching the fluid routes by switching the valve positions. Fluids are drawn and expelled through fluid tubing 99 connected to the syringe.
• a water jacket 101 surrounds the syringe, and temperature controlled water is passed through the jacket via tubes 103 and 105 which lead to suitable manifolding (not shown) which is in turn connected to water supply and control unit 57 (Fig. 3).
• the plunger shaft 107 passes through a frame plate 109 and is attached to a drive carriage 111 by means of a conventional set screw 113 in the first preferred embodiment.
• Carriage 111 has an adjustable bearing slide 115 attached which guides on a guide rod 117.
• the guide rod is fixed in upper plate 109 and in a lower frame plate 119.
• Carriage 111 has a nut 121 attached at one end, and a threaded shaft 123 engages the nut.
• Motor 131 drives shaft 123 through a reducer 129, resulting in a very small rotational movement of the threaded shaft for a larger rotation of the motor, and hence a very small linear movement of the syringe plunger for motor rotation.
• a stepper motor is used so a specific volume of solution may be related to a single motor pulse, depending on syringe size.
• Plates 109 and 119 are spaced apart by a side plate 133, forming a rigid frame unit for the syringe unit.
• a microswitch 135 is fastened to plate 133 in a position to sense and report the forwardmost travel of the syringe plunger.
• syringes In the Assayomate, all of the syringes have a stroke of about 6 cm.
• the lead of the threaded shaft and nut is 1mm. per turn, and the gearing ratio is 16.66 : 1.
• the stepper motor requires 48 pulses for a single revolution, so a full stroke of a syringe requires 48,000 stepper pulses.
• the volume delivered (or drawn in) by a single pulse is 1 micro-liter, 0.1 micro- liter, and 0.05 micro-liter respectively.
• a substrate syringe has to perform minimally a stroke of .005 mm. and maximally a stroke of 10 mm. within 1 second.
• each syringe unit there are typically three or four syringe units comprising syringe array 17, and the framing is designed so that the discrete syringe units may be mounted side by side in a rack-mount fashion in cabinet 53.
• Tubing from each syringe connects to specific ports of the valve block, water lines connect to suitable manifolds, and there are power and control wiring connections from each syringe unit to the microprocessor control unit.
• Fig. 5 is a schematic showing the valve block in the first preferred embodiment, showing also the connection to the syringe array and the autosampler module. For this example, four syringe units are depicted. There are 8 solenoid activated valves in the valve module, two serving each of the four syringes, and all are shown in Fig. 5 in the deactivated state.
• the valves used are LFYA 120 and LFYX valves from the Lee Company of Westbrook, CT., although there are other suitable valves commercially available.
• the Lee valves have a very small internal volume (7.4 ml for the LFYA) and inert internal surfaces, both of which are
• Operation is at 12V and switching is accomplished within 10 msec.
• Valve 151 in the deactivated state, directs fluid from syringe 153 to mixer 19; valve 147 deactivated directs fluid from svringe 149 to the mixer; valve 143 deactivated directs fluid from syringe 145; and valve 137 deactivated directs fluid from syringe 139 to the mixer.
• the pertinent valve 151, 147, 143, or 137 has to be
• valve 151 When valve 151 is activated and valve 167 remains deactivated, syringe 153 is connected to a reservoir in the autosampler module through line 169; when valve 147 is activated and valve 163 remains deactivated, syringe 149 is connected to a reservoir in the autosampler unit through line 165; when valve 143 is activated and valve 159 remains deactivated, syringe 145 is connected to a reservoir in the autosampler through line 161; and when valve 137 is activated while valve 155 remains
• deactivated syringe 139 is connected to a reservoir in the autosampler through line 157.
• Fluid line 171 is connected to pipette 83 (Fig. 3) at the automatic sample changer in the autosampler module, and splits to four lines, one each going to valves 155, 159, 163, and 167.
• any one of the syringes may draw fluid from a probe on the stage of the automatic sample changer, or return material to a container on the stage.
• syringe 145 typically draws from the automatic sample changer pipette through valves 143 and 159 activated, although this is but one of the many arrangements that can be made by switching valves off and on.
• lines from each of valves 137, 143, 147, and 151 extend to mixer 19.
• syringes are filled with fluid samples that together make up an assay.
• syringe 139 may draw in buffer, syringe 145 substrate solution, and syringe 147 an enzyme solution from the automatic sample changer.
• syringe pistons By driving the syringe pistons forward simultaneously at programmed rates and distances, a pre-programmed amount of each of the components of an active assay are delivered to the mixer, where the enzyme catalyzed reaction begins immediately.
• the mixer assures complete homogeneity of the mix, which passes through the mixer into cuvette 173, where data is collected by means of a spectrophotometer or other analytical instrument.
• the syringes are of a size that a single draw is adequate in most cases to provide all of the assays needed, in some cases 100 or more, to determine accurately the kinetic constants for a particular enzyme and substrate. Effluent (completed assays) is forced on through the cuvette, and
• variable probes and as many as four stock solutions in dedicated reservoirs allows for a very broad range of assays to be mixed and measured, and constants can be automatically determined for several enzyme and substrate combinations without pause for major service.
• the larger syringe 139 allows for such operations as flushing and cleaning of the lines and equipment.
• fluid line connections shown in Fig. 5 are exemplary of those of the first preferred embodiment, as are the number of valves and syringes.
• FIG. 6 is a broken section illustrating the construction and operation of mixer 19 shown elsewhere in Figs. 1 and 5. This drawing is meant to show the general arrangement of detail elements to one another rather than specific construction of each element.
• a generally cylindrical outer body 175 is a fluid -tight enclosure with four incoming ports, and these each are connected to one of the lines 141, 177, 179, and 181 from valves 137, 143, 147, and 151 respectively.
• the incoming lines penetrate body 175 around the periphery, so that incoming fluid from each of the lines (injected by the syringes) will enter the mixer in an annular space 185 between the outer body and an internal cylindrical drum 187.
• the tubes need not be perfectly orthogonal to the mixer body or exactly equally spaced as shown in Fig. 6, but it is important that they enter the annular space, and it is advantageous that they enter near the end of the mixer away from outlet line 183.
• the body is inert plastic material, such as fluorocarbon material
• the drum is the same plastic material with imbedded
• ferromagnetic elements although there are a number of other suitable materials that may be used. It is advantageous that the material be inert with the reagents used with the apparatus.
• Drum 187 floats within the outer body of the mixer and is driven rotationally within the outer body by an external magnetic drive (not shown in Fig. 6) that also urges the drum against the top of the enclosure.
• an external magnetic drive not shown in Fig. 6
• the drum is shorter than the cylindrical cavity of the body, leaving a space 193 between the drum and the end with the grooves.
