Patent Application
20080072665
In the following description, preferred embodiment examples are used as explanatory models for the attached drawings. There is shown in:
As is shown in
The measuring probe 4 is designed as a cantilever extension, which cantilever serves to allow resilient properties to the said probe 4. At the free end thereof, in particular, at the tip of the cantilever a probe (not shown) can be integrated as a unified component. The presentation in
In particular, the measurement apparatus 2 can possess two, four, six or eight probes. The number of probes 4 can extend itself even to 100 or more. To incorporate this extended number of probes 4 to be used within the measurement apparatus recourse may be made to the use of the so-called “cantilever chips” (see
Giving consideration to the sample to be analyzed and relative to the current analysis being carried out, it can be necessary to set a probe into vibration at a predetermined amplitude. For this reason, the measurement apparatus includes a force producing unit which coacts with the measuring probe 4 and with the resilient unit. This force producing unit is not illustrated. The force producing unit, however enables the measuring probe to move in such a manner, that a desired quality-factor (hereinafter “Q-factor”) appropriate to the analysis can be achieved.
Additionally, with measurement apparatus possesses the sample positioning device 18 (not shown in detail), which serves the purpose of positioning a probe 4 in relation to the sample 8. Relative to the characteristics of the sample 8 to be analyzed, as well as relative to the analysis itself, it is possible that by means of the sample positioning device 18, a probe 4 can assume a correspondingly appropriate position relative to the sample 8 and likewise follow a desired path thereon.
By means of a first detector unit 12, which is included in the assembly of the measurement apparatus 2 or, otherwise, is assigned thereto, it is possible that the positions and travel paths of the sample 8 can be determined. The first detector unit 12 is made as an optically based detector and encompasses a source of radiation 14, which can be a laser beam emitter, and includes further a light first receiver 16 which collects the light from the said radiation source 14 after its interaction action with a probe 4. The said radiation source 14 targets a probe 4, particularly in the area inclusive of the free ends thereof, while interactive actions, specifically here reflections of the light from the light source 14, are collected by a second receiver 16. These collected reflected light rays are then conducted to the evaluation unit 42 for the determination of positioning and movements of the measuring probe 4.
The measuring apparatus 2 possesses further the sample-carrier positioning element 10, which allows the said sample-carrier positioning element to be integrated in, and be come available to, the said measuring apparatus 2, thus being a component thereof The sample-carrier 6 and the sample-carrier positioning unit 18 can also be designed a as separate and discrete components.
The sample-carrier positioning unit 18 here provided is actuated by a piezo-electrical electrical element, which energizes the movements and positioning of the sample-carrier 6 and therewith the thereon superimposed measuring probe 8 in its relative space.
The sample chamber 20 encapsulates the measuring probe 4 and the sample 8 or encloses at least that area of the sample 8 which is to be analyzed, for example, this said area can be the upper surface thereof. The sample chamber 20 is closed in a fluid tight manner, absent the in and out lines, which lines are described in the following, and on this account presents a closed-off space in relation to its ambient surroundings. The sample chamber 20 can be totally dedicated to the acceptance of incoming fluid or as is shown in
By means of a second detector unit 24, the level of fluid in the sample chamber 20 can be determined. The second detector unit 24 is designed as an optical measuring instrument, which includes in its assembly a light source 26, for instance, this being a laser beam emitter and a receiver 28. The light from the said laser source 26, after its interaction with the fluid in the sample chamber 20 is collected by the receiver 28. Readings in regard to the level of the fluid in the sample chamber 20, which readings are activated by the received laser light are possible to obtain.
The sample chamber 20 is connected with a feed line 30 by means of which, fluid can be introduced into the sample chamber 20. By means of a discharge line 32, fluid from the sample chamber 20 finds an outlet from the said sample chamber 20.
