EP2257788A2

EP2257788A2 - Device for measuring transport systems

Info

Publication number: EP2257788A2
Application number: EP08856201A
Authority: EP
Inventors: Stefan Hummel; Matthias Pirsch
Original assignee: Synentec GmbH
Current assignee: Synentec GmbH
Priority date: 2007-12-06
Filing date: 2008-12-05
Publication date: 2010-12-08
Also published as: WO2009071065A3; US20100274496A1; WO2009071065A2; DE102007059166A1; JP2011519017A

Abstract

The invention relates to a device (1) for optically measuring properties of transport systems (50) in membranes (40), particularly carrier or channel proteins. In order to be able to measure the properties of biological transporter molecules (50) with a high throughput, the invention proposes that the device (1) comprise an optical measurement unit (2) and a data processing unit (6) having a process control (7) and a data capture.

Description

Title: Device for Measuring Transport Systems Applicant: SynenTec GmbH

The invention relates to a device for the optical measurement of properties of individual transport systems in membranes, in particular of carrier,

Channel proteins or other systems for transporting substances through biological membranes, such as secretory mechanisms.

Biological membranes separate cells from the outer medium and the individual cell compartments of the cells. Transport systems such as transport proteins and channels selectively control the mass transfer through these membranes. Dysfunctions of these transporters and channels are responsible for many common diseases. Among the top 100 drugs sold in the US in 2004, membrane transporters were the most abundant group. Overall, more than 100 transporter targets are currently being researched by the pharmaceutical companies, which shows what immense economic importance they have.

For the development of such drugs, measurement methods are needed to evaluate properties such as the transport rates of specific substrates through the transporter target and the influence of drug candidates. In this case, in particular methods are needed, the individual

Target molecules can even be automated in high-throughput characterization.

For the analysis of transport rates of ions and charged particles electrical measurements can be used. This process is already being used in high throughput biotechnological and pharmaceutical research. However, it is limited to charged transport substrates and is therefore usually used for the group of ion channels.

For the transport of uncharged molecules such as amino acids, peptides, sugar compounds and fatty acids, but also biological macromolecules such as RNA, DNA and proteins, a fluorescence analysis method is suitable, which is referred to as fluorescence analysis of individual transporters (nanoFAST). in this connection For example, a lipid membrane or biological membrane or cells containing the transport systems is applied to a sample chamber structured surface of a support. Suitable transport systems are, for example, membrane proteins, which may be channels or carriers. A substrate is then added to the transport system or produced by the cells, which is labeled with a fluorescent dye or provides intrinsic fluorescence. The transport across the membrane can then be optically measured by fluorescence. The optical measurement can be carried out, for example, by confocal laser scanning microscopy, wide-field fluorescence microscopy or by TIRF microscopy (Total Internal Reflection Fluorescence). In TIRF microscopy, excitation light is irradiated at a total reflecting angle so that fluorescent dyes are selectively excited within the spatial extent of an evanescent field.

Since no suitable devices exist, these measurements can be carried out so far only on a laboratory scale. However, there is a great need to use the process also in the industrial context, for example in biotechnology research and drug development.

The object of the invention is therefore to propose a device by which the properties of biological transporter molecules can be measured with high throughput.

This object is achieved in that a device for optically measuring properties of individual transport systems in membranes, in particular of carrier or channel proteins, is proposed, which has an optical measuring device and a data processing electronics with a process control and a data acquisition and evaluation. Through the process control, the functions of the microscope are controlled and the measurement is carried out automatically. After the process-controlled acquisition of the measured data, their automatic evaluation takes place. This automation advantageously allows a high sample throughput.

In a preferred embodiment, a TIRF microscope is provided as an optical measuring device. By the TIRF measurement, those are preferred Fluorescent dyes excited, which were transported by the respective transport system via the membrane in a measuring chamber. In contrast, fluorescent dyes outside the measuring chamber are not excited. This allows a more accurate measurement possible.

A further increase of the sample throughput is possible by providing a sample manipulator which can be controlled by the process control. This can take over several tasks, u. a. the preparation of the biochip for the measurement, the loading of the biochip with samples and substrates and the feeding into the measuring apparatus.

