WO1996014571A1 - Osmometrie a gradient thermique - Google Patents

Osmometrie a gradient thermique Download PDF

Info

Publication number
WO1996014571A1
WO1996014571A1 PCT/US1995/014352 US9514352W WO9614571A1 WO 1996014571 A1 WO1996014571 A1 WO 1996014571A1 US 9514352 W US9514352 W US 9514352W WO 9614571 A1 WO9614571 A1 WO 9614571A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
temperamre
die
phase transition
zone
Prior art date
Application number
PCT/US1995/014352
Other languages
English (en)
Inventor
Boris Rubinsky
Original Assignee
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to AU41463/96A priority Critical patent/AU4146396A/en
Publication of WO1996014571A1 publication Critical patent/WO1996014571A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N25/00—Investigating or analyzing materials by the use of thermal means
    • G01N25/02—Investigating or analyzing materials by the use of thermal means by investigating changes of state or changes of phase; by investigating sintering
    • G01N25/04—Investigating or analyzing materials by the use of thermal means by investigating changes of state or changes of phase; by investigating sintering of melting point; of freezing point; of softening point

Definitions

  • the present invention relates to a freezing/melting point osmometry device and methods to perform freezing point osmometry.
  • the invention is referred to as temperature gradient osmometry (TGO).
  • TGO temperature gradient osmometry
  • the devjce and methods find application in the fields of determining the compositions of materials, such as alloys, and also in zone purification processes.
  • the freezing temperature depression of a solution is a thermodynamic property indicative of the solvent free energy and is considered colligative because equal numbers of chemically active particles per mass of solvent produce equal freezing temperature depression values independent of their composition, shape, or mass.
  • Osmometers are devices used to measure the effects of solute concentrations on the thermodynamic properties of solutions. Essentially, osmotic measurements are based on changes in the free energy of solvent molecules that occur when a solute is dissolved in the solvent. Osmometers generally fall into three categories: 1) freezing point osmometers, 2) vapor pressure osmometers, and 3) membrane osmometers. The focus of the present invention concerns, particularly, freezing point osmometers.
  • freezing point osmometry There are two basic designs and procedures that are used in freezing point osmometry.
  • One such device is available from Wescor (Logan, Utah).
  • the solution is then agitated (nucleated) which causes the solution to freeze.
  • the sample releases heat (as the heat of fusion) and causes the sample to rise in temperature to thermodynamic equilibrium at the freezing point.
  • the freezing point can be observed as a plateau in the temperature of the sample before the sample cools again.
  • the temperature is detected and read with thermocouples.
  • the second type of freezing point osmometer is generally based on the principles described by Ramsey and Brown in “Simplified apparatus and procedure for freezing point determinations upon small volumes of fluid. " J. Sci. Instrum. 32:372 (1955).
  • the general principle involves observing a solution sample with a magnifying device, lowering the temperature of the sample uniformly in space, in a controlled manner, and measuring the temperature of the sample with a measuring device, such as a thermocouple.
  • the sample is first frozen, then remelted, partially, util a few ice crystals are left.
  • the temperature of the sample is then dropped again, uniformly in space.
  • the sample is continuously monitored with the magnifying device and the observations correlated to the temperature measurements.
  • the temperature at which the sample is observed to start freezing (i.e., where the remaining ice crystals begin to grow) is taken to be the freezing point.
  • a commercial version of a device using this principle is the Clifton osmometer which employs small microliter wells in a copper plate to hold the sample.
  • Another scientific version is described in DeVries et al. "Freezing resistance in some antarctic fishes. " Science 163:1074 (1969).
  • AFP antifreeze proteins
  • THPs cold tolerance and freeze tolerance on animals and plants.
  • New species of animals and plants that produce THPs in response to low temperature exposure are being identified continuously.
  • proteins are identified by gathering biological fluids from plants or animals after low temperature exposure and measuring the fluid freezing and melting temperatures to find hyper-colligative freezing temperature depression.
  • THPs THPs
  • osmometer To measure freezing and melting temperatures in the same sample, to determine thermal hysteresis. Because many organisms, such as insects, have limited quantities of biological fluids and the THPs are often purified in small quantities, the osmometer must be able to measure phase transition temperatures of small volume samples.
  • THP activity is about 30 milliosmols (mOsm). This is determined through use of the relationship of the depression of the freezing temperature depression and osmolality of ⁇ T « 1.86 • C, where C is the solute concentration in osmols and ⁇ T is the freezing temperature depression in °C.
  • THPs occur in nature in low millimolar concentrations with colligative freezing temperature depression of 10 "3 -10" 4o C. Most of the known THPs are not miscible in water at higher concentrations. Therefore, the THPs anomalous freezing temperature depression can be detected with available osmometers only because the depression is several orders of magnitude greater than the colligative freezing temperature depression. However, smaller anomalies in freezing temperature depression or colligative melting temperatures cannot be detected in THPs with existing devices.
  • thermocouples employed. Indeed, the nature of heat transfer problems is that they are inherently boundary value problems and unless one can specify the boundary, one cannot specify the precise heat transfer relationship or the error. Thus, thermocouple inaccuracies can lead to unknown imprecisions in measurement of osmometric relationships. At best, the resolutions of the devices are limited by the resolution of the temperature measurement apparatus. In addition to thermocouple imprecision, most commercial freezing point osmometers (with the exception of the Clifton-type osmometer) require relatively large sample sizes.
  • Clifton-type osmometers rely for their determination of the freezing and melting temperature on a relatively subjective visual determination by an operator of the onset of crystal growth processes. Temperature gradients have been utilized to cause phase transformation in samples. For example, a procedure by Bridgman has been used to grow crystals across a temperature gradient. See Fleming "Solidification Processes" (McGraw Hill (1974)). Temperature gradients have also been employed by the present inventor in several contexts. For example, in U.S. Patent No. 4,531,373, a directional freezing device for the controlled freezing of biological samples was disclosed. The device generally included a first base and a second base which could be maintained at independent temperatures.
  • the bases were separated by a gap and were connected by a substrate spanning the gap.
  • T H and T c first and second temperature
  • the substrate would experience a temperature gradient T H - T c .
  • Samples could be moved across the substrate and cooled across the gradient.
  • the apparatus and method allowed highly controlled freezing of biological samples.
  • the inventor proposed a method to perform studies on the solid-liquid interface and ice crystal formation using a similar apparatus to that just described. Rubinsky et al. "Experimental Observations and Theoretical Studies on Solidification Processes In Saline Solutions". Experimental Thermal & Fluid Science 6(2): 157 (1993).