The input side of feed line 30 is connected with a feed unit 34, by means of which the individual, combined, mixed and blended fluids of the input line 30 can be introduced into the sample chamber 20. The feed unit 34, for this purpose, includes one or more pumps or multichamber pumps and apparatuses (not shown) for the mixing and blending of the said fluid mixtures. By means of the fluid supply lines 361 . . . 36n the feed unit 34 obtains various fluids from their appropriate reservoirs (not shown). In the case of embodiment 1, which is the total diagram of
The measurement apparatus 2 and the additional, above described components of the embodiment—absent the fluid sources connected with the supply lines 361 . . . 36n and parts of the supply lines 361 . . . 36n—are housed in a temperature controlling enclosure 38. This temperature enclosure 38 possesses, at least where temperature is concerned, a temperature directed closure against the ambient environmental conditions.
The embodiment form 40 serves for the control of the components which find themselves housed within the temperature enclosure as well as the conditions of the temperature enclosure itself. In
The evaluation unit 42 contains principally all components which are confined within the temperature chamber 38, insofar as such components are designed to be so enclosed. Further, the said evaluation unit also obtains from the temperature enclosure 38, data, measurement signals, and the like, in order to transmit and evaluate the actual operational conditions, i.e., the actual results, obtained from the said measurements. Representing the connections which permit the above operation,
Further, the embodiment designated as 1 encompasses a data storage memory 54, which is connected to the control device 40 and the evaluation unit 42. The data storage memory 54 serves for the storage of data, measurement signals, and the like, which have been obtained from the evaluation unit. These data are available from the evaluation unit 42 along with parameters, which can be used by the control device 40 for the regulation of the said embodiment 1. These and other data are so stored, in accord with the following described information:
In particular, the data storage apparatus 54 is so designed, that it may also be employed as a data bank, in which storage is provided for already evaluated data and for outside data which has a relation to the embodiment 1.
In the case of the execution of a raster probe microscopic measurement, the control unit regulates, besides the true force-spectroscopic experiment, also all experimental conditions, such as temperature, the active pH value of the sample 8, the coacting electrolyte(s) of the sample 8 as well as the supply of pharmaceutical, biochemical and chemical additives. Further the control device monitors the control device 40 for predetermined operational points of time, also checks the again predetermined duration periods or active parameters for the on-going measurement and also supervises the side conditions and finally controls the measurement in such a manner, that specifications intended for that measurement are held.
The evaluation unit 42 analyzes the force-spectrum acquired by a probe 4 during the investigation in regard to sample 8 and is able to interpret this force-spectrum. Relative to this operation, the possibility also exists, that for example, upon the attainment of a preset value, the measurement then in course can be brought to a termination and a new measurement initiated with new specifications (for instance, ambient condition changes). The control device 40 and the available data from the evaluation unit 42 also enable iterative measurement cycles being carried out, in order that concurrent reactive effects are determined, which could enhance specified interactive operations.
During the measurement, a probe 4 is in a crisscrossing relationship with the sample 8, whereby, in the presence of interactions between a probe 4 and the sample 8, (biological) molecules situated on the surface of the sample 8 can be detected. Relative to the formulation of the measuring probe 4 and the type of sample 8, defined contact duration times and/or frequencies of contacts between a probe 4 and the sample are required. These parameters are applied and adjusted by the control device 40, which also supervises and if necessary corrects the ongoing procedure. In this matter, it can be advantageous, especially in the execution of a completely automatic measurement, to prepare the sample 8 to an optimum condition. More detailed methods for this preparation are to be found in the following description.
During the measurement procedure, the control device 40 regulates a plurality of relevant experimental conditions, such as, for example, maximum/minimum tension and compressive forces between a probe 4 and the sample 8, also the speeds with which the relative movements between a probe 4 and the sample 8 are carried out, likewise, the number of measurement points (resolution) and the maximum/minimum separating distances between a probe 4 and the sample 8.
As this is done, it is possible to proceed with the measurements, by means of which individual, several or all experimental conditions are held constant and/or may be systematically and/or chaotically changed. Thus it is, for example, possible to execute a measurement in which, except for the travel speed of a probe 4, all experimental conditions are held constant.