The German patent application DE 10 2007 016 699.2 has already been filed by the applicant, who proposes as a sample carrier a biochip, which is designed essentially as a transparent layer with a plurality of measuring chambers. The biochip allows more accurate and reproducible measurements. Advantageously, the device according to the invention is designed to measure such or similar biochips.

In a preferred embodiment of the biochip, a gold layer with smaller openings than the measuring chambers below is provided on its upper side, so that the measuring chambers are partially covered by the gold layer. When measuring such biochips, it is advantageous if the optical measuring device has a beam guidance which is close to the TIRF angle, but has not yet reached it. The TIRF angle is defined here as the limiting angle of the total reflection, which can be calculated from the arc sinus of the ratio of the refractive indices of two optical media by means of Snell's law of refraction. For water / glass, water / quartz or water / polycarbonate TIRF angles of 61, 7 °, 64.7 ° or 56.2 ° are obtained. The angle of the beam guide is smaller than the TIRF angle, so for example 55 ° instead of 61.7 ° for water / glass. Preferably, the angle of the beam guide is at most less than 20% of the TIRF angle. As a result, the excitation light is not totally reflected at the underside of the measuring chambers but a part of the light penetrates the carrier and the measuring chambers directly and another part additionally reflects on the gold layer in order to penetrate the measuring chamber. This results in a stronger suggestion of Fluorescent dyes. Since the excitation light is coupled close to the TIRF angle, ie obliquely into the transparent support, it enters the openings in the region of the openings of the gold layer at an angle with which it can not leave it. This results in a more favorable signal / noise ratio that the overlying substance is not irradiated with the excitation light.

The device has an incubation station for the biochips to be measured. As a result, biochips and proteo-liposomes are stored at a specific temperature for an adjustable period of time so that a membrane containing the transport system can form above the measuring chambers.

The described features of the device enable measurement cycles to be carried out in a process-controlled and automated manner, with one measurement cycle essentially comprising the preparation of the biochip for the measurement, a subsequent optical measurement and an evaluation of the measurement data following thereon.

In order to prepare the measurement on the biochip, the sample manipulator successively performs the following steps: First, the biochip is equilibrated with a buffer solution. Equilibration heats the biochip to a desired temperature that ensures the fluidity of the lipid membrane. Only with sufficient membrane fluidity are the lipids homogeneously distributed in the membrane. This is followed by addition of proteo-liposomes.

Subsequent washing with the buffer solution removes excess lipids. In the case of biological membranes or cells, addition and incubation are sufficient to allow their membranes to occlude the measuring chambers.

Then a drug candidate is added. An active substance candidate is a substance, for example an organic molecule, which is suspected to have certain effects on transport, for example to inhibit transport. Subsequently, the labeled with a fluorescent dye or intrinsically fluorescent transport substrate is added, which passes specifically by means of the transport system through the membrane. In addition, a spectrally separable, fluorescently labeled control substrate is added, which can not get through the membrane or the transport system. Finally, the biochip is fed into the measuring range of the microscope.

After the preparation of the biochip, a time-resolved fluorescence measurement of the substrate in the measuring chambers of the biochip is carried out in a process-controlled manner and the measured data acquired thereby are processed. The measurement can also be multispectral at different wavelengths.

After fluorescence measurement, the time-resolved fluorescence intensity for the individual measuring chambers is determined by the data processing unit by means of pattern recognition. The measured data determined in this way is adjusted by means of a subroutine a mathematical curve. The mathematical curve allows a classification of the measuring chambers into three categories, in dense measuring chambers with fluorescence signal, dense measuring chambers without fluorescence signal and open measuring chambers. The automatic distinction is made on the basis of the parameters of the mathematical curve. Measurement data from dense measuring chambers without fluorescence signal and from open measuring chambers are discarded. For the non-discarded measurement data, ie for dense measurement chambers with a fluorescence signal, the rate constant for the transport is calculated.