  • thermocouples to detect and measure temperatures of freezing and/or melting.
  • thermocouples are not highly accurate.
  • the systems required relatively large sample volumes. Accordingly, a need exists in the art for a freezing point osmometer that has enhanced resolution and accuracy. Further, it would be advantageous to provide an osmometer that required small sample sizes. In particular, it would be desirable to have a device meeting these objectives for the study of antifreeze proteins.
  • a freezing point osmometer that is capable of high precision and degrees of accuracy.
  • the osmometer of the invention utilizes very small sample sizes (i.e., on the order of microliters or nanoliters of sample or below).
  • the invention includes both apparatus for achieving these objectives as well as methods for performing osmometry.
  • apparatus in accordance with the present invention includes a substrate generally separated into three zones: a first zone, a second zone, and a gradient zone.
  • the gradient zone generally extends between the first and second zone.
  • the first and second zone are adapted to be independently varied in temperature such that a temperature gradient can be established across the gradient zone.
  • the gradient zone includes a plurality of sample rows or channels extending generally from the first zone to the second zone.
  • the osmometer further comprises a first temperature controller in communication with the first zone and a second temperature controller in communication with the second zone, the first and second temperature controller being adapted to independently vary the temperature of the first and second zone.
  • the osmometer further comprises a detector for detecting a solid-liquid interface in the sample rows.
  • the detector comprises an imaging device.
  • the detector comprises a magnifying device.
  • the magnifying device comprises a microscope.
  • the osmometer further comprises a video camera in optical communication with the magnifying device.
  • the osmometer further comprises a computer for capturing a video image from the magnifying device.
  • the osmometer further comprises at least three sample rows, comprising a first, a second, and a third sample row, wherein, the first and second sample rows comprise standard solutions having a first and a second known phase transition temperature and the third sample row includes a solution whose phase transition temperature is desired to be quantified.
  • the first phase transition temperature is higher than the phase transition temperature of the unknown solution and the second phase transition temperature is lower than the phase transition temperature of the unknown solution.
  • the first zone further comprises a first base and the second zone further comprises a second base, the first and second bases being separated by the gradient zone and each of the first and second bases being in heat transfer relation with the substrate.
  • the sample rows comprise capillary tubes.
  • a thermal-gradient osmometer comprising a substrate having a first end, a second end, a first surface, and a second surface, the second surface having a plurality of sample rows extending generally from the first surface to the second surface, a first base having a first heat transfer surface adapted to sit in heat transfer relation with the first surface of the substrate, a first temperature controller in communication with the first base for controlling a temperature, T H , of the first base to be above the phase transition temperature of samples contained in the sample rows, a second base spaced a distance, d, from the first base to define a gap therebetween and having a second heat transfer surface adapted to sit in heat transfer relation with the first surface of the substrate, and a second temperature controller in communication with the second base for controlling a temperature, T c , of the second base to be below the phase transition temperature of samples contained in the sample rows.
  • the osmometer further comprises a substrate mover for moving the substrate generally longitudinally across the first base in the direction of the second base with the first surface in heat transfer relation with both of the first heat transfer surface and the second heat transfer surface.
  • the temperature, T H is controlled to be substantially constant
  • the temperature, T c is controlled to be substantially constant
  • the substrate mover moves the substrate at a substantially constant velocity.
  • the temperatures, T H and T c are so chosen and the substrate mover moves the substrate at a velocity chosen such that the samples in the sample rows undergo phase transformation when opposite the gap.
  • the osmometer further comprises a magnifying device positioned opposite the gap and being adapted for viewing solid-liquid interfaces in the sample rows.
  • the magnifying device comprises a microscope.
  • the osmometer further comprises an imaging device in optical communication with the magnifying device.
  • the osmometer further comprises a computer for capturing a video image from the imaging device.
  • a method to perform freezing point osmometry comprising providing in a first sample row a first solution having a first phase transition temperature, wherein the first freezing point is desired to be quantified, providing in a second sample row a second solution of a second known phase transition temperature, the second phase transition temperature being different than that of the first solution, providing in a third sample row a third solution of a third known phase transition temperature, the third phase transition temperature being different than that of the first and second solution, exposing the first, second, and third sample rows longitudinally across a substantially linear temperature gradient that extends from a first temperature that is higher than the phase transition temperature of the first solution to a second temperature that is lower than the phase transition temperature of the third solution so as to cause a portion of the first, second, and third solutions to undergo phase transformation and to generate respective first, second, and third solid-liquid interfaces at a respective first, second, and third positions in the sample rows, detecting the respective first, second, and third solid-liquid
  • a method to perform osmometry comprising: providing a substrate having a first zone, a second zone, and a gradient zone, the first and second zone being adapted to be independently varied in temperature such that a temperature gradient may be established across the gradient zone, the gradient zone having at least three sample rows extending generally from the first zone to the second zone, each of the sample rows being adapted to be filled with a sample, providing in a first sample row a first standard solution having a known first phase transition temperature that is different than that of an unknown sample whose phase transition temperature is to be determined, providing in a second sample row a second standard solution having a known second phase transition temperature that is different than that of the unknown sample and the second sample, providing in a third sample row the unknown sample, creating a temperature gradient across the gradient zone, the temperature gradient extending from a first temperature that is higher than the first phase transition temperature to a second temperature that is lower than the second phase transition temperature, allowing the substrate and the sample rows to come
  • the sample rows comprise capillary tubes.
  • the sample rows are contained within a substrate.