• drum 187 is typically rotated about its central axis at a rotational velocity of from about 1000 to 10,000 revolutions per minute while small amounts of fluids are injected into annular space 185, less than .2 mm in width.
• the rotational speed is adjustable (programmable) through the software
• the translation speed of the drum surface rotationally should be typically about 20 times the translational axial velocity of liquid mixture in the annular space to assure adequate mixing of solutions under all expected conditions.
• one fluid may have a viscosity very much larger than another, and in many cases the required amounts of fluids may vary by as much as 2000 to 1.
• the rapid shearing action in the anullar space during injection assures that homogeneity is accomplished extremely rapidly, while only minimally slowing the flux of material through the mixer.
• the liquids can be injected with low pressure, unlike known passive mixing devices. In most cases, the injection step and complete mixing is done within 1 second.
• the dead time of the apparatus which is the time between mixing and measuring, varies from .075 seconds to 1 second, due to different injection volumes and injection speeds. For assays with low substrate concentration the deadtime is kept as short as possible because substrate is used up quickly by the enzyme reaction, whereas for assays with high substrate concentration the dead time can be longer.
• annular space 185 has an axial length of about 8mm and the gap width is about .2 mm.
• the total active volume of this annular space is thus about 25 microliters.
• 400 microliters are injected in 1 second. Under these conditions, materials remain in the shear field for about .0625 seconds.
• the axial velocity is 128 mm per second, and for the peripheral speed of the drum to be 20 times the axial fluid velocity, the rotational velocity of the drum must be more than 6000 revolutions per minute.
• Space 193 is especially useful if the stepper motor driven syringes add volumes in a non- continuous manner. In this case the space acts as a buffer zone to level out sharp pulses to a more steady concenteration.
• Fig. 7 (A) is a section view of mixer module 20 (also shown in Fig. 2) which includes mixer 19.
• Mixer 19 is driven in the module by magnetic drive rotor 195 which is driven by motor 197.
• the motor, the magnetic rotor, and the mixer are located in separate
• compartments of an inner structure 199 of the module, and the magnetic nature of the mixer drive does not require a penetration of the mixer volume.
• the only- physical penetrations of the mixer are the fluid line penetrations.
• the mixer module has an outer shell 201, and the space between the inner and the outer shell is a water jacket for temperature-controlled water, circulated to control the temperature of solutions delivered in the fluid lines.
• Connector 205 is for connection to tubing 55 (Fig. 2) within which other fluid lines are routed.
• Line 141 is one of the lines from a valve in the valve block to the mixer, and is shown extending behind the inner shell and connecting to the mixer at connection 207. In the first preferred embodiment there are four lines from the valve block to the mixer for injection of solutions to form assays for measurement, but only one is shown in Fig. 7 (A) to avoid confusing clutter on the drawing.
• Fluids injected are mixed in mixer 19 and the mixture is forced through the exit line and connection 209 into cuvette 21, where flow is stopped by virtue of the further injection of fluids being stopped, and measurement of optical density vs. time is made using the spectrophotometer (not shown in Fig . 7 ) .
• the spectrophotometer not shown in Fig . 7
• readings from 10 photodiodes are taken at 100 msec intervals, and the time window is adjustable through an input variable.
• a second cuvette 215 is mounted within the water jacket to the inside sidewall of the outer shell, and an analytical instrument 217 may be mounted in a well from outside the mixer module into this cuvette.
• Instrument 217 is typically either a temperature measuring instrument or an instrument for measuring acidity (pH). Effluent urged through the second cuvette exits the mixer module via fluid line 33 back to the autosampler module, where waste is captured in a reservoir for that purpose.
• tubing 55 In addition to the fluid tubes described passing through tubing 55, there is additionally a supply tube for incoming water at a controlled temperature, which empties into the water jacket.
• the circulating water flows around the water jacket surrounding the mixer and the fluid feed lines, and backflows through tubing 55, returning the water to a manifold (not shown) that routes it back to water control unit 57.
• the overall height of the mixer module is about 8 cm., and the diameter is also about 8 cm.
• the various parts are made to fit together with suitable o-ring seals and fasteners, as is known in the art, to be demountable for service.
• Control lines and electrical power lines are typically routed to the mixer module outside and alongside tubing 55, and the lines are such that the module extends about 25 cm. from the valve module on a flexible tether, and may be easily moved and
• Fig. 7 (B) is an enlarged section view of
• Opening 237 will be a minimum during stopped flow, but under the influence of injection pressure while fluid is injected, will expand.
• the volume that can be washed out during stopped flow is reduced to the volume 239 of line 141 between the annulus of the mixer and the o-ring restricted position.
• the length of this volume is 1mm and the inside diameter of the tubing is .3 mm, so the wash-out volume is minimized to less than .1 microliter.
• the adjustable o-ring connection is typical of all of the tubing connections to the mixer, not just the connector shown for illustration.
• the mixing system There are several important features of the mixing system that bear emphasis.
• the mixer can be built with absolutely inert surfaces and is essentially sevice free. There is no contamination with lubricant or other substances in the mixer.
• the driving magnet fulfills three functions. It drives the mixing body, it pulls it ot one end of the mixing chamber, and it constantly adjusts the position of the mixing body, keeping it in the center of the mixing chamber.
• the design further allows a very narrow shear slit, which although is 0.2mm in the illustrated preferred
• the embodiment can probably be reduced to 0.05 mm. This is possible because the driving magnet and the fluids tend to center the mixing body, the mixture being a lubricant because of the Teflon outer coating of the mixing body. Also, the secondary mixing chamber below the shear mixing area allows pulsed operation of the stepper motor drive systems without causing pulses in the output of the mixing chamber. Also, the design of the input tubing with elastic orifices is unique in preventing leakage from the supply lines during stopped flow operations without interrupting flow during other times. Finally, the system allows the active volumes to be very small compared to typical prior art mixing systems.
• Microprocessor unit 35 in the first preferred embodiment comprises a Z-80 microprocessor in a single- task operating system. It treats driving the syringes as a single-task procedure. It also comprises Readonly memory (ROM), Random-Access memory (RAM), and a variety of other elements for accomplishing direct control of the machine elements of the apparatus. The other elements include such devices as tranceivers, decoders, stepper motor drivers, logic elements, and the like. All of the electronic elements are mounted on printed circuit boards mounted in cabinet 53. In building and testing the apparatus of the first preferred embodiment, it was discovered that accuracy of results is better achieved when timing inaccuracy does not exceed 10 microseconds between syringe drives, and that the accuracy is limited with a single
• a microprocessor is dedicated to each of the syringe drives and the dedicated
• microprocessors are slaved to an additional
• microprocessor allowing parallel control functions to be performed during the driving of the syringe units.