The data processing unit then preferably creates a histogram in which all calculated speed constants for the transport are plotted against their frequency. From the histogram, the respective speed constants for the transport are assigned a corresponding number of transport systems per measuring chamber. The assignment makes it possible to normalize all rate constants for transport to a transport system per measuring chamber. The maximum of the histogram for a transporter is determined by a mathematical function and reproduces with high accuracy the speed specific to the transport system with which it transports the measured transport substrate across the membrane. If the rate constant for transport in the presence of a drug candidate has been lowered or increased, then the drug candidate has inhibited or accelerated the transport system, for example, as a potential drug in question. The invention will be described by way of example in a preferred embodiment with reference to a drawing, wherein further advantageous details are shown in the figures of the drawing.

Functionally identical parts are provided with the same reference numerals.

The figures of the drawing show in detail:

Figure 1 is a schematic representation of the measuring device 1;

Figure 2 is a flow chart of the most important steps of a measurement;

FIG. 3 shows measuring curves of the time-dependent fluorescence;

Figure 4 is a histogram with different rate constants for transport; and

FIG. 5 Detailed view of a biochip 9 in vertical section.

Figure 1 shows a schematic representation of the measuring device 1. As optical measuring device is a TIRF microscope 2 (Total Internal Reflection Fluorescence), also referred to in the figure as FM. However, a conventional fluorescence microscope can also be used. The functions of the TIRF microscope 2 are automatically controlled by a process controller 7 (PS). The microscope 2 is set up for multispectral measurements. This makes it possible to simultaneously measure a transport substrate 60 (see FIG. 5) and a control substrate (not shown) in parallel. The measurement of the control substrate determines whether the measuring chambers 30 (see FIG. 5) are tight with the membrane 40 stretched over them (see FIG. 5).

The preparation, preparation and feeding of a sample 40, 50, 60 (see Figure 5) and a biochip 9 (see Figure 5) also takes place automatically. For this purpose, a mechanical sample manipulator 4 (PM) is provided, which is likewise controlled by the process controller 7. The sample manipulator 4 has a receiving device 5 for the biochip 9 (see FIG. 5). After the preparation and preparation of the sample 40, 50 (see FIG. 5), the biochip 9 (see FIG. 5) is incubated in an incubation station 8, which is also supplied by the process controller 7 is controlled. After an adjustable period of time, the sample manipulator 4 moves the receiving device 5 with the biochip 9 (see FIG. 5) into the measuring beam path of the TIRF microscope 2 and the measurement is started. The fluorescence images are recorded by a CCD camera 3, stored in the data processing unit 6 (DV) and automatically evaluated.

Figure 2 shows a flow chart of the most important steps of a measurement. The entire measuring process runs automatically and is controlled by a process controller 7 (see FIG. 1). The process controller 7 first queries a variable as to whether a new measurement cycle should be started. If this is the case, a biochip 9 (see FIG. 5) is prepared for the measurement. First of all, the biochip 9 (see FIG. 5) is guided by the sample manipulator 4 from a storage container (not shown) into the receiving device provided for this purpose and then brought into a preparation region of the device 1. A suitable biochip (see FIG. 5) consists of at least one layer 20 (see FIG. 5) which is transparent to the excitation light or the fluorescent light. It has upwardly open measuring chambers 30 (see FIG. 5).

The biochip 9 (see FIG. 5) is then equilibrated by the sample manipulator 4 with a buffer solution having a fixed pH. In addition, the sample manipulator 4 pipettes previously prepared proteo-liposomes onto the biochip 9 (see FIG. 5). The artificial proteo-liposomes contain carrier proteins or pore-forming channel proteins as a transport system. Thereafter, the biochip 9 (see FIG. 5) is stored in the incubation station 8 for an adjustable period of time. By incubation at a certain temperature, the lipids in the proteo-liposomes become fluid and form membrane layers 40 (see FIG. 5), which tightly close the individual measuring chambers 30 (see FIG. 5) of the biochip 9 (see FIG. 5). Subsequent washing with buffer solution removes excess lipid vesicles and transport systems.

However, it is also possible to use native biomembranes or cells, which, in addition to the determination of transport rates, also makes it possible to measure secretion rates from cells. For biological membranes or cells, the steps are reduced to addition and subsequent incubation through the biological membranes to close the individual measuring chambers 30 (see Figure 5), and a washing step.