  • the method further comprises the steps of: raising the second temperature so as to cause a portion of the first, second, and third solutions to melt and to generate respective fourth, fifth, and sixth solid-liquid interfaces at a respective fourth, fifth, and sixth positions in the sample rows, detecting the respective fourth, fifth, and sixth solid-liquid interfaces in the sample rows and quantitating the first phase transition temperature from the position of the fourth solid-liquid interface in relation to the positions of the fifth and sixth solid-liquid interfaces.
  • the sample rows comprise capillary tubes.
  • the sample rows are contained within a substrate.
  • a method to screen for molecules having antifreeze (AF) or thermal hysteresis (TH) activity comprising: providing a thermal-gradient osmometer, comprising a substrate separated into a first zone, a second zone, and a gradient zone, the gradient zone generally extending between the first and the second zone, the first and the second zone being adapted to be independently varied in temperature such that a temperature gradient can be established across the gradient zone, wherein the gradient zone includes a plurality of sample rows extending generally from the first zone to the second zone, providing in a first sample row a first standard solution having a known first phase transition temperature that is different than that of an unknown sample which is to be screened for AF or TH activity, providing in a second sample row a second standard solution having a known second phase transition temperature that is different than that of the unknown sample and the second sample, providing in a third sample row the unknown sample, creating a temperature gradient across across the gradient zone, the temperature gradient extending
  • the temperature can be changed through either physically raising and/or lowering the temperature or through physically moving the samples or the substrate relative to the temperature gradient.
  • a molecule possessing antifreeze or thermal hysteresis activity identified according to the screening process.
  • FIGURE 1 is a schematic representation of the temperature gradient osmometer of the present invention showing a transverse view of the device.
  • the figure shows two independently variable temperature zones marked T H and T c separated by a gap with a substrate (i.e. , a microslide) extending therebetween which creates a temperature gradient T H -T C across the substrate.
  • Samples in the sample rows i.e. , capillary tubes
  • the optical image is recorded by a video camera and is displayed to a monitor.
  • FIGURE 2 is a top perspective view of an alternative design of the substrate which, in a single, unitary piece includes a first zone, a second zone, and a gradient zone.
  • FIGURE 3 is a top perspective view of a substrate having in situ sample rows.
  • FIGURE 4 is another top perspective view of a substrate having in situ sample rows.
  • FIGURE 4A is another top perspective view of a substrate having in situ sample rows, wherein the sample rows are discontinuous.
  • FIGURE 5 is a laser printed picture of a captured image of a sample run on the TGO using light polarized microscopy.
  • the freezing interfaces appear as horizontal lines inside the capillary tube, separating the frozen lower region from the unfrozen upper region.
  • FIGURE 6 is a laser printed picture of a captured image of a sample run on the TGO using light polarized microscopy.
  • Five capillary tubes containing (labelled from left to right (a) to (e)) distilled water samples (HPLC quality, 1 ppm residue) (tubes (a) and (e), 1.7 mM Alanine in water (tube (b)), 0.17 mM Alanine in water (tube (c)), and 0.017 mM Alanine in water (tube (d)) are shown with respective solid-liquid interfaces.
  • the freezing interfaces appear as horizontal lines inside the capillary tube, separating the frozen lower region from the unfrozen upper region.
  • FIGURE 7 is a laser printed picture of a captured image of samples run on the TGO using light polarized microscopy. Seven capillary tubes containing
  • FIGURE 8 is a laser printed picture of a captured image of samples run on the TGO using light polarized microscopy. The samples are the same as those depicted in Figure 7, but are shown during melting. To effect melting, the temperature gradient was changed, i.e., the T H and T c were set to different values, which alters the optical resolution through changing the distances separating the solid-liquid interfaces.
  • FIGURE 9 is a laser printed picture of a captured image of samples run on the TGO using light polarized microscopy.
  • the freezing interfaces appear as horizontal lines inside the capillary tube, separating the
  • FIGURE 10 is a laser printed picture of a captured image of samples run on the TGO using light polarized microscopy. The samples shown in Figure 9 are shown during melting as described in Figure 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the osmometer of the invention is referred to herein as a temperature-gradient osmometer (TGO).
  • TGO temperature-gradient osmometer
  • the TGO of the invention can perform with extremely small sample volumes, can measure both freezing and melting temperatures in a single sample, and has a resolution many orders of magnitude greater than that of existing osmometers.
  • anomalies in the depression of the freezing temperature and thermal hysteresis in aqueous solutions of hydrophilic amino acids, poly-amino acids and lectins These anomalies would not have been possible to detect with currently used technology.
  • Hydrophilic amino acids, poly-amino acids and lectins have been reported in the literature as having the ability to bind to cell membranes.
  • Hydrophilic amino acids, poly-amino acids and lectins have been reported in the literature as having the ability to bind to cell membranes.
  • thermal hysteresis and hyper- colligative freezing temperature depression a relationship between a molecule's ability to bind to cell membranes and its anomalous freezing temperature depression is indicated.
  • Threonine and Lysine have thermal hysteresis and hyper-colligative freezing temperature depression, albeit with smaller freezing temperature depressions than those of previously identified THPs.
  • An earlier study showed that Threonine and Lysine can interact with plant cell membranes and contribute to freeze tolerance in plants. Finale et al. in Cryopreservation of Plant Cells and Organs pp. 75-113 (Kartha ed., CRC Press, Florida (1985)).
  • poly -Lysine has thermal hysteresis and specific colligative coefficients comparable to those of fish THPs. Poly-Lysine is also widely used as a strong cell adhesion protein.
  • THPs may have first formed in animals and plants, their ability to induce thermal hysteresis may have found direct functions in such animals as the Antarctic an North Atlantic fish.
  • THPs affect freezing temperatures by binding to ice crystals.
  • Raymond et al. “Adsorption inhibition as a mechanism of freezing resistance in polar fishes.
  • Cell membranes like ice crystals, interact with the surrounding water.
  • An organic molecule that binds with membranes will replace the water adjacent to the membrane. The binding process must be thermodynamically advantageous for these same molecules to bind to ice crystals while replacing the liquid water surrounding the ice.
  • the TGO of the present invention is uniquely adapted to carry out studies to further demonstrate the "antifreeze” activity of additional membrane binding proteins. Accordingly, the TGO of the invention provides a superb assay for the relation between a protein's ability to bind to ice crystals and to adhere to cell membranes leading to a new method to study adhesion protein function and structure and a better understanding of protein structure and the interaction between proteins, cell membranes, and water.
  • Apparatus in accordance with the invention operate by establishing a space-temperature correlation in a domain.