• Operation of the apparatus includes indexing of the rotary stage of the automatic sample changer, lifting and lowering of the pipette at the sample changer, operation of the solenoids of each of the valves in the valve block to switch the direction of fluid flow, independent drive of each of the stepper- motor driven syringe units of the syringe module, operation of the motor-driven mixer drum of the mixer module, and operation of the spectrophotometer unit. All of these control functions are performed through the microprocessor control unit, and the status of various parts of the apparatus is monitored through the position of microswitches.
• the microprocessor used is a Z-80
• microprocessor There are, in addition, a number of data transfer, manipulation, and output functions performed by computer elements.
• Fig. 8 is a closer view of the module, which consists of an LED status display 221 that is, in the first preferred embodiment, designed to show the positions of the valves and limit switches in real time; a dot-matrix LCD digital display 223, and a matrix of 32 keys in two 16-key patterns.
• the keys are for inputs to the control unit, and the two-line digital display is for messages and for display of each of a number of control menus that are used for input of variables at appropriate times, and for directing the flow of control programming. Many keys are dually identified, once on the key and once above. For example, the "B" key is also used for "J", and there is a shift key to accomplish the differentiation.
• the output shown on the digital display in Fig. 8 is the first two lines of a Syringe Control Menu.
• the unsynchronized mode is accomplished in the first preferred embodiment through system software programmed in Z-80 assembly language and resident at the microprocessor module in ROM. In this mode a use can enter a comprehensive set of single keystroke commands from the built-in keyboard, controlling all of the functions of the apparatus in a manual mode. For example, the "F" keystroke activates a function called "EMPTY". This function empties the currently active syringe back to the reservoir immediately.
• the complete set of keystroke commands is presented in Appendix A.
• the manual-mode operations the first preferred embodiment are divided into three groups for control of the functions of the detector instrument, the automatic sample changer, and the syringes of the syringe array.
• the groups are access through prefix keys: (-) for the detector, (+) for the sample changer, and (*) for the syringes.
• prefix keys With the prefix, softkey labels appear on the display, arrange to correspond to the eight keys labeled "A" through "H” on the front panel. Pressing the corresponding key initiates the function. Additional functions in each group are accessed through the shift key.
• the master processor is an 80286, the automatic operations are controlled by programming written in "C" language, which is resident at the master processor.
• the master processor is an 80286
• Determination of kinetic constants according to the model of the Michaelis-Menten model as described in the background of the invention is one of a number of useful applications of the present invention, and the Michaelis-Menten model of kinetic behavior is one of the models for data manipulation and display.
• the kinetic software for collecting reaction velocity data is useful for a broad range of kinetic models, some of which are programmed and selectable by the user at his option.
• Syringe constants and updated status are stored in predetermined control memory blocks, and another memory block is designated as the "active" block.
• constants and status include position of the piston in pulses; maximum pulses for a full length move; number of wait cycles after a pulse; number of functions executed for functions B, C, and D; maximal fill level in pulses; and the conversion factor for the particular syringe for motor pulses into volume.
• a Syringe Control Menu may be displayed. This menu is also displayed while in the local mode, so syringe data may be followed by an operator.
• the complete Syringe Control Menu for the first preferred embodiment is shown as Fig. 9. Two lines at a time may be displayed, and other lines are displayed by rolling the menu up or down with the "up arrow” and the “down arrow” keys. There are in addition to the information lines on the window, "softkey” lables presented by pressing the space key. Pressing the softkeys in the local mode will activate the named function (see appendix A).
• the softkeys in the first preferred embodiment are the keys on the front panel labeled "A" through "H", and correspond respectively to the softkey designations f1 through f8.
• the main program of the Kinetic software is started when the command "/" is received by the
• the Main Menu allows the user to jump to other menus and to perform other functions, such as accessing information in files, by using the softkeys.
• Softkey A “Measure” jumps to the Measure Menu.
• Softkey B “Dacom” jumps to a menu called the Dacom Menu.
• Softkey C “Read Pa” allows the user to read Measure Parameters from files , and prompts for filename .
• Softkey D “Meas Pa” jumps to a Measure Parameter menu.
• Softkey E “Direct” shows a directory of connected disk drives, and the user is prompted for drive specification.
• Softkey F "Test M” jumps to a Test Menu.
• Softkey G Store Pa” stores the current measure parameters and prompts for a filename.
• Softkey H “Setup M” jumps to a Setup Menu which is use to change the Assayomate setup.
• the measurement Parameter menu (2 pages) is presented in Fig. 10 (C) and Fig 10 (D).
• the functions in the softkey portion of the menu allow values in the menu to be changed.
• the association of the softkey labels with the actual keys (A through H) are the same for all of the menus, and will not be repeated.
• the user is prompted to enter three numbers. The first is the number of points, the next is the number of repetitions to be done at each point, with the
• Valid ranges are (2 -40), (2 - 10), and (0- 8) respectively.
• LRange is used to enter 3 wavelength ranges in a valid range from 180 to 820 nm. The first is the main measure range, the second is for an additional substrate or product, and the third is internal reference used to compensate for lamp
• Page 2 jumps to Page 2 of the Parameter Menu.
• Conc is for entry of the concentration of the stock solutions. The user is prompted to enter the enzyme concentration in nM and the substrate
• concentration in microM Name is for entry of the name of the enzyme and the substrate in a maximum of 20 characters with no commas. [S]max. prompts to enter the substrate maximum concentration, and there is a requirement that the ratio to the stock concentration be a minimum of 4 and a maximum of 10. Epsilon prompts for four extinction coefficients for the substrate and product; 1 for each at each of two wavelengths. These are for conversion factors.
• Optical Density is plotted vs. time. DOD Sea prompts for similar information when difference in OD vs. time is to be plotted.
• Type is for entering the type of data format according to one of six implemented models. Again, for more detailed description, the Software Documentation and listings are appended, as is the complete software code for the implementation of the first preferred embodiment. (Note: The software appendices reflect implementation with three syringes. The functions coded and called are the same.)
• the Setup Menu is accessed from the Main Menu by the Setup M softkey, and is shown in Fig. 10 (E). Exit jumps back to the Main Menu. Volume prompts to enter an assay volume in the range of 0.3 to 1 ml. Time is for the entry of a stopped-flow addition time of from 100 to 600 ms. Needle lets the user enter the distance the pipette must move vertically at the automatic sample changer to clear tubes supported in a rack on the stage, and the speed the drive is to travel. Syr 1 is for entry of the volume, fill level, and drive speed for the first syringe. Syr2 enters the same
• the menu has a softkey for each. Furthermore the software is easily extended to the four syringe case. The mode of operation and entry is the same.
• automatic sample changer tray such as the speed for the drive and the number of positions in the tray for tubes (probes).
• the Measure Menu is shown in Fig. 10 (F) and 10 (G), and is the menu where a series of assays may be initiated, and the results stored and printed.