As soon as a predetermined number (for example, a thousand) of measurements have been undergone by a single sample, it is possible to send the acquired data to a data set. Additional data sets can be made by means of changed experimental conditions, and then comparisons can be carried out. This makes it possible to analyze different biological and/or medicinal relevant experimental conditions in regard to their influence on molecular interactivity.
A given value, which can influence the analyses of biological samples, would be thermal alterations.
The said temperature enclosure 38 is provided in order that thermal drift during experimental conditions can be minimized. This might be, for example, changes arising from buffers coacting with sample 8. Further it is possible that a heating or a cooling element (for instance, a Peltier-element) can be employed, in order that the temperature of the sample 8 itself can be controlled. Such a heating/cooling element can, for example, be placed under the sample-carrier 6.
Bimolecular interactivities are, as a rule, very dependent upon the governing, physiological ambient conditions of the measurement. These, on this account, should be monitored during a measurement procedure and accordingly provided with control. In this way, during the measurement, the desired surrounding conditions are maintained, which, generally, stimulates the inherent properties of the sample 8. Thus, for example, provision is made, that during a measurement procedure, the liquid level of a buffer solution in the sample chamber 20 is monitored and controlled by means of the second detection unit 24 at predetermined intervals or is supervised continually and, if necessary, the supply equipment 34 is so operated, that a desired liquid level is correctly held or can be properly reached. In this way, it is possible, that during a measurement procedure a measurement of a lessening of the buffer level due to evaporation can be compensated for. In this way also, pH-variations as well as changes regarding the employed electrolyte or other materials which can interact with the sample 8 can be placed under monitoring supervision and, if required, also controlled. By means of an intended control of buffer solutions in the sample chamber 20, evaporation losses and salting-out occurrences can be avoided.
In the control of buffer solutions which are in the sample chamber 20, the possibility exists that fluid movements, that is, for example, a turbulence or swirling may occur, which can influence the accuracy of the measurement. For instance, fluid movements in the sample chamber 20 can activate vibratory resonances in the measuring probe 4. In order to prevent this, provision is made, to the effect that the control device 40 interrupts a procedure which is already in operation, if such disturbances are detected and/or predicted.
The speed, with which a measurement can be carried out with, plays an essential role. In any case, quickly executed measurement procedures are subject to question. In order to achieve a high degree of quality and at the same time perform a measurement in a short time, the invention allows, that the measuring probe 4 can be moved with a greater than normal speed and likewise quickly assume a desired position. When this occurs, then provision is made, that during a measurement procedure, as measurements are taken at different and/or the same points, different speeds for the movement and/or positioning of a probe 4 may be employed. This control of varying speeds and/or positioning enables, in the case of force spectra (i.e. curves of force-intervals) permits the acquisition of detailed, automatic reporting in regard to molecular interactions. In addition in this way, measurements are optimized, in that the resolution, with which the force-spectra were acquired, is increased. This situation can be achieved, in that even the smallest force, which lies within the detection capability of a probe 4, can be minimized. The smallest detectable force depends on, among other conditions, the resilient properties of the measuring probe 4. In order to capture the smallest possible, detectable force, it is advisable to make use of such probes, which exhibit the greatest degree of spring-related characteristics and which show a high resonance-frequency.
A further possibility exists, in increasing the tractive velocity of a probe 4. High speeds of movement can induce special hydrodynamic turbulences, which in turn create undesirable displacements of the measuring probe 4. As a result, in a case of selected high speed operations, undesirable noise reactions can be infused into measurement data, whereby the sensitivity for the said lesser forces between a probe and the sample 8 is diminished. This disadvantage can be avoided, by the use of probes, which possess the shortest possible length along with a minimum expanse of elasticity therein. Such probes exhibit, in comparison with conventional probes, a clearly superior hydrodynamic character and permit obviously increased tractive speeds. In any case, such probes deflect from their chosen paths to a lesser extent that the conventional probe. On this account, the detector unit 12, in the invented case, can be designed as the greatest possible, optical assembly with special optical features.