  • the space-temperature correlation can be established by introducing samples with a known phase transition temperature in the domain and recording the location of the phase transition interface of these samples.
  • the phase transition temperature of a sample with an unknown composition can be determined from the known space-temperature correlation by introducing that sample in the domain, observing the spatial location of the phase transition interface and inferring (through interpolation or extrapolation) the temperature from the space-temperature correlation.
  • a “solution” refers to a pure liquid compound, a mixture of pure liquid compounds, a pure liquid compound or mixture of pure liquid compounds having a solute dissolved therein, a mixture of liquids having a solute dissolved therein, and the like.
  • “Liquid,” as used herein, can refer to solids which become liquids at a given temperature, i.e., metals, alloys, and the like. Referring now to Figure 1, there is provided a schematic representation of an apparatus that can be used to implement the above general principles.
  • the TGO of the invention includes an elongated substrate 20 spanning two independently variable temperature zones or bases 21 and 22 (labeled T c and T H respectively) separated by a gap 23.
  • a plurality of sample rows 24 are provided on or in the substrate 20.
  • the sample rows 24 in a simple embodiment are square or rectangular capillary tubes.
  • T C -T H a temperature gradient
  • T C -T H a temperature gradient
  • the temperatures in base 21 and base 22 are selected so that the temperature gradient established crosses the phase transition temperature of samples provided on the substrate 20, a clear demarcation between the solid and liquid phase in the samples is visible.
  • Figure la There, the solid phase is shown in dark and the liquid phase is shown in light in the sample rows 24a-24d. The differences between the phase transition temperatures in sample rows 24a/24d and 24b and 24c are clearly visible.
  • the bases 21 and 22 in a simple embodiment are prepared from copper blocks through which a row is drilled, serving as a coolant passage (not shown).
  • liquid nitrogen can be used as a coolant and passed through the bases 21 and 22.
  • the temperatures of the bases 21 and 22 can be independently controlled through a temperature controller 25 which may be in communication with thermocouples or thermistors 28 and 29 through lines 26 and 27 which can be utilized for relative feedback on the temperatures in bases 21 and 22.
  • the controller 25 is also in communication and control, through lines 30 and 31, of temperature controllers or heaters 21a and 22a for the bases 21 and 22.
  • thermofoil heaters Minco Products, Inc.
  • a metal plate i.e., a 0.16 cm thick copper plates may be placed on the top surface of the bases 21 and 22.
  • the bases 21 and 22 can be maintained at different constant or variable temperatures.
  • the control system in a preferred embodiment works by connecting the two thermocouples 28 and 29 to two controllers (Fuji Electric, PYZ4) which in turn are connected to the thermofoil heaters 21a and 22a. This arrangement allows control of the temperature of both bases 21 and 22 by providing the required heating to compensate for the cooling from the coolant flow.
  • one base 22 is set at a temperature (T H ), which is higher than the phase transition temperature of the given sample, and the other base 21 is set at a temperature T c , which is lower than the phase transition temperature of such sample.
  • the bases 21 and 22 are, in a preferred embodiment, adapted to be placed on a microscope stage for optical examination of the sample rows 24.
  • the substrate 20 may be either optically transparent or not. Where the substrate 20 is transparent, a transmitting illuminator may be used. Where the substrate 20 is not transparent, a reflecting illuminator can be used. In a preferred embodiment, the substrate 20 is transparent and the system is set on a microscope 32 (i.e., Zeiss Universal) with a transmitting illuminator and light polarization.
  • the bases 21 and 22 are mounted on the horizontal stage of the microscope 32 separated by a gap 23.
  • the bases 21 and 22 are preferably arranged in the same plane to within a few ⁇ m.
  • the substrate 20 is transparent and comprises a glass microslide (26 mm x 76 mm x 1 mm) (Objecttranger, Germany) which spans the bases 21 and 22 across the gap 23.
  • Thermal contact between the substrate 20 and the bases 21 and 22 can be enhanced through a variety of known methods, such as clipping the substrate 20 to the bases 21 and 22.
  • clipping the substrate 20 to the bases 21 and 22 For example, brass clips can be used.
  • the temperature of the substrate 20 will vary from T H to T c across the gap 23 between the bases 21 and 22.
  • the apparatus is designed to generate a one dimensional temperature distribution in the substrate 20 across the gap 23.
  • the gap 23 is preset to a predetermined distance (i.e., approximately 2.5 mm).
  • the gap 23 between the bases 21 and 22 is generally centered around the focal line of the microscope 32.
  • Thermocouples 28 and 29 are located in thermodynamic contact with each of the bases 21 and 22 on each side of the gap 23. In a preferred embodiment, 66.72 ⁇ m type T thermocouples (Omega, Stamford, CT) are used.
  • the sample rows 24 (24a-d; Fig. la) in a preferred embodiment include a plurality of rectangular capillary tubes (0.1 mm x 0.1 mm x 40 mm) (Vitro Dynamics, Inc., NJ) which rest on the substrate 20 and are in good thermal contact with the substrate 20.
  • the sample rows 24 may be attached to the substrate with brass clips to enhance thermal contact.
  • the top view of the substrate 20 (microslide) with the sample rows 24a-24d (capillaries) is shown schematically in Figure la.
  • the samples to be tested are introduced into the sample rows 24.
  • the system allows the simultaneous analysis of multiple samples, for example, six or more samples.
  • the sample rows 24 are chosen to be large enough to avoid surface tension effects on the phase transition temperature.
  • the freezing point depression due to surface tension effects is equal to the product of the Gibbs-Thomson coefficient and the inverse of the radius of curvature.
  • the bases 21 and 22, the gap 23, the substrate 20, and the sample rows 24 are preferably confined within a housing (not shown) which has either a quiescent controlled atmosphere or a partial vacuum.
  • the system is designed in such a way that the sample rows 24 easily take the temperature of the substrate 20. Consequently, the temperatures of the substrate 20, the sample rows 24, and the samples within the sample rows 24 vary one-dimensionally in the horizontal direction, across die gap 23 from temperatures of the bases 22 and 21 (T H to T c , respectively).
  • the TGO apparatus provides a unique one-dimensional correlation between position on the substrate 20 and temperature.
  • the 20 carrying the sample rows 24 with the samples can be translated across die gap, horizontally either from the high temperature base 22, T H , to the low temperature base, T c , or in reverse, to freeze or melt the samples in the sample rows 24, respectively.
  • the temperatures of the bases 21 and 22 can be raised or lowered to effect freezing or melting.
  • the TGO apparatus may be equipped with a motorized substrate positioner (not shown) which can be used to move the substrate 20 with a controlled velocity V in either direction relative to the bases 21 and 22.
  • a motorized substrate positioner (not shown) which can be used to move the substrate 20 with a controlled velocity V in either direction relative to the bases 21 and 22.
  • Such movement of me substrate acts to change the temperature gradient and temperature acting on a given point (i.e., in the image area of the gap 23) of the substrate 20 and can be used to either melt or freeze the samples in the sample rows 24 depending on the direction of movement.