• Main M jumps to the Main Menu.
• Page 2 jumps to the second page.
• Syringe jumps to the syringe control functions. Wash is used to wash a selected syringe, and the operator is prompted to enter the number of cycles and the syringe.
• DiVar displays the printer header so that a parameter set may be checked before a series is started.
• Test M jumps to the Test Menu.
• [ E,S ] allows reentry of the enzyme and substrate concentration, in case they may have changed. Points allows reentry of the points
• Page 1 jumps to the first page.
• Print is a toggle that selects a different amount of printout.
• Content shows a directory of the data disk. Comment allows a user to enter 64 characters of comment without a comma. KmMeas starts the measure for a kinetic constant, after prompting the user for series, filename, and the enzyme concentration. Blank allows the entry of a blank file to be subtracted from all of the following
• Fig. 10 (H) is the Single Assay Submenu below the Measure Menu.
• the softkey functions are: Exit jumps to the Measure Menu (1 level up). Go On proceeds to the next task if in manual operating mode. Graph stops automatic operation and shows a graphic display. Alpha shows an alphanumeric display. [E] prompts the user to enter a new enzyme concentration, then mixes and measures a single assay, and proceeds with automatic operation. Activ. prompts the user to enter an
• Fig. 10 (I) shows the debug submenu. It can be invoked during automatic operation, which is then suspended, and offers several functions designed to cure problems that might develop.
• the softkeys are inactive for the Debug Menu. A number entry performs the function listed . Repeat Single Assay prompts the user for the assay to repeat. Next probe ready prompts for concentrations, and allows the user to build up a job queue.
• control goes to the End submenu, which displays the data for all the points that have been meaasured.
• the End Menu is active for about 10 seconds, then data is stored and the next enzyme probe is sucked in.
• Appendix B provides a more detailed description of the End Menu and associated softkey functions.
• Test Menu (second level) is provided to test baselines, wash syringes, and activate third-level menus.
• a softkey is provided in this menu to inject non-absorbing buffer to generate a zero baseline for the analytical instrument in use. Further detail is available in the Appendices to this specification.
• Test Measure submenu is a third-level menu under the Test Menu, and its function is the evaluation of assay condition for later use in the Auto Measure Menu, or for the automatic determination of kinetic constants.
• a typical menu and softkey functions are described in detail in the appendices. There are two pages to this menu in the first preferred embodiment.
• the Auto Measure submenu is also a third-level menu under the Test Menu, and its function is the acquisition of a batch of assays with the same probe and the repetitive application of the batch measurement to a series of probes.
• the softkey functions and detailed description are in the appedices to this specification.
• the Measure Concentration Submenu is a third-level menu for measuring the concentration of substrates and products. It is also used for measuring spectra of samples at different dilutions. Again, detail is provided in both the software documentation and
• the Dacom menu is a second-level menu for Data Communication to a host computer, and is also used for showing the header of the data curve in memory, editing the data curve, copying blank files, and plotting data
• the Data Transfer Submenu under the Dacom Menu (third- level), has control functions for initiating and aborting data transfer.
• the softkey functions and additional detail are in the Appendices.
• the Display Variable Submenu under the Dacom Menu displays variables and provides softkey functions for data editing a transfer.
• the Edit Data Submenu is a third level menu under the Dacom Menu, anb provides softkey functions to peruse and edit data files before plotting or other output function.
• the Plot Submenu allows the user to Title and prepare a plot before execution. There are two sets of softkey functions, and additional detail is available in the appended documents.
• the "local" controller is a Z-80 microprocessor, and there is a limit to the sophistication and the range of software that can be resident and operable at that level.
• Attachment to the HP-85 provides a second level for programmed control, and communication to a larger host, such as the HP-150 allows even more sophisticated post-acquisition data processing to be done.
• the Kinetic Software described above runs on the HP-85.
• the Fit Software described in the present section resides on the larger host, which, in the first preferred
• the master processor for the microprocessor controller is an 80286, and the functions of the Kinetic Software are programmed on the 80286, while the Fit functions resid on a built-in Mac II or PS/2 computer.
• the broad function of the Fit software is data analysis and display. Functions are accessed, as in the Kinetic Software, through a menu structure, and ar described in detail in Appendix C, the Michfit Softwar Documentation. The software is written around a workfile of 100 records in the first preferred
• the records are used as operands of mathamatical operations or as sources for direct curve fits or graphical presentations.
• the records are those transferred by the Kinetic software typically as a result of Assayomate operations, although data from other sources may be received and processed as well.
• the Menus include:
• Input Menu which is responsible for all inputs from outside of the workfile.
• Output Menu which is responsible for sending data to several output devices.
• Data Handling Menu provides functions to edit the workfile, to perform operations with registers, and to exchange the active workfile with stored workfiles.
• interpreter and a macros function for automating sets of procedures that have to be done regularly.
• Direct Curve Fit provides a new fitting program for the Fit Software. It is started, as are other direct curve fit routines, by entering first estimates. It may also use a grid search technique covering the entire meaningful range of all parameters. A binary gradient search routine is used to optimize the first estimate found by the grid search.
• Plotsize Menu has the major function of the graphical presentation of data on a screen, plotter, or printer.
• Display Menu provides a comprehensive set of functions to create and annotate displays of data.
• a principle use of the apparatus of the first preferred embodiment is the determination of kinetic constants for enzymes by measuring large numbers of assays quickly, accurately, and efficiently, as has been described above. This is, however, not the only application of importance.
• the apparatus as described may be employed as well for screening operations. For example, by stocking one reservoir with a particular substrate, or even more than one reservoir with more than one substrate, and providing a plurality of enzymes in probes at the automatic sample changer, which may be constituted to have a large number of probe positions, a user may investigate, quickly and automatically, the specificity of enzymes for the substrates. Many similar screening investigations may be arranged and performed with the apparatus and software, such as the effects of a plurality of
• dilutions may be performed to prepare solutions over a very broad range of concentrations, which has not heretofore been possible automatically.
• line 169 (Fig. 5) extends to a reservoir containing a stock enzyme solution, one might draw from the enzyme reservoir with valve 151 activated and valve 167 deactivated, to an extent of one one-thousandth of the volume of syringe 153 (48 motor pulses in the
• the dilution could be compounded by ejecting the 1000 to 1 diluted solution to another empty probe on the stage of the automatic sample changer, and the 1000 to 1 dilution could be performed again, drawing the originally diluted solution in place of the stock enzyme.
• the result would be a solution accurately diluted by a factor of 1,000,000 to 1.
• preprocessing that is important is automatic dilution.
• the system will automatically dilute the reagent until it is brought into a useful range. With this feature, the apparatus becomes truly automatic.