In order to carry out raster probe, microscopic measurement along with static measurements, a probe 4, during the operation of a measurement, is set into vibrations of low amplitude, namely 0.1 to 10 nm. A difficulty related to dynamic raster probe, microscopic measures, can be found therein, in that governing low quality factors (Q-factors), which can be attributed to measuring probes 4 which are immersed in puffer containing solutions. Normally, molecular interactions can be captured by means of resonance displacement, possibly at the maximum possible value thereof and these can be determined by a probe. The resonance characteristics of a probe are proportional to the Q-factor, whereby a lesser Q-factor can lead to a broadly spread resonance maximum. On this account, a reduced sensitivity of the detection of force under circumstances of a reduced Q-factor can be brought about. Additionally, such characteristics evoked by (for example) hydrodynamic turbulence of fluid surrounding a probe or by the elastic action of a probe itself, (for instance, damping means) can contribute to dissipative interactions in the analysis. Accordingly, in the case of embodiment 1 (see
To obtain an automatic analysis from the results of measurement, provision is made, that during a measurement procedure, individual force-curves are made under the use of the evaluation unit 42. As far as obtaining statistical analyses, the procedure would be as follows:
To begin with, the force-curves are so compensated among themselves, that they can be compared with one another. This can be done, for example, by establishing a common unit separation along the zero line (reference value or abscissa) and/or by accordingly extending or compressing the given curves to match.
Thereafter, it is possible the entire measurement procedure can be statistically analyzed, in order that a determinative view may be obtained regarding proteins into the forces necessary for the probable apportioned degree of individual processes for folding and/or unfolding.
In addition, it is possible that the force-curves can be classified, viewed in real or supposed superimposition, and so determined as to characteristics. For the classification of force-curves, for example the length of the individual force-curve can be seen, and number and position of the therewith evident maximum forces can be determined. The length of a force-curve discloses the directions in which the expected interactions proceed. The number and positioning of force-maxima permit statements to be made in regard to the collective and/or individual results of interaction procedures. By means of a classification of force curves, it is possible that data, especially graphically illustrated data, can be obtained in regard to different interaction processes.
By means of an overview of class of classified force-spectra, the noise of the individual curves is reduced. Thereby the actual interactive procedures, which are illustratively disclosed by the force-curves, become obvious to the observer. In addition, statements about possible variations of the interactive procedures can be made, which statements are based on standard deviations from already determined force-curves.
Comparisons of interactive procedures, carried out at the same experimental conditions on samples under the same experimental conditions are made possible, when the following conditions are present:
In this way, concerning the embodiment 1, three mutants of the same receptor, which differentiate themselves from one another in point-mutations, are identified by means of their interactive spectra and can be compared with one another. Also statements in regard to the effects of an input of mutations into local interactions of a protein as well as the interaction of the given protein or proteins with other molecules can be carried out.
By means of the data storage memory 54, it becomes possible to create a data-bank for force-spectra, in order, for example, to characterize typical interactive procedures of various samples under different experimental conditions. For access to a data bank of the data storage memory 54, provision is made to employ different search strategies. For example, the structure data of an unfolded protein can be used, in order that structurally related proteins can be localized and the characteristics of their unfolded outline can be compared. In order to compare different unfolded spectra, it is possible, by means of the evaluation unit 42, to evaluate data in the said data memory bank, in order that, for example, different force-spectra to superimpose on one another and thus to compare. This makes it possible, to make statements in regard to dependencies of interaction procedures where experimental conditions are concerned. Furthermore, interactive procedures of different samples can be judged, as to whether or not the stored data indicates that their interactive procedures are comparative, similar or have the same characteristics.
Further, provision has been made, that that databank access can be made in relation to the presence of forces, physiological dependencies, interactive spectra and experimental conditions.
| Number | Date | Country | Kind |
|---|---|---|---|
| DE102004048971.8 | Oct 2004 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP05/10604 | 9/30/2005 | WO | 00 | 4/5/2007 |
