Thermal measuring and testing – Thermal testing of a nonthermal quantity
Reexamination Certificate
1999-06-17
2001-02-27
Bennett, G. Bradley (Department: 2859)
Thermal measuring and testing
Thermal testing of a nonthermal quantity
C374S043000, C374S045000, C436S147000, C436S157000
Reexamination Certificate
active
06193413
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to a system and method for an improved calorimeter, and more specifically, to a system and method for an improved calorimeter for determining thermodynamic properties of chemical and biological reactions.
2. Related Art
Heat absorption and/or heat release is ubiquitous to chemical and biological reactions. Thermodynamic information that characterizes these reactions is directly measurable through calorimetry. The thermodynamic information provides insight into the molecular forces that are driving the reactions.
Conventional calorimeters used to measure chemical and biological reactions generally comprise a fluid cell and an injection syringe. The fluid cell is loaded with a liquid receptor sample. One example of a receptor sample is a protein solution. The fluid cell is then placed within a cylindrical liquid filled chamber, where temperature measurements are subsequently made.
The injection syringe is loaded with a ligand, such as a drug that binds to the receptor sample when injected into the fluid cell. A known volume of the ligand solution is then injected into the fluid cell containing the receptor sample solution. When this occurs, the ligand and receptor sample solutions bind, which causes heat to be liberated.
Thermometers, within the cylindrical liquid filled chamber, precisely measure the amount of heat released during this process. This information is recorded, and the injection and measuring steps are repeated. This process continues until heat is no longer released. This indicates that all binding sites have been filled. Once the entire process is complete, scientists can determine thermodynamic properties associated with the two interacting molecules.
That is, because the exact volumes of the samples are known, as well as the precise amount of heat liberated, scientists can determine properties such as the equilibrium binding constant, the ratio of the participating molecules in the reaction (stoichiometry), and the heat of binding. Typically, these properties are determined by constructing a binding curve comprising multiple data points that are derived from each of the reactions as described above.
The problem with conventional chemical/biological calorimeters is that the above process is very meticulous and extremely time-consuming. In addition, the sensitivity of current systems is quite limited. For example, typically current systems cannot measure dissociation binding constants below 10
−8
(or affinity binding constants above 10
8
). It is noted that the term “binding constant” is hereinafter defined as the dissociation constant. It would be desirable to increase the sensitivity of chemical/biological calorimeters so that lower binding constants can be detected and measured.
Further, current state of the art calorimeters require relatively large sample volumes on the order of one milliliter. Using these large sample volumes can be very expensive, especially for large-scale operations, such as high-throughput pharmaceutical drug screening and the like. Still further, current systems require that solutions are more dilute as the binding constants of the systems increase.
In addition, the large sample volumes required by current calorimeters preclude the study of certain phenomena. For example, many proteins, such as transcription factors, exist in relatively small amounts in the cell. Further, amplification is not possible until a gene is cloned and an expression system is developed. Consequently, scientists are precluded from studying the thermodynamic properties of such proteins using current systems.
Accordingly, what is needed is a system and method for determining thermodynamic properties of biological and chemical reactions that can be performed using lower volumes, can detect lower binding constants, and can be performed more efficiently and economically than conventional chemical/biological calorimeter systems.
SUMMARY OF THE INVENTION
Accordingly, the present invention is directed toward a system and method for determining thermodynamic properties of biological and chemical reactions that can be performed using much lower volumes and can detect lower binding constants than current systems. Further, the system and method of the present invention can be performed more efficiently and economically than conventional chemical/biological calorimeter systems. For example, to determine the equilibrium binding constant using conventional titration techniques involving multiple injections can take on the order of one hour to complete. Using the thermodynamic profiling technique as described below, equilibrium binding constants are calculated in seconds or minutes.
In operation, the present invention dramatically increases the sensitivity of calorimeters used to study chemical and biological systems. This includes thermodynamic study of protein-protein and protein-DNA interactions, the thermodynamics and kinetics of drug binding for both proteins and DNA, drug occupancy studies and studies of protein folding.
An advantage of the present invention is that it can measure samples that are at least 1000 times smaller than conventional systems. Further, an advantage of the present invention is that it can determine thermodynamic properties of samples with very high relative and absolute accuracy and from time resolved measurements.
Another feature is that many calorimeters can be made inexpensively due to the intrinsically parallel nature of semiconductor manufacturing techniques. Many novel applications lie in the ability to make large numbers of matched devices on a single wafer with interconnections built in. For example, in one embodiment, 24 devices fit on a four-inch wafer. In another embodiment, the devices are scaled down ten-fold, so that 2400 individual devices fit on a single wafer. Miniaturization of the devices to even smaller sizes is straightforward and depends solely on the fabrication techniques used to implement the present invention.
An advantage of the present invention is that it can measure both the heat capacity and the enthalpy of chemical and biological systems in a single experiment using a single device. In this fashion, binding constants can be derived much faster than conventional systems that require multiple experiments, such as conventional titration techniques using binding curve analysis.
As stated, the present invention requires much lower sample volumes for determining thermodynamic properties of chemical and biological systems. Specifically, sample volumes are in the nanoliter and smaller rather than the milliliter range as found in conventional systems. Thus, an advantage of the present invention is that it can accept sample volumes three orders of magnitude lower than is currently possible using conventional systems.
The smaller sample volumes and consequently faster thermal equilibration allow probing in shorter time domains than is presently possible. Unlike current chemical/biological calorimeters, one advantage of using the present invention is that it provides the ability to probe the time domain of heat release. This advantage allows for direct time-resolved thermodynamic characterizations. Thus, for example, the present invention can be used to determine the thermodynamic properties associated with protein folding, heat release of a single cell, the thermodynamic properties of molecular motors, molecular polymer assembly dynamics and enzymatic substrate turnover.
The present invention improves upon traditional applications such as differential scanning calorimetry and stopped-flow calorimetry. In addition, the present invention can be implemented using calorimeter arrays to facilitate and make possible large-scale operations, such as high-throughput pharmaceutical drug screening and the like.
The microcalorimeter of the present invention builds on existing microcalorimeters used in the electronics industry for measuring thin film samples of magnetic and superconducting materials. The present invention provides major imp
Bennett G. Bradley
Lyon & Lyon LLP
Verbitsky Gail
LandOfFree
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