• Figs. 11-12 show the results of a determination of the Mikaelis constant for cytochrome c. These figures show the printouts provided by the Assayomate as a result of that determination. In the printout shown in Fig. 11, a header is shown in the upper portion which indicates the file name, the name of the disc volume, and the date of the measurement. Below that the measure parameters are printed. Line 1 indicates the type of measurement (Michaelis constant initial
• Lines 2-4 indicate time window at lowest and highest substrate concentration.
• Line 5 indicates measuring interval and integration time for each measurment.
• Line 6 indicates noise, and determines the standard deviation within which single measurements must fall so that the average is calculated.
• Lines 7-9 indicate the two monitoring wavelength ranges and wavelength range for normalization.
• Lines 12-13 indicate concentration of enzyme in stock and in assay.
• Lines 12-13 indicate concentration of substrate in stock and highest substrate concentration in assay.
• Line 14 indicates the number of different substrate concentration (points) and number of repetitions for single substrate concentration.
• Lines 15-16 indicate extinction coefficient for wavelenght ranges on line 7- 8.
• Line 17 indicates volume of assay (sum of buffer, enzyme and substrate). Fig.
• Fig. 13 is shown a plot of the derivative of absorption (DELTA OD/sec) as a function of time at 20 different substrate concentrations.
• Fig. 14 is a printout showing the turnover numbers at 20 different substrate concentrations calculated from data of Fig. 12 with the difference extinction coefficients and the enzyme concentration given in Fig. 11. Shown in the first column is the point number. The second column is the turnover number determined from the absorption change at 548 to 552 nm. In the third column is turnover number determined from th eabsorption change at 416 to 420 nm, and in the fourth column are the substrate concentrations in uM.
• Fig. 13 is shown a plot of the derivative of absorption (DELTA OD/sec) as a function of time at 20 different substrate concentrations.
• Fig. 14 is a printout showing the turnover numbers at 20 different substrate concentrations calculated from data of Fig. 12 with the difference extinction coefficients and the enzyme concentration given in Fig. 11. Shown in the first column is the point number
• microprocessors communication protocols, data
• the number of driven syringes can vary, as can the number of stock reservoirs and the number of probe positions on the automatic sample changer stage.
• the stage itself need not be a rotary device, as there are a number of other ways that a plurality of probes might be presented to a pipette. There could be more than one automatic stage and pipette, too. The variety of such possible
• the message displayed is 'Please enter the volume in (ML)', it expects the input of a number between 0 and the maximal volume.
• E WASH This function washes the active syringes by repetative suck in and give out cycles. It displays "PLEASE ENTER THE NUMBER OF CYCLES' and expects the input of a positive number in the range from 0 to 99. The first cycle empties the syringe, the next cycle sucks in 2% of the maximal volume, the third empties the syringe and so on. An odd number of cycles, stops washing with an empty syringe, an even number stops washing with a filled syringe. The message 'WASHING OF ACTIVE SYRINGE' is displayed during the entire operation.
• H STOPFL This function mixes an assay and displays the message 'STOPPED FLOW'.
• the amounts of buffer, enzyme and substrate have to be defined by the function 'K' before this function is executed. If one of the syringes runs out of volume, the function is immediately aborted. Approximately 100 milliseconds before the assay is finished, the busy bit in t he status byte of the IEC interface is cleared, and after the termination of the assay, the menu is updated.
• I SPEED This function is used to change the speed of piston movement of the active syringe. It displays 'PLEASE ENTER THE SPEED IN ML/MN' and expects the input of one number in the range of 0 to 99 ml/min.
• composition of the assay It displays
• substrate spacing the number of points, the number of repetitions, the volume of the assay (in ml), the amount of enzyme (in ml), and the maximal amount of substrate (in ml) e.g. *M 1,20,3,0.400,0.040,0.100.
• M FUBED This function calculates the need of buffer, enzyme, and substrate according to the settings of function 'M'. Then it executes a fill command for all syringes, but only the syringes containing not enough volume are actually moved. If a syringe has to be moved, it displays 'FILLING SYRINGE'.
• LEVEL IN ML' and expects one number in the range from 0 to the total volume of the active syringe.
• P SFSET This function is used to set the volumes for an assay by specifying which point of the active rowtype and number of points shall be prepared. Before this function can be executed, proper parameters for function 'M' have to be entered. It prompts 'PLEASE ENTER POINT (ASSAY)' and expects an integer in the range from 0 to the maximal number of points.
• HEXADECIMAL ADDRESS and expects a hexadecimal number in the range of 0 to FFFF.
• the hexadecimal address can be followed by to 32 pairs of hexadecimal numbers each separated by a space from the next pair.
• V RECASC This function expects a hexadecimal address and a string of ASCII characters from the IEC talker.
• the ASCII string is stored to the ASSAYOMATE memory.
• W SEASC This function epects a hexadecimal address and sends the content of the ASSAYOMATE memory to the IEC listener until a carriage return is encountered. The longest string that will be sent is 128 bytes. If the IEC listener is not ready to receive data, the ASSAYOMATE displays 'IEC TIMEOUT' after 1 second and aborts the operation. X MSTAT This function reads the status of the microswitches and copies it to the status byte.
• Z NUWFU This command executes the RAM based routine that has been loaded to the ASSAYOMATE memory before.
• the MICHKIN user software is the interface between the user on one hand and the ASSAYOMATE and the photometer
• the determination of a kinetic constant is executed by simply pressing a softkey in the Measure Menu.
• Menu They can be stored on disc to be used at a later time.
• MICHKIN software calculates the amount of buffer, enzyme, and substrate of inhibitor for the data point i and sends the command to the ASSAYOMATE (functions *O and *H). When the mixture is finished, the
• spectrophotometer is activated and the MICHKIN software accepts series of spectra from the spectrophotometer.
• the software calculates a normalized time dependence of the optical density at one or two wavelength ranges. It then differentiates the data and checks the stability and the noise of the derivative.
• the computer controllable ASSAYOMATE and a commercial photometer are used by the MICHFIT software to determine kinetic constants from a set of steady-state activities measured under different conditions
• the MICHKIN software is divided into four overlays, a start overlay and three overlays, that can be activated from the start overlay. Data is exchanged between the different overlays with the aid of a common data block .
• the MICHKIN software has been written in HP BASIC
• Measure overlays Measure Menu
• Testmeasure overlay Test Menu
• syringe parameter e.g. fill speed, fill-level, assay volume
• the Syringe Control Menu is used to move the syringes under manual control. This menu offers the functions fill, empty, suck in, give out, give back and change amount, described in the ASSAYOMATE Documentation and in the MICHKIN User
• the syringes are activated one after the other by pressing softkey #8 in this menu.
• An assay is mixed and measured by activating the single assay function in the Measure Menu. After the softkey ⁇ Single Assay> has been pressed, the user is asked to enter the substrate and the enzyme concentrations. If the entered concentrations are valid, the assay is mixed and the photometer starts measuring. The optical density is plotted on the screen as a function of time, then the derivative, the linearity and the noise are printed.