  • the substrate 20 in the colder direction i.e., toward base
  • the samples will freeze. Conversely, through moving the substrate 20 in the warmer direction (i.e., toward base 22 (T H )), die samples will thaw.
  • the image in the area of the gap 23, where the analyzed events occur may be recorded through an image capturing device, such as the microscope 32 with a video camera 33 (for example, a CCD/RGB model DXC-151 , Sony) attached to the microscope 32.
  • the video image from the camera 33 may be recorded on video cassette with a video cassette recorder 34 and/or displayed through a monitor 35.
  • video images may be captured witii a computer (not shown).
  • a PC based video capture board (Aitech Int. Corp. Vimager Pro
  • Image processing software (such as NIH-Image v. 1.54) can be used for image processing analysis.
  • the NIH Image v.1.54 software is a Macintosh based software package. However, image files, captured on PC based systems, may be transferred from such systems to Macintosh systems through networks, or the like. Graphical analysis can then be performed on the Macintosh utilizing the image processing software. Micrographs may be printed using a laser printer. The method of d e invention may be understood more particularly from Figure la. There, four sample rows 24a-24d are loaded with various samples.
  • sample rows 24a and 24d are loaded wim a sample that is a standard which has a higher melting point than an unknown sample contained in sample row 24c.
  • sample row 24b a second standard sample which has a lower melting point than the unknown sample contained in sample row 24c.
  • the samples contained in sample rows 24a and 24d can be used to ensure that the gradient is one dimensional across the substrate 20, varying only linearly across the gap 23, and not laterally across d e substrate 20. Thus, a baseline isotherm in a direction normal to that of the one-dimensional temperature variation can be established.
  • These two exterior sample rows (24a and 24d) together with the interior sample row 24b with the other sample with a known composition are used to establish the space-temperature correlation.
  • the solid-liquid interface in the unknown sample contained in sample row 24c can be compared in distance to die solid-liquid interfaces of the known samples contained in sample rows 24d and 24b to determine me melting point of the unknown sample in sample row 24c with a high degree of accuracy.
  • the axial temperature distribution in the space bounded by the phase transition interfaces in the standards contained in sample rows 24 is determined from knowledge of each control solution's composition, and by assuming a one-dimensional, linear temperature distribution between the phase transition interfaces.
  • the phase transition temperature of the sample with the unknown composition is determined by linear interpolation from measurements of me axial distance between the phase transition interface locations of the tested sample and me controls.
  • the distances in die axial direction between the phase transition interfaces in the various sample rows 24 can be measured wim light microscopy resolution.
  • the temperature measurement resolution can be continuously increased by replacing the control solutions with compositions more closely matching the phase transition temperature of the tested sample and by generating shallower temperature gradient across die gap 23.
  • me substrate can be movable (i.e. , with a slide mover) so mat the sample rows can be moved across the temperature gradient between the bases 21 and 22.
  • d e substrate 20 having the sample rows 24 are loaded on die high temperature side of the gradient (i.e. , on base 22).
  • die high temperature side of the gradient i.e. , on base 22.
  • T H to T c die base 21
  • phase transition temperature during melting is determined similarly by translating or moving the sample rows 24 on the substrate 20 in the reverse direction (i.e. , T c to T H ).
  • the temperature of the bases 21 and 22 can be initially established to be same (i.e. , bom at a temperature above the phase transition temperature of the samples in the sample rows 24). Over time, the temperature of a base (i.e. , base 21) can be lowered slowly to effect freezing of the samples in the sample rows 24 as observed in the image area of the gap 23. Phase transition temperatures during melting can be measured through simply raising the temperamre of the base (i.e., base 21) until melting is observed in the image area of the gap 23.
  • TGO system of the present invention can be manufactured in a variety of different manners. A variety of these additional embodiments are described below:
  • the substrate of the TGO of the present invention can be designed as a single piece including me temperature control features and die like.
  • An exemplary embodiment of a single piece substrate is shown in Figure 2 which is a top perspective view of an alternative design of the substrate.
  • apparatus in accordance widi the present invention includes a substrate 40 generally separated into three zones: a first zone 41, a second zone 42, and a gradient zone 43.
  • the gradient zone 43 generally extends between the first and second zone 41 and 42.
  • the first and second zone 41 and 42 are adapted to be independently varied in temperamre such diat a temperature gradient can be established across die gradient zone 43.
  • the gradient zone includes a plurality of sample rows 44 extending generally from the first zone 41 to the second zone 42.
  • the first zone 41 is typically separated from the second zone 42 by a recess 45 under me gradient zone 43 which assists in generation of the temperamre gradient across die gradient zone 43.
  • the substrate 40 can be formed of a single piece of material (as shown) or can be manufactured from a variety of materials.
  • me substrate is manufactured from several materials and components.
  • the bases 21 and 22 wim me substrate 20 (in Figure 1) operate equivalentiy to die first zone 41, the second zone 42, and me gradient zone 43 (in Figure 2).
  • the first zone 41 and die second zone 42 are typically connected to a heating/cooling system and/or controller as is discussed in connection with Figure 1.
  • d e sample rows utilized in the present invention can be as simple as capillary mbes (Figs. 1 and la) or can be etched or imbedded into die substrate. All that is generally required for a sample row to be effective in accordance with the invention is that the sample row extend generally longitudinally along the substrate in good thermal contact with the temperamre gradient. The sample rows, therefore, are generally elongate but their particular geometry is not exceedingly important. One requirement of me sample rows is that a sample is exposed to a range of temperatures across the temperamre gradient, whatever geometry of the sample row that is chosen.
  • an etched or imbedded sample row design is provided in Figure 3 which is a top perspective view of a substrate having in situ sample rows.
  • the sample rows 54 comprise rows within the substrate 50 extending generally from the first zone 51 to the second zone 52 across the gradient zone 53.
  • sample rows 54 preferably include sample wells 56 which allow for easy loading of me sample rows 54 with sample. Samples may be pipetted into the sample wells 56 and will fill me sample rows 54 through capillary action.
  • the sample rows 54 are preferably covered from the sample wells 56 over a portion of their length (i.e., extending over the gradient zone 53 of me substrate 50). This assists in limiting any air effects as well as creates very fine resolution of me solid-liquid interface upon freezing across a temperamre gradient.
  • the sample rows 54 may include at the opposite end from the sample wells 56, vent holes 57.