Subsequently, the sample manipulator 4 pipettes the transport substrate 60 marked with a fluorescent dye (see FIG. 5) and the control substrate onto the biochip 9 (see FIG. 5). So that both substrates can be measured in different wavelength ranges in parallel, they are labeled with different, spectrally separable fluorescent dyes. In general, a drug candidate is also added. This may, for example, be an inhibitor which binds to the transport system 50 (see FIG. 5).

After the preparation phase, the sample manipulator 4 introduces the biochip 9 (see FIG. 5) into the measuring range of the fluorescence microscope 2. In the measuring range, the substrate dyes are excited with a laser in one point of the biochip 9 (see FIG. 5) and the fluorescence emission is measured with a CCD camera 3. The recorded fluorescence images are provided with a time stamp and stored in the data processing unit 6. In this way, an image stack is generated which contains information about the change in fluorescence in time.

After the change in fluorescence has been measured over a defined period of time, the images are evaluated by means of a pattern recognition routine and the fluorescence signals are assigned to individual measuring chambers 30 (see FIG. 5). From the time stamp and the time-dependent fluorescence intensity of each measuring chamber 30 (see FIG. 5), which is stored in the image stack, the data processing unit 6 then calculates measurement data points which reproduce the fluorescence course in the measurement period.

Thereafter, the data processing unit 6 adjusts a mathematical curve to the measurement data points for each measurement chamber (see FIG. 3).

In the ideal case, a single measuring chamber 30 (see FIG. 5) is tightly closed by a membrane 40 (see FIG. 5) in which a transport system 50 (see FIG. 5) is currently located. However, there is also the possibility that a measuring chamber (see FIG. 5) is not tight but open and therefore both the Substrate 60 (see Figure 5) and control substrate in the measuring chamber 30 (see Figure 5) can penetrate. In addition, there is the possibility that a measuring chamber 30 (see FIG. 5), although dense, does not contain a transport system 50 (see FIG. 5) and thus neither substrate 60 nor control substrate penetrates into the measuring chamber 30. A classification of the measuring chambers 30 in the three categories mentioned is then based on the curve through a subroutine. The measurement data of open measurement chambers or of measurement chambers without fluorescence signal are rejected (the course of typical measurement curves is shown in FIG. 3).

The remaining measurement curves are further evaluated by the data processing unit 6 for determining the specific rate constant. In this case, a histogram is preferably created by a subroutine in which all calculated rate constants for the transport are plotted against their frequency (see FIG. 4). From the histogram, each speed constant for transportation is given a certain number of

Transport systems assigned per measuring chamber and normalized to one transport system per measuring chamber. The maximum of the histogram for a transport system results in the speed constant specific to this transport system for transporting the specified substrate (see FIG. 4).

This completes a complete measuring cycle. The measured biochip is moved out of the measuring range of the fluorescence microscope 2 by the sample manipulator 4 and, if appropriate, another biochip is prepared for the measurement.

The described device can typically be used for the screening of potential drugs in drug development. If the rate constant for transport in the presence of a drug candidate is lower (higher) than without drug, then this indicates that the drug candidate has inhibited (accelerated) the transport system and may be considered, for example, as a potential drug. In such cases, the device 1 may automatically measure the drug in several cycles at various concentrations to automatically determine the binding constant and other properties. Figure 3 shows typical but exemplary time-dependent fluorescence traces measured with the device. There are five types of different fluorescence curves A, B ₁ C, D, E that typically occur when measuring a biochip. Each measuring curve is assigned to a specific measuring chamber on the carrier of the biochip.

Curve A shows the time course of the fluorescence in a measuring chamber 30 (see FIG. 5) which is not or not completely covered by a membrane 40 (see FIG. 5). The measuring chamber 30 is therefore not dense and both the substrate 60 (see Figure 5) and the control substrate with the spectrally separable fluorescent dyes can diffuse unhindered in a very short time in the measuring chamber. The fluorescence in the wavelength range of the control substrate has therefore already reached its maximum intensity after a very short time.

Curve B shows an exemplary time course of the fluorescence in a measuring chamber 30, which contains no transport system or no active transport system. The measured fluorescence intensity of the labeled substrate is very low and shows only a small change during the measurement time. The curves A and B contain no usable measurement data and are therefore discarded automatically by the data processing unit 6.