• the test overlay has three major functions.
• Concentration Menu the concentration and spectra of substrate and product can be measured.
• the Testmeasure Menu is used to test assay conditions for new
• Serial Assay Menu a set of assays can be performed on a large number of probes, either manually or automatically by means of an
• This overlay The main function of this overlay is the transmission of data via RS-232 interface to a host computer. Data files that have been stored on disc are read in and sent to the host. The data transfer is controlled by this overlay if the softkev 'Master' is selected in the Data Transfer Menu. The data transfer has to be
• this overlay is responsible for editing of result files, and
• the computer and the ASSAYOMATE are switched on, they have to be connected by the IEC bus.
• the photometer and the ASAYOMATE are then switched on, they perform the initiation simultaneously.
• a measurement parameter file has to be read in. Then the concentration of the substrate is determined with the Testmeasure overlay.
• the Testmeasure overlay is executed for the first time, syringe parameters are loaded into the ASSAYOMATE memory. But before the photometer is able to perform the first measurement, a 'Reference Measurement' has to be performed. To do that, buffer is sucked into syringe 1 (either with functions from ASSAYOMATE keyboard or with the Syringe Control Menu).
• concentration is measured in the Concentration Menu. Then the concentration of the enzyme is determined with the Testmeasure Menu.
• Printouts of result from the determination of kinetic constants can be generated in two modes: If the print flag is on, a full printout is generated a) - h), otherwise only a) and h) are printed.
• a typical full length printout consists of 8 parts.
• the accuracy of kinetic constants is increased when the measurements are corrected for a blank reaction.
• a blank can be measured by setting the enzyme stock concentration to zero. It is then stored as
• the common data area of the MICHKIN software is a portion of RAM which is reserved in all overlays. It is used to exchange spectra, measurement parameters, flags, and pointers between the overlays.
• the entries in this block are:
• the measurement parameter files are generated after the
• 'Sto Pa' softkey has been pressed in the Main Menu of the MICHKIN software. They are used to store the set up, conversion factors, and enzyme names. Such a file is generated for each different enzyme or substrate.
• the MICHKIN software measures and stores velocities of enzyme reactions at different substrate concentrations
• the content of the file 'INHALT' is shown by the procedure on line 2000. This file contains 12 lines of information about the content of the data files on this diskette.
• a special parameter file 'DEFAULT' is then read by the READ_PARAMETER procedure (lines 4410ff). If this has been completed successfully, the Main Menu is shown (line 4000).
• 8 softkeys are defined by the ON KEY#X, ' ⁇ Label>' GOTO YYYY (lines 4150-4220). The softkey are then shown on the last two lines of the screen with the KEY LABEL command. Line 4230 forms an indefinite loop that can only be left by pressing one of the softkeys. If, however, another key is pressed the program stops.
• the routine from line 4250 to 4317 checks if all parameters are in a valid range (procedure on line 4600) and stores the axes and labels of the three plot types in three files (procedures on lines 5000, 5100, and 5200). Two hidden temporary files are generated on lines 4290 and 4295. All
• spectrophotometer data has to be erased before the KINX overlays can be CHAINed.
• different measure overlays are loaded ('KINl', 'KIN2'. 'KININ', 'KINSU'). All these overlays contain the same Measure Menu and the same data
• Pressing softkey #2 displays '
• the datatransfer program is loaded ' and loads the data transfer overlay from the system disc (CHAIN 'DACOM. KSYS', (lines 4320 and 4330).
• Pressing softkey #3 shows the directory of the system disc (CLEAR @ CAT '.KSYS') and prompts for the file name of a parameter file (line 4405).
• the file is opened and the parameters are read (lines 4410-4430). If an error occurs because the file is too short, the message 'Old file : Please update syringe parameters ' is shown.
• the files continuing the information for the plots are updated (line 4440) before the routine ends with the GOTO 4000 statement.
• Softkey #4 activates the Parameter Menu (GOTO 500).
• Files can be erased (PURGE), spaces left in the directory from earased files can be removed (PACK), all files can be erased (INIT), or the routine can be left without anv action (N or no input) (lines 1600-1640). If INIT has been selected the user has to confirm his intention to erase all files (line 1657).
• the diskette is then initialized (formatted) using the name provided by the user on line 1660, and the drive specification (H1).
• the INHALT file has to be reentered using the EDIT INHALT procedure on line 2200.
• Pressing softkey #6 checks if all parameters are in a valid range, updates the stored axes of the three plots, checks if the ASSAYOMATE has been initialized and loads the testmeasure overlay (CHAIN
• Lines 500 to 540 are used to generate the following screen:
• Range 1 from 548 to 552 nm
• Exit Points LRange Page 2 The softkeys are defined on lines 550-564 and shown on the screen using the KEYLABEL command on line 566.
• the indefinite loop can only be left when a softkey is pressed.
• Softkey #2 is used to input the number of
• Softkey #3 is used to edit the wavelength ranges for the data acquisition (lines 760- 820).
• Softkey #5 is used to edit the values of the enzyme concentration (in uM) (lines 880-885).
• Softkey #6 is used to edit the names of enzyme
• the second screen of the Parameter Menu is generated on lines 600-640. Again a set of 8 softkey labels is defined and shown on the last two lines by means of lines 670 to 686:
• Softkey #1 selects among several types of data
• the data spacing variable T(19) is incremented and set to 1 if it is larger than four.
• the current spacing type is shown in the menu (lines 620-626) depending on the value of T(19).
• Softkey #2 is used to modify the selection criteria for the kinetic data acquisition
• Softkey #3 is used to set the duration of the
• the duration at [Slmax (start T(6), stop T(7)) is different from the duration at [S]min (start B(13), stop B(14)).
• the measure interval (B(12) ⁇ 2) and the integration time (B(11) ⁇ 1) can be set. Intervals and integration time are only accepted if the interval is larger than the integration time and if they are truncated to one digit in the fractional part.
• the time windows are further checked if the stop time is larger than the start time and if the number of data points (T(7)/ ⁇ 2) is not larger than 80.
• the stop time at [S]min should not be larger than the stop time at [S]max (lines 745-750).
• the axes for the OD plot and the delta OD plot are adjusted in the AXES_OD and AXES_DELOD
• Softkey #5, #6, and #7 are used to modify the Y-axes of the TN vs. plot, the OD vs. time plot and the delta OD vs. time plot.
• the program sequences can be found on lines 960, 940, and 980.
• the axes definitions minimum A( ,1), maximum A(,2),
• increment A( ,3) and first increment A( ,4) are input and checked. Then the axes are updated with the appropriate
• a set of 8 softkeys is provided to modify the
• Softkey #1 exits to the Main Menu (GOTO 4000).