  • the substrate as described above, can be manufactured from one or more components. For example, where temperamre control of the gradient is accomplished utilizing bases (Fig. 1; 21 and 22), d e substrate may be prepared as shown in Figure 4. Or, alternatively, the substrate can be manufactured as a single piece as shown in Figure 3. An advantageous construction is to manufacture the substrate as a single piece of glass including me temperature control apparams.
  • the sample rows 54 in such embodiment can be etched or embedded using common techniques known in die art.
  • sample rows are made (i.e., etched or embedded) directly in the substrate diere is a greater ability to line up the samples to ensure consistency and uniformity. Moreover, greater heat transfer control is facilitated.
  • die solid-liquid interface is detected optically.
  • alternative methods to detect the solid-liquid interface are contemplated in accordance widi the invention. Essentially, all that is necessary in accordance widi the invention is to be able to detect die relative positions of the solid-liquid interfaces of the samples.
  • any detection system which enables such detection will be useful in the present invention.
  • ultrasonic imaging techniques are highly effective at detecting interfaces between materials.
  • liquid crystal technology With advances in solid state physics and chemistry, there are practically daily developments in liquid crystal technology. Certain liquid crystals can be designed to change color in response to changes in temperature. Accordingly, we expect that liquid crystal type designs might be incorporated into the TGO of the present invention for aiding in edge detection or as replacements for sample rows containing standard solutions.
  • Figures 5 is a laser printed picture of a captured image of a sample run on die TGO.
  • five capillary tubes containing (labelled from left to right (a) to (e)) a distilled water sample (HPLC quality, 1 ppm residue) (mbe (a)), followed by a 4.25 mM aqueous NaCl solution (mbe (b)), a 8.5 mM aqueous NaCl solution (mbe (c)), a 12.75 mM aqueous NaCl solution (mbe (d)), and a 17 mM aqueous NaCl solution (mbe (e)).
  • the axes of the capillary tubes are in the Figure's vertical direction.
  • Example 2 we measured me freezing point of water in comparison to several aqueous solutions of me amino acid Alanine (Spectrum, Gardena, CA) to view the freezing point depressions in me solutions and to gadier insight into the resolution of the TGO of me present invention.
  • An embodiment similar to that described in conjunction wim Figure 1 was used.
  • the results are shown in Figure 6 which is a laser printed picture of a captured image of a sample run on die TGO.
  • d e mbes in the precise direction of the temperamre gradients.
  • d at the line which was computer generated by connecting the midpoints of me exterior freezing interfaces, passes through, and closely coincides with, bodi of d e flat freezing interfaces in the exterior capillaries.
  • the computer generated line defined by die freezing point interfaces in mbe (a) to mbe (e), defines die 0°C isotherm baseline.
  • the distance of die phase transition interfaces in the interior capillaries (mbes (b) through (d)) can be measured from the 0°C baseline in a normal direction as described in Example 1.
  • Figure 6 also illustrates the resolution of the TGO since the Alanine solution freezing temperamre depression measured here cannot be measured wim conventional osmometers.
  • the freezing point interface observed in the capillaries can be either straight as in Figure 5 or concave as in Figure 6.
  • the shape of the line changes in the same sample with (i) the rate of freezing and melting that is used to achieve the equilibrium conditions and (ii) die temperamre gradient.
  • slower freezing and melting rates and shallower temperamre gradients produce a straighter freezing interface line.
  • the interface curvature may be caused by die difference in thermal conductivity between glass and ice. Therefore, regardless of the reason for the curvature, for standardization purposes, whenever die experiment produces a curved surface we use the center of die ice column for measuring distances.
  • Example 4 me TGO is particularly useful in the study of thermal hysteresis in thermal hysteresis protein (THP) solutions and odier solutions.
  • THP thermal hysteresis protein
  • Bom of Figures 7 and 8 show me typical spicular ice crystal structure that forms in the presence of THPs.
  • Figure 8 shows die phase transition interface after melting and demonstrates that, as anticipated, diere is thermal hysteresis in THP solutions. That is, the phase transition melting temperature is much higher than the phase transition freezing temperature.
  • a different temperamre gradient was used in the freezing experiment shown in Figure 7 and the melting experiment shown in Figure 8. The different gradient occurred because, rather than move the substrate to change die sample location in the gradient, die temperature across me gradient was changed on only one side. This was accomplished dirough raising the temperamre on me low temperamre side of die gradient (i.e., in Figure 1 the temperamre of base 21 was raised while the temperamre of base 22 was kept constant). Therefore, the distance between the phase transition interface in the NaCl capillary (mbe (b)) and in die pure water capillary (mbes (a) and (g)) changed.
  • Example 5 In an effort to establish negative controls for use in studying THPs, we measured die freezing temperamre depression in several amino acids and poly- amino acids. The results were surprising.
  • v is a solvent-dependent, experimentally determined coefficient which is a function of concentration and normally has values of less than one. Values of v and n for NaCl solutions are well established. Partanen et al. "Freezing point depression of dilute aqueous sodium chloride solutions.
  • v 0 is defined as die ratio between the osmotic coefficient of a substance v and die osmotic coefficient of a NaCl solution widi die same product (n m) and pressure. Widi limited exceptions a substance that produces hyper-colligative freezing temperamre depression will have a specific osmotic coefficient greater than one.
  • Phe Phenylalanine
  • Example 6 In addition to amino acids, we investigated die freezing temperature depressions of aqueous solutions of two commercially available poly-amino acids, poly-Proline (Pro (Sigma, St. Louis, MO)) and poly-Lysine (Lys (Sigma, St. Louis, MO)), each of which have good solubility in water. We found no hyper- colligative freezing temperamre depression or thermal hysteresis in poly-Pro. However, the poly-Lys behaved very differendy.
  • FIGS 9 and 10 are laser printed picmres of capmred images of samples run on the TGO.
  • the capillary mbes in Figures 9 and 10 contain, as labelled from left to right mbes (a) through (f), water (HPLC quality, 1 ppm residue) (mbe (a)), 0.625 mM aqueous poly-D-Lys (6.7 kDa) (mbe (b)), 1.25 mM aqueous poly-D-Lys (6.7 kDa) (mbe (c)), 2.5 mM aqueous poly-D-Lys (6.7 kDa) (mbe (d)), 5 mM aqueous poly-D-Lys (6.7 kDa) (mbe (e)), and 1 mM aqueous AFGP 1-8 (DeVries, University of Illinois at Urbana-Champaign) (mbe (f)).
  • Example 8 we describe a method for screening for molecules having antifreeze or thermal hysteresis activity. Basically, there are two approaches with reference to Figure 1. In each, at least one sample and two "controls" are used. In die first screenining process phase transformation is accomplished through changing the temperamre across me gradient. 1. raise temperamre of zone 3 or lower temperamre of zone 1 to cause freezing across the gradient.
  • tiiis screening process allows one to quickly screen for thermal hysteresis and, consequently, likely antifreeze activity. Compounds can be quickly screened and identified through this process.