The curve C is similar to the curve B, but is measured in the spectrally separated wavelength range of the labeled control substrate. The fluorescence intensity of the labeled control substrate is very low and shows only a small change during the entire measurement time. The control substrate is thus excluded from the measuring chambers, so they are tight.

Curve D shows the time course of the fluorescence of the substrate in one

Measuring chamber 30, which is dense and also contains an active transport system.

Curve E shows the time course of the fluorescence of the substrate in a dense measuring chamber 30 as in curve D. However, it can be seen that the time-dependent intensity of the fluorescence increases faster than in curve D. This is due to the fact that in the membrane section above this measuring chamber 30th two or more transport systems 50 are present. To calculate the specific Speed constant for the transport 70, therefore, the number of transport systems 60 must be considered. How this happens is shown in FIG. 4.

Figure 4 shows a histogram with different rate constants for transport. In this case, all measured values of the rate constant for the transport k are plotted on the abscissa. On the ordinate the relative number of all measured rate constants for the transport k, ie their frequency, is plotted. The first peak I represents all the rate constants measured in measuring chambers with a transport system. The second peak II then represents all the rate constants measured in measuring chambers with two transport systems and the third peak III accordingly reproduces all the rate constants measured in measuring chambers 30 (see FIG. 5) with three transport systems 50 (see FIG. 5). From the histogram, therefore, the number of transport systems 50 per measuring chamber 30 can be assigned to each speed constant for the transport.

The histogram makes it possible for the data processing unit 6 to use a mathematical function to determine the velocity constants associated with the peaks of the peaks and to normalize these and thus all measured rate constants to a transport system per measuring chamber. This speed constant for transport for a transport system 50 thus corresponds with high accuracy to the specific rate constant of this transport system 50 for the transport substrate 60 under the selected experimental conditions.

FIG. 5 shows in vertical section a detailed view of a biochip 9, as it can be used for the measuring device 1. The biochip 9 has a carrier 10 which consists of a layer of glass transparent to exciting fluorescent light. On the carrier 10, a further layer 20 is disposed of silicon dioxide. This has recesses which are formed as upwardly open measuring chambers 30. The measuring chambers 30 have an inner diameter of about 200 nm, the depth is about 500 nm. For measurement, a lipid membrane 40 is applied to the surface of the biochip 9, so that the measurement spaces 30 are closed. The lipid membrane 40 is a lipid layer containing transport proteins 50. One or more fluorescence-detectable substrate molecules 60 labeled with a fluorescent dye are added to the lipid membrane 40. The substrate molecules 60 are then transported by the transport proteins 50 via the membrane 40 into the measuring chambers 30. In one measurement, excitation light (not shown) is radiated obliquely from below at a TIRF angle. At the transition from the carrier 10 to the measuring solution (not shown) in the measuring chamber 30, an evanescent field is generated in total reflection of the light, which excites the substrate molecules 80 in the measuring chamber 30, but not the substrate molecules 60 above the lipid membrane 40. In biochips with a reflective Top (not shown), the angle of the beam guide of the measuring device 1 is smaller than the TIRF angle.

The time-dependent transport 70 of the substrate molecules 80 by means of the transport proteins 50 contained in the membrane 40 into the measuring chambers 30 is specific to the transport system 50 contained and can be determined by time-resolved fluorescence measurements, as described above. By means of the measuring device 1, the specific speed constant of a transport system 50 for the transport substrate 80 is measured. To develop new medicines, drug candidates (not shown) are added. For example, they bind to the transport protein 50, thereby changing the rate constant of the transport 70 across the membrane 40. The measured change in the rate constant demonstrates an effect of the drug candidate that may be therapeutically significant. As a result of the measuring device 1 and the measuring method described, the development of new medicaments can be decisively improved both quantitatively and qualitatively. LIST OF REFERENCE NUMBERS

1. Measuring device

2. Fluorescence microscope

3. CCD camera

4. Sample manipulator

5. Recording device for biochip

6. Data processing unit

7. Process control

8th incubation station

9. Biochip

10. carrier layer

20th shift

30. measuring chamber

40. Lipid layer

50. Transport protein

60. Substrate molecule

70th transport

80. Transported Substrate Molecule

Claims

claims

1. Device (1) for the optical measurement of properties of transport systems in membranes, in particular of carrier or channel proteins and secretion mechanisms, characterized in that it comprises an optical measuring device (2), a data processing unit

(6) for the acquisition and processing of measurement data and a process control (7).