• Softkey #2 is used to enter the stopped flow
• B(7) W1(2) + W1(3).
• Softkey #3 is used to enter the acceleration ramp angle for stopped flow operations
• Softkeys #4, #5, #6, #7, and #8 are used to enter the parameters for the syringes.
• the table above is given for the first syringe (Softkey #5) the variables for the second syringe belong to the same array but with each index increased by one. The same applies for syringe 3.
• the fourth syringe or the tray is controlled in the same way.
• the fifth syringe or needle is controlled using the same array. Tray and needle are the labels because the stepper motors can be used to move a tray and a needle of an autosampler.
• This procedure reads the file INHALT on the diskette with the name B9$. It clears the screen and displays a header, then it opens the file (lines 2025 and 2030).
• the file INHALT is erased and a new file with the same name is opened. It is opened and the prompt 'Please enter max . 12 l ines of commen t ' is displayed. Up to 12 lines with a max. length of 32 bytes are accepted
• the measure ranges are checked by the CHECK_RANGE procedure.
• the array L(1) to L(10) is loaded with the wavelength of the active diodes.
• Thev are spaced with 2 nm on even numbers.
• the diodes necessary to cover range 1 are activated. The same is done for the range 2 on lines 4640 to 4665. If range 2 overlaps the diodes need not be activated twice (line 4650). The total number is limited to 10.
• the remaining diodes are used for the normalizing range (lines 4670- 4680). If 10 diodes are already exceeded in the second range the procedure is left after having displayed the error message: 'Too large wavelength ranges ' (line 4655).
• the number of the variables exceeding the limits is passed to the procedure on lines 4795-4799. It displays the array type (T B or W) and the number of the
• variable for 2 seconds e.g. variables T3 or 4 are invalid
• flag H2$ "F"
• the flag is tested after all variables have been checked. If one or more variables have been found exceeding limits the Main Menu is displayed (GOTO 4000).
• the Y-axis has been entered by the user (A(1, ) array).
• the increment X2 is determined from [S]max. so between four and seven digits are written to the X-axis (lines 5030 to 5048).
• the X-axis is labeled with the name of the substrate (E2$) plus (micro molar) and the Y-axis is labeled with ' Turnover number ( 1/sec . ) ' .
• the file is generated with the STORE,AXES procedure.
• the axes of this plot are stored in a file with the file name 'PFOD.KSYS' on the kinetic system diskette.
• the Y-axis is stored as it has been entered by the user.
• the X-axis is generated using the duration variable T(7). The minimum of the axis is set to zero; the maximum is equal to T(7), the first increment is set to zero.
• the increment ( X2 ) is set to 2, 5 or 10 seconds depending on the value of T(7) (lines 5110- 5130).
• the X-axis is labeled with ' Time ( sec . ) ' and the Y-axis is labeled with 'Absorption '.
• the file is generated with the STORE AXES procedure.
• AXES DELOD procedure (lines 5200ff) The axes of this plot are stored in a file with the file name 'PFAB.KSYS' on the kinetic system diskette.
• the Y-axis is stored as in the procedure above but with 'Speed (Del ta OD/sec . ) ' as the label.
• the X-axis is identical as in the AXES_OD procedure.
• the plotter is set to default status and pen 1 is selected "IN: SP1;”.
• the size of the plot area is defined with the "IP” command. This area is then scaled by the SC command.
• On line 6140 the title is plotted.
• the frame is plotted on lines 6180 and 6190.
• the loop from line 6200 to 6220 plots the ticks and the digits to the X-axis:
• the digits are plotted with a size of 0.2 * 0.25 cm, the origin being
• the scaling is set to user units using the plotted frame as the maximum plotting area "IP” and "IW” commands.
• the file is closed and made invisible to the user (line 6360).
• the routine on line 6400 adds string delimiters to the E5$ string before it is written to disc.
• This procedure is activated by the 'ON ERROR GOTO 9000' commands if a BASIC command results in an error.
• ERROR numbers ERRW are used to inform the user about the type of error that has occured (see lines 9100-
• the program stops after having displayed the error number and the error line.
• the program can be restarted by pressing the 'CONT' key.
• Softkey #1 displays the header information as it is printed on the printer. To do that, the output to the printer is redirected to the display (PRINTER IS 1) and the
• PRINT HEADER procedure is called (line 9545).
• the program pauses so that the screen can be studied by the user.
• the Measure Menu is shown again, when the 'CONT' key is pressed,
• Softkey #4 is used to wash the syringes. To do that the WASH SYR procedure is called,
• Softkey #8 is used to change the number of data
• the second page of the Measure Menu is displayed using lines 1000 to 1260.
• Softkey #2 changes the amount of information that is printed for each automatic determination of a kinetic constant. If the fifth byte of the D4$ string is "1", all
• Softkey #4 calls the EDIT_COMMENT procedure to edit the comment (GOSUB 1700 on line 1690).
• the series character is incremented (A —> B a.s.o.). If the enzyme concentration has been entered with a negative sign, the highest substrate concentration is measured first and the lowest substrate
• the selected enzyme concentration in the assay (B(5)) has to be given. This concentration is identical to the actual concentration (B(5)).
• the enzyme concentration in the assay (B(5)) is used for the first
• the actual concentration B(3) is then adapted so that the activity in the assay is between 90% and 110% of the desired activity (B(10)).
• Softkey #8 performs a single assay by calling the
• concentration of substrate in the assay has to be given (B(6)).
• the display of the time dependent data is switched off (MODE 0,1) and the single assay is started. If a substrate concentration smaller than zero has been entered, the SINGLE ASSAY procedure performs a BASELINE or REFERENCE
• This procedure displays 'Comment 2 lines' and inputs up to 64 characters (2 lines). If the string is shorter, a space is added (line 1610). The prompt is placed in a way so that the cursor is just on the first character of the old version of the comment so that it need not be typed entirely.
• D4$[4,4] is set to "L", that means when a blank has been measured.
• a file with a length of 5 records is created on the system disc and opened. First a part of the header variables is stored (line 1560). Then the delta OD/second information of the two wavelength ranges (arrays T1 ( ) and T2 ( ) ) is stored in this file. The file is closed and the message 'Blank stored in file ................................. « ' is added to the printout.
• this procedure is called before the first measurement is performed by the data acquisition procedure. It opens the blank file using the name stored in the header of the data file.
• a variable in the file header can be changed or the difference can be ingored. Certain variables must not be changed: If variables 1, 2, 10, 11, 12, 13, or 19 are different the measurement is aborted.
• a blank has to be measured with the identical
• the blank is read in and inverted (lines 1850 to 1870). The file is closed and the message 'Blank subtracted' is printed.