Abstract

Un osmomètre à gradient thermique comprend d'une part un substrat (20) couvrant deux zones (21, 22), dont la température varie de façon indépendante, séparées par un espace (23), et d'autre part une pluralité de rangées (24a-24d) situées sur ou dans le substrat (20). Les zones (21, 22) établissent un gradient thermique sur l'ensemble du substrat (20). Lors de l'utilisation, une solution inconnue, dont on souhaite déterminer la température de transition de phase, est placée dans une première rangée d'échantillons. Ensuite, une première et une deuxième solution, présentant des températures de transition de phase respectivement supérieure et inférieure à celle de la solution inconnue, sont placées respectivement dans une deuxième et une troisième rangées d'échantillons. On expose ensuite les trois rangées d'échantillons au gradient thermique, de façon à provoquer une transformation de phase dans une partie des trois solutions, tout en créant respectivement une première, deuxième et troisième interfaces solide-liquide dans leurs rangées d'échantillons respectives. La température de transition de phase de la solution inconnue est déterminée d'après la position de la troisième interface par rapport à la première et à la deuxième interfaces.
PCT/US1995/014352 1994-11-08 1995-10-25 Osmometrie a gradient thermique WO1996014571A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU41463/96A AU4146396A (en) 1994-11-08 1995-10-25 Temperature-gradient osmometry

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US33591694A 1994-11-08 1994-11-08
US08/335,916 1994-11-08

Publications (1)

Publication Number Publication Date
WO1996014571A1 true WO1996014571A1 (fr) 1996-05-17

Family

ID=23313768

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/014352 WO1996014571A1 (fr) 1994-11-08 1995-10-25 Osmometrie a gradient thermique

Country Status (3)

Country Link
AU (1) AU4146396A (fr)
IL (1) IL115900A0 (fr)
WO (1) WO1996014571A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004017050A1 (fr) * 2002-08-06 2004-02-26 The Regents Of The University Of California Osmometrie de film lacrymal
US7905134B2 (en) 2002-08-06 2011-03-15 The Regents Of The University Of California Biomarker normalization
DE102010011613A1 (de) * 2010-03-16 2011-09-22 Herbert Knauer Gefrierpunkts-Osmometer
US9335243B2 (en) 2006-12-11 2016-05-10 Tearlab Research, Inc. Systems and methods for collecting tear film and measuring tear film osmolarity
US11536707B2 (en) 2014-09-23 2022-12-27 Tearlab Research, Inc. Systems and methods for integration of microfluidic tear collection and lateral flow analysis of analytes of interest
WO2024086215A1 (fr) * 2022-10-18 2024-04-25 Massachusetts Institute Of Technology Estimation d'état basée sur une imagerie thermique d'un problème de stefan avec une application à la décongélation de cellules