2. Device (1) according to claim 1, characterized in that the optical measuring device (2) is a TIRF microscope.

3. Device (1) according to claim 1 or claim 2, characterized in that for the device (1) by the process control

(7) controllable sample manipulator (4) is provided.

4. Device (1) according to one of the preceding claims, characterized in that the device (1) for measuring biochips (9) which have a transparent layer (20) with a plurality of measuring chambers (30) is arranged.

5. Device (1) according to one of the preceding claims, characterized in that the optical measuring device (2) has a beam guide whose angle is smaller than a TIRF angle, preferably at most less than 20% of the TIRF angle.

6. Device (1) according to one of the preceding claims, characterized in that the device (1) by the process controller (7) controllable incubation (8) for the biochips to be measured (9).

7. Device (1) according to one of the preceding claims, characterized in that the device (1) is formed, measuring cycles are process-controlled and automated, each one measuring cycle substantially a preparation of the biochip (9) for the measurement, one on it includes the following optical measurement and subsequent evaluation of the measurement data.

8. Device (1) according to one of the preceding claims, characterized in that the sample manipulator (4) is designed to perform on the biochip (9) the following steps: a) equilibration with a buffer solution, b) addition of proteo-liposomes, biological Membranes or cells c) washing with buffer solution, d) addition of substrates and / or drug candidates d) delivery into the measuring range.

9. Device (1) according to one of the preceding claims, characterized in that the device (1) is designed to perform a time-resolved fluorescence measurement of the measuring chambers (30) of the biochip (9) after preparation of the biochip (9) and the measurement data capture and process.

10.Vorrichtung (1) according to any one of the preceding claims, characterized in that the data processing unit (6) is arranged to perform the fluorescence measurement following steps: a) determination of the time-resolved fluorescence intensity respectively for the individual measuring chambers (30) by means of pattern recognition, b) Adapting a mathematical curve to the time-resolved fluorescence intensity, c) Classifying the measuring chambers (30) using the mathematical curve in one of the following three categories: i) dense measuring chambers with fluorescence signal, ii) sealed measuring chambers without fluorescence signal, iii) open measuring chambers and d) discarding the measurement data from dense measurement chambers without fluorescence signal and from open measurement chambers, and e) calculating a parameter for the rate of transport, preferably the rate constant for transport, for each dense measurement chamber with fluorescence signal.

11. Device (1) according to one of the preceding claims, characterized in that the data processing unit (6) is set up to carry out the following steps: a) Plotting of all calculated rate constants for the transport against their frequency in a histogram, b) Assignment of a corresponding number of transport systems per measuring chamber (30) to the respective rate constants, c) Determining a specific rate constant of a used transport substrate (60) for a transport system ( 50) per measuring chamber (30).

12. A method for the optical measurement of properties of transport systems (50) in membranes (40), in particular of carrier or channel proteins and secretion mechanisms, comprising the following steps: a) Determining the time-resolved fluorescence intensity in each case for individual measuring chambers of a biochip (9) by means of pattern recognition , b) fitting a mathematical curve to the time-resolved fluorescence intensity, c) classifying the measuring chambers (30) in one of the following three categories using the mathematical curve: i) dense measuring chambers with fluorescence signal, ii) dense measuring chambers without fluorescence signal, iii ) open measuring chambers.

13. The method according to claim 12, characterized in that it comprises the following steps: d) discarding the measurement data of dense measuring chambers without fluorescence signal and of open measuring chambers, and e) calculating a parameter for the speed of transport, preferably the rate constant for the Transport, for each dense measuring chamber with fluorescence signal.

14. The method according to claim 12 or claim 13, characterized in that it comprises the following steps: f) plotting all calculated rate constants for the transport against their frequency in a histogram, g) assigning a corresponding number of transport systems (50) per measuring chamber (30 ) to the respective rate constants, h) determination of a specific rate constant of a used transport substrate for a transport system (50) per measuring chamber (30).