• This procedure flushes the cuvette twice with 0.5 ml of buffer and performs a REFERENCE 10 measurement.
• the command "*1" is sent by the SEND_CMD procedure (line 7000). It selects syringe 1 as the active syringe.
• the command "*A0.5” determines the relative amount of buffer to be moved.
• the command "*B” (GIVE OUT) moves 0.5 ml buffer from the buffer syringe through the mixer into the cuvette.
• This command is sent a second time after a delay of 2 seconds (line 1905).
• the reference is measured ( line 1920 ) followed by a test measurement (MEASURE .1).
• the loops on line 1930 and 1940 wait until the measurements have been completed. The results of the 10 photodiodes are then evaluated.
• SETUP_KIN procedure is called to test if the file already exists and to multiply the standard substrate concentrations with a factor (L(20)). If a blank has to be used, the subtract blank procedure is called by this procedure. Then the PRINT_HEADER routine is called to print the header information to the printer.
• T(0) is loaded with the current number of repetitions. This number is higher for the data points with low substrate or inhibitor concentrations because the noise of the average is larger at lower concentrations (see MICHKIN User Manual).
• the assay is performed T(0) times with the FOR-NEXT loop on lines 2305-2500. In the normal mode, the repeat assay flag (f6$) is set to "0".
• the time dependent data is added up in the S3( ) buffer.
• R8 and R9 add up the noise information of the single assays.
• the procedure AVERAGE_VEL is called to calculate the average of 2-10 initial velocities (line 2530).
• the plot delta OD versus time is shown on the screen with the PLOT_DATA procedure. The standard deviation among the assays is determined by the
• ASSAYOMATE This command is used to read the fill levels of all syringes into the MICHKIN software.
• the softkeys of the End Menu are defined and shown on the screen:
• a next sample can be added to the job queue, or the last output can be started. If the job queue is not empty (C(6)>1) the next sample is analyzed after the End Menu has been shown for six seconds (line
• Softkey #1 exits directly to the Measure Menu. This softkey is active during the entire data acquisition and processing,
• Softkey #3 repeats one of the averages of several assays with the same substrate
• the same routine can read all values of the blank starting from the point K if it was called by the resume measure softkey. (#5). After the blank has been read the file is closed and the resume measure softkey. (#5). After the blank has been read the file is closed and the resume measure softkey. (#5). After the blank has been read the file is closed and the resume measure softkey. (#5). After the blank has been read the file is closed and the resume measure softkey. (#5). After the blank has been read the file is closed and the
• Softkey #4 is used to build up the job queue (next probe ready). This is done by the
• Softkey #5 is needed if a measurement has to be
• the blank is read again by the subroutine on line 3110. Depending on the state of the Q2 flag (1:
• Softkey #8 activates the SYRINGE_ CONTROL procedure and then jumps back to the End Menu ( see below).
• This procedure calculates the sum of the squares of the differences between the average value (S9) and the delta OD vs. time data (S1(J1)) for the current time window T8 to T9.
• the root of the mean square is then compared to a minimal value and a relative value.
• the minimal value (0.00001 * (1+K/T(5)) * B(2)) is
• this routine calculates the time window (start time and stop time) for the data point (K) which is measured.
• Two time windows are defined by the user: The time window at the largest substrate concentration and the time window at the smallest substrate concentration. Using these values, the time windows for intermediate concentrations are calculated by linear interpolation:
• This procedure tests if the file name already exists in the disc directory (lines 3600 and 3605) if it exists, the Measure Menu is shown and the determination of the kinetic constant is stopped. The disc drive is checked if it is switched on and if the proper data diskette is inserted. Then the output devices are selected
• This procedure prints and displays the column headers according to the value of the printer flag. It
• the printer flag (D4$[5,5]) is set, the Delta OD vs. time plot is labelled (E4$) and the graphic screen is copied to the printer.
• the PRINT_HEADER procedure is called once more to repeat file name and date of measurement (line 4202).
• the columns for the turnover number versus substrate concentration table are labelled.
• the labels and axes for the turnover number versus substrate concentration plot are read from the file and shown on the screen (lines 4230 and 4250).
• the data file is opened by the OPEN_DFILE procedure.
• the procedure CALCTN is called to return the substrate (H4) and enzyme (H2) concentrations, and to convert the velocity data into turnover numbers (H3 and J1).
• On line 4290 both ranges are plotted.
• the result is printed in the following format:
• This routine enables the user to influence the data acquisition. This menu is called after the softkey #2 'INPUT' has been pressed. It offers 8 functions:
• This procedure derivates the absorption versus time data so that the S1() and S2( ) arrays contain delta OD versus time information (delta OD/per second).
• the average velocities are added up for the current time window (S9 S8 line 4700) and divided by the number of data points.
• the velocities for the two ranges are shown on the display as follows:
• the velocity are then copied into the arrays S4() and S5( ).
• ASSAYOMATE by means of the SETSF procedure and the assay is executed with the "*H" command (line 4860).
• the ASSAY procedure is called for an intermediate data point and the time window is set to B(13) resp. T(7).
• the average absorption of the first four data points is stored in the H1 and H2 variables.
• the axes of the OD versus time plot are loaded.
• the optical density is used to calculate the substrate concentration which is shown on line 5057.
• the derivative procedure is called to calculate the derivative of the absorption which is then checked by the LINCHECK procedure.
• the relative noise is calculated from the absolute noise 56 and the average velocity S9. The absolute and the relative noise are shown on line 5160.
• the linearity is calculated by comparing the velocity of the first five data points (A1 and H4 ) with the velocity of the last five data points H2.
• the relative nonlinerarity (H3) is then displayed on line 5190. If the print flag is set the Single Assay Menu is shown:
• Softkey #1 is used to exit to the Measure Menu. It is active during the entire AUTOKIN and
• Softkey #2 switches back to automatical mode after the user has changed anything manually in the Single Assay Menu.
• Softkey #3 is used to show the graphic screen of the menu and to wait in manual mode for the next user command (line 5420).
• Softkey #4 is used to show the alphanumeric screen of the menu and then waits in manual mode for the next user command (line
• Softkey #5 is used to change the enzyme
• the variable J1 is set to 9; that causes the single assay to be repeated (lines 5310 and 5390).
• Softkey #6 is used to change the desired velocity
• Softkey #7 is used to change the content of one
• the repeat assay flag is checked and the assay is repeated if necessary. Then the delta OD versus time plot is read from diskette and shown on the screen. The data is added by means of the PLOT_DATA procedure and the menu subroutine is called once again (line 5310-5390).
• OPEN_DFILE procedure (lines 5500ff) This procedure creates a file on the data diskette
• the file is opened and the header information is written (line 5570), see data structures).
• H3 intercept (Sum X*Y/N - Sum X/N * Sum Y/N ) / HI

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