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2691885A (en) * 1952-04-07 1954-10-19 Standard Oil Dev Co Apparatus for detecting thermoplastic properties
US2730892A (en) * 1952-05-22 1956-01-17 American Home Prod Melting point bar
JPS5223380A (en) * 1975-08-18 1977-02-22 Terumo Corp Temperature indicator
US4138889A (en) * 1977-04-26 1979-02-13 Laboratorio Farmacologico Milanese S.N.C. Ready-reading, liquid-crystal-display thermometer
US4383770A (en) * 1981-07-09 1983-05-17 Boschung Mecatronic Ag Apparatus for determining the freezing point of a liquid on or from a road surface
US4400096A (en) * 1980-07-16 1983-08-23 Molloy Robert E Osmometers
US5278284A (en) * 1992-05-14 1994-01-11 Miller Brewing Company Protein purification method

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2691885A (en) * 1952-04-07 1954-10-19 Standard Oil Dev Co Apparatus for detecting thermoplastic properties
US2730892A (en) * 1952-05-22 1956-01-17 American Home Prod Melting point bar
JPS5223380A (en) * 1975-08-18 1977-02-22 Terumo Corp Temperature indicator
US4138889A (en) * 1977-04-26 1979-02-13 Laboratorio Farmacologico Milanese S.N.C. Ready-reading, liquid-crystal-display thermometer
US4400096A (en) * 1980-07-16 1983-08-23 Molloy Robert E Osmometers
US4383770A (en) * 1981-07-09 1983-05-17 Boschung Mecatronic Ag Apparatus for determining the freezing point of a liquid on or from a road surface
US5278284A (en) * 1992-05-14 1994-01-11 Miller Brewing Company Protein purification method

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9217702B2 (en) 2002-08-06 2015-12-22 The Regents Of The University Of California Biomarker normalization
US7017394B2 (en) 2002-08-06 2006-03-28 The Regents Of The University Of California Tear film osmometry
AU2003233449B2 (en) * 2002-08-06 2009-07-02 The Regents Of The University Of California Tear film osmometry
US7574902B2 (en) 2002-08-06 2009-08-18 The Regents Of The University Of California Tear film osmometry
US7905134B2 (en) 2002-08-06 2011-03-15 The Regents Of The University Of California Biomarker normalization
US7987702B2 (en) 2002-08-06 2011-08-02 The Regents Of The University Of California Tear film osmometry
EP2299255A3 (fr) * 2002-08-06 2014-05-14 The Regents of the University of California Osmométrie de film lacrymal
US9217701B2 (en) 2002-08-06 2015-12-22 The Regents Of The University Of California Biomarker normalization
WO2004017050A1 (fr) * 2002-08-06 2004-02-26 The Regents Of The University Of California Osmometrie de film lacrymal
US9335243B2 (en) 2006-12-11 2016-05-10 Tearlab Research, Inc. Systems and methods for collecting tear film and measuring tear film osmolarity
DE102010011613A1 (de) * 2010-03-16 2011-09-22 Herbert Knauer Gefrierpunkts-Osmometer
US11536707B2 (en) 2014-09-23 2022-12-27 Tearlab Research, Inc. Systems and methods for integration of microfluidic tear collection and lateral flow analysis of analytes of interest
WO2024086215A1 (fr) * 2022-10-18 2024-04-25 Massachusetts Institute Of Technology Estimation d'état basée sur une imagerie thermique d'un problème de stefan avec une application à la décongélation de cellules

Also Published As

Publication number Publication date
IL115900A0 (en) 1996-01-31
AU4146396A (en) 1996-05-31

Similar Documents

Publication Publication Date Title
Ramsay A new method of freezing-point determination for small quantities
Rubinsky et al. A cryomicroscope using directional solidification for the controlled freezing of biological material
Diller et al. A cryomicroscope for the study of freezing and thawing processes in biological cells
Gulik‐Krzywicki et al. The use of low temperature X‐ray diffraction to evaluate freezing methods used in freeze‐fracture electron microscopy
US4531373A (en) Directional solidification for the controlled freezing of biomaterials
Fahey et al. Lateral diffusion in phospholipid bilayer membranes and multilamellar liquid crystals
Briegel et al. Correlated light and electron cryo-microscopy
Brown et al. Using monomolecular films to characterize lipid lateral interactions
Elder et al. Optimum conditions for cryoquenching of small tissue blocks in liquid coolants
Mader The thermal behaviour of the water-vein system in polycrystalline ice
Toner et al. Transport phenomena during freezing of isolated hepatocytes
JP2006507540A (ja) 凍結乾燥顕微鏡ステージ装置及びそれを用いた方法
Grandum et al. Characteristics of ice slurry containing antifreeze protein for ice storage applications
Nail et al. An improved microscope stage for direct observation of freezing and freeze drying
WO1996014571A1 (fr) Osmometrie a gradient thermique
JP2004113092A (ja) 細胞培養チップ
Beckmann et al. Redefining cooling rate in terms of ice front velocity and thermal gradient: first evidence of relevance to freezing injury of lymphocytes
Lorber et al. A versatile reactor for temperature controlled crystallization of biological macromolecules
EP2623892A2 (fr) Congélation à vitesse régulée
Bayer-Giraldi et al. Characterization of ice binding proteins from sea ice algae
Bearce et al. Measuring osmotic pressure of sap within live cells by means of a visual melting point apparatus
Gulik-Krzywicki et al. The use of freeze-fracture and freeze-etching electron microscopy for phase analysis and structure determination of lipid systems
WO2002043856A2 (fr) Structures ordonnees bi- ou tridimensionnelles de molecules amphyphiles
Tanaka et al. Algae Permeability to Me2SO from− 3 to 23 C
Ishiguro et al. Influence of fish antifreeze proteins on the freezing of cell suspensions with cryoprotectant penetrating cells

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA UG UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase