Abstract:
Provided is a method for determining one or more kinetic parameters of binding between a first binding member and a second binding member. The method includes adsorbing the first binding member to a surface at a plurality of microspots. The second binding member is then presented to the first binding member at each of the microspots, there being a plurality of combinations of first binding member surface density and second binding member concentration among the plurality of microspots. Data indicative of a binding reaction between the first of microspots are then obtained and analyzed so as to obtain one or more kinetic parameters of the binding between the first and second binding members. Also provided is a system for carrying out the method. A method for localizing a molecular species at microspots on a surface, and a probe array produced by the method are also provided.

Description:
This is a Continuation Application filed under 35 U.S.C. §120 as a continuation of U.S. patent application Ser. No. 10/578,860, filed on Jun. 30, 2006, which was a National Phase under 35 U.S.C. §371 of International Application No. PCT/IL2004/001043, filed on Nov. 14, 2004, which was an application claiming the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/518,878, filed Nov. 12, 2003, the content of each of which is hereby incorporated by reference in their entirety. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to methods for carrying out multiple binding reactions between bio-molecules in an array-format and more specifically to such systems and methods using biosensors and more specifically using optical detection methods such as surface plasmon resonance (SPR). 
     BACKGROUND OF THE INVENTION 
     In the new era of genomics, proteomics and bio-informatics, a vast number of proteins, new drug targets and small molecules are being investigated intensively and in high-throughput fashion. Although the full mapping of the human genome is done, genomics cannot provide a complete understanding of cellular processes which involve functional interactions between proteins and other molecules as well. Therefore, proteomics may be considered as a cutting-edge area of research today, bridging genomics and cell function. 
     Current technological methods for analyzing a large number of functional interactions between bio-molecules (especially proteins) include well-plate based screening systems (e.g., ELISA), cell-based assays, soluble reactants screening (e.g., radio immunoassays) and solid-phase assays (e.g., DNA-chips). Today, there is an obvious lack of high throughput technology which enables real-time, label-free monitoring of kinetics of multiple bio-molecular interactions (especially proteins). 
     The major current limitation in developing such solid-phase based-assays stems from the complexity and variability of proteins. Proteins, in contrast to DNA molecules which are used in producing DNA-chips, are less stable, and generally must kept hydrated and in an active structure and conformation. Also, proteins are very sensitive to chemical and physical changes (e.g., temperature). Finally, with regard to solid-phase kinetic studies, the amount or capacity of an immobilized protein must be known in order to perform an accurate, full kinetic study. 
     As used herein, the term “biosensor” refers to combination of a receptor surface for molecular recognition and a transducer for generating signals indicative of binding to the surface. 
     Various related optical methods can be used to measure kinetic binding interactions between bio-molecules. These include, among others, Surface Plasmon Resonance (SPR), total internal reflection fluorescence (TIRF) and evanescent wave elipsometry. It is known in the art to use biosensors and mainly SPR for such purpose. A kinetic binding reaction involves a first molecular species referred to herein as “the probe”. The probe is adsorbed to the sensor surface, and a solution containing a second molecular species, referred to herein as “the target” is then allowed to flow over the probe molecules adsorbed onto the sensor surface. As is known in the art and in commercially available devices, a standard kinetic binding interaction measurement can be described by the following procedure:
         (1) Chemical activation of solid-phase surface with a chemical activator (e.g., EDC/NHS); (2) Immobilization of a ‘probe’ molecule on a chemically-activated surface; (3) Washing and blocking of un-occupied activated groups with a blocker such as 1M ethanolamine; (4) Addition of one concentration of a ‘target’ molecule; (5) Washing and regeneration of the ‘probe’ with appropriate regenerating chemicals (e.g., 50 mM NaOH, 0.05% SDS); (6) Addition of another concentration of ‘target’; (7) Repeat stages 4-6, at least five times, each time with a different ‘target’ concentration.       

     In one aspect of this invention, the invention provides a method, referred to herein as “One-Shot Kinetics” (OSK). for obtaining one or more kinetic parameters of a binding reaction As shown below, this method allows carrying out a plurality of binding reactions without the need of the regeneration stage which is known to be harmful to the ‘probe’. 
     In general, any binding event between probe and target molecules can alter an SPR detection parameter which is than is used to monitor the binding reaction. The change in the detection parameter over time is used to determine a characteristic of the binding reaction, such as an association or dissociation constant rates as well as affinity. It is known to use surface Plasmon resonance (SPR) as the method of detection. SPR devices and methods are very sensitive to changes in an optical property of a probe layer and have proven to be useful in detecting changes in an optical property of a probe layer generated by relatively small stimuli. 
     An SPR probe layer may be configured as a multi-analyte “microarray” in which at each of a plurality of discrete regions, “microspots” on the sensor surface a probe material for interaction with a target material is adsorbed. Berger et al., describes a method for preparing a probe array and for presenting targets to the probe array so as to monitor the binding of the targets to the probes (“Surface Plasmon Resonance Multi-sensing”, Anal. Chem. Vol. 70, February 1998, pp 703-706). 
     PCT publication WO 02/055993, discloses the use of electrostatic fields and chemical cross-linking for binding probes to a sensor surface. 
     SUMMARY OF THE INVENTION 
     The present invention provides a system and method for determining kinetic parameters of one or more binding reactions between one or more probes and one or more targets. The probes and targets may be, for example, peptides, proteins, nucleic acids or polysaccharides. The probes and targets may be of the same species. For example, both of them may be proteins. Alternatively, the probes and targets may be of different species. For example, the probes may be nucleic acids, while the targets are proteins. 
     The system of the invention uses any detection method suitable for use in biosensors. More specifically, it uses a detection method based on an evanescent wave phenomenon such as surface plasmon resonance (SPR), critical angle refractometry, total internal reflection fluorescence (TIRF), total internal reflection phosphorescence, total internal reflection light scattering, evanescent wave elipsometry or Brewster angle reflectometry. The detection method makes use of a surface that allows a plurality of binding reactions to be monitored simultaneously. The method comprises adsorbing the probes to the sensor surface at different locations on the surface, for example by means of micro-fluidic methods using a chemical surface activator, or using a localized electric field. Each target is then presented to its respective probe adsorbed to the surface. The binding reactions between each pair of probe and target are monitored simultaneously. 
     In its first aspect, the present invention provides a system and method for determination of the kinetic parameters of a binding reaction, referred to herein as “One-Shot Kinetics” (OSK). This method allows carrying out a plurality of binding reactions without the need of the regeneration stage and without the need of repeated experiments which is known to be harmful to the ‘probe’. In this preferred embodiment of the method of the invention, a single probe species is adsorbed to microspots on a surface such as an SPR surface under a plurality of conditions, for example at different concentrations or pH, in order to obtain different probe densities. Some conditions may be repeated in order to obtain density duplicates. A single target species is then presented to the microspots at a plurality of concentrations. A plurality of probe density and target concentration combinations is thus obtained. The pluralities of reactions are monitored simultaneously and signals indicative of the binding reactions are obtained and analyzed so as to produce a kinetic analysis of the binding. The kinetic analysis may comprise of, for example, calculating an association constant or a dissociation constant or affinity constant for the binding of the probe to the target. 
     In its second aspect, the invention provides a method, referred to herein as “array-screening”, for simultaneously monitoring a plurality of binding reactions between a plurality of probes and one or more targets so as to obtain analysis of many binding reactions. In one embodiment of this aspect of the invention, a specific probe species is adsorbed to the surface at different one of a plurality of microspots so that each probe in each microspot may be selected independently of the probes on the other microspots. A target species is then presented to the probe in each microspot. Binding of the targets to the probes in the plurality of microspots is monitored simultaneously and signals indicative of the binding reactions are analyzed so as to produce analysis of the binding. The analysis may comprise of, for example, determining the existence of a detectable interaction at each microspot or calculating an equilibrium constant for the binding of the probe to the target at each microspot or determining the kinetics of binding. 
     The probes may be localized at different locations on the surface, for example, by means of micro-fluidic methods. The location on the surface may be activated, for example by using a chemical activator, or by applying an electric field, or by exposure to light (photo-activation). In order to achieve, localization, it is known to form a chemical thin layer covering a specific region of the surface, frequently referred as a binding layer. The binding layer may include different functional groups that are chemically activated, either by contact with chemical reagents, by applying an electric field, or by exposure to light (photo-activation). 
     Activation by an electric field may be carried out in two principal ways: (A) inducing an electrochemical reaction (reduction or oxidation) of functional groups in the binding layer. (B) applying an electric field so as to attract charged bio-molecules to the surface, and thus enhance the immobilization reaction; thus forming a higher local concentration of the probe molecules at the surface. 
     The most common binding layers for protein immobilization contain carboxylic groups. These carboxylic groups are activated by exposing the surface to accepted chemical activators, generally a mixture of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide)) in an aqueous solution. As a result, active NHS esters are formed. When the activated surface is contacted with a protein solution, the NHS esters react efficiently with nucleophilic groups on the protein backbone, mainly with amino groups to form stable amide bonds. Thus, covalent immobilization of proteins is achieved. Other methods for chemical activation include attachment of a molecule that exhibits a high affinity to the candidate for immobilization, e.g. attachment of avidin or an avidin derivative for immobilization of biotin-labeled molecules. 
     The invention also provides a method for preparing a probe array for use in the method of the invention for monitoring binding reactions. 
     Thus, in its first aspect, the invention provides a method for determining one or more kinetic parameters of binding between a first binding member and a second binding member comprising:
         (a) adsorbing the first binding member to a surface at a plurality of microspots;   (b) presenting the second binding member to the first binding member at each of the microspots, there being a plurality of combinations of first binding member surface density and second binding member concentration among the plurality of microspots;   (c) simultaneously obtaining data indicative of a binding reaction between the first and second binding members at each of the plurality of microspots by a biosensor detection method; and   (d) processing the data so as to obtain one or more kinetic parameters of binding between the first and second binding members.       

     In its second aspect, the invention provides a method for localizing a molecular species at each of two or more microspots on a surface, comprising, for each of one or more localization regions:
         (a) activating the surface in the localization region;   (b) for each of one or more microspots in the localization region, adsorbing a molecular species to the microspot; and   (c) optionally deactivating the localization region.       

     In its third aspect, the invention provides a probe array produced by the method of the invention. 
     In its fourth aspect the invention provides a system for simultaneously monitoring a plurality of binding reactions between one or more probe species and one or more target species comprising
         (a) A surface;   (b) An applicator capable of applying probe species to microspots on the surface so as to allow the probe species to be adsorbed to the microspot, the applicator being further capable of presenting a target to each probe species adsorbed to the surface;   (c) A photosurface receiving light reflected from the surface and generating signals indicative of the binding of the targets to the probes; and   (d) A processor configured to receive the signals generated by the photosurface and to analyze the signals so as to produce a kinetic analysis of the binding.       

    
    
     
       BRIEF DESCRIPTION OF FIGURES 
       In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described by way of non-limiting example only, with reference to the following accompanying drawings, in which: 
         FIGS. 1A and 1B  show a system for performing multiple binding reactions in accordance with one embodiment of the invention; 
         FIG. 2  shows a system for performing multiple binding reactions in accordance with another embodiment of the invention; 
         FIGS. 3A-3D  show a method for preparing a probe array in accordance with one embodiment of the invention; 
         FIGS. 4A and 4B  show a method for preparing a probe array in accordance with another embodiment of the invention; 
         FIGS. 5A-5F  show binding curves of IL-4 to anti-IL-4 antibody obtained by the method of the invention; and 
         FIGS. 6A-6E  show binding curves of five antigen targets to six antibody probes. 
         FIGS. 7A-7F  show binding curves of various compound targets to six CYP450 enzyme probes. 
     
    
    
     DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
       FIGS. 1A and 1B  schematically show a system  10  for simultaneously carrying out multiple binding reactions in accordance with one embodiment of this aspect of the invention. The system  10  includes an SPR device  80  comprising an array  24  of light sources  26  and a prism  30  having a sensor surface  32 . The light sources  26  provide light at a wavelength appropriate for SPR applications as is known in the art. The light array  24  is positioned at the focal plane of an optical system schematically represented by a lens  46  having an optical axis  48 . Lens  46  collects and collimates light from each light source  26  into a beam of parallel light rays and directs the collimated light so that it is incident on an “input” prism surface  50  of prism  30 . Light directed by collimator  46  that is incident on input surface  50  enters prism  30  and is incident on sensor surface  32 . 
     All light incidents on the sensor surface  32  from a given light source  26  is incident on the sensor surface at substantially a same incident angle and light from different light sources  26  is incident on the sensor surface at different incident angles. The angle at which light from a given light source  26  is incident on sensor  26  on sensor surface  32  is determined by the position of the given light source along the axis of the array  24 , the focal length of the lens  46  and the index of refraction of the material from which prism  30  is formed. The SPR device  80  may include a “displacement plate” (not shown) formed from a transparent material that is positioned between light source array  24  and prism  30 . The angular orientation of displacement plate is set so that the normal to the displacement plate is oriented at a desired angle with respect to the optic axis  48 . 
     Light incident on sensor surface  32  that is reflected from the surface exits prism  30  through an output prism surface  52  and is collected and imaged by a camera  55  having a lens  53  and a two dimensional photosurface  54  such as a CCD. A polarizer (not shown) is positioned between the array  24  and the prism  30  or preferably between the prism  30  and the camera  55 . The polarizer linearly polarizes light received by photosurface  54  so that relative to sensor surface  32  it has substantially only a p component of polarization. 
     The camera  55  outputs signals  57  that are indicative of images formed on the photosurface  54 . The signals  57  are input to a processor  59  having a memory  63  for storing signals  57 . The processor  59  is configured to analyze the signals as described below. Any of the signals  57  or results of the analysis performed by the processor may be displayed on an associated display screen  65 . 
     The system  10  includes a flow cell  34  having m microchannels  36  for flowing liquid across and in contact with the sensor surface  32 . In the device  80 , m=5 microchannels  36   a  to  36   e  are shown. This is by way of example only, and the method of the invention may be carried out using flow cell having any number m of microchannels. The outer form of flow cell  34  is shown in ghost lines and details of internal features, such as microchannels  36 , of the flow cell are shown in solid lines for clarity of presentation. Each microchannel  36  has at one end an inlet (not visible in the perspectives shown in  FIGS. 1A and 1B ) and, at its other end, an outlet  61  through which fluid flowing in the microchannel exits the microchannel. Each of the inlets is adapted to be independently connected to a suitable pumping apparatus (not shown) in order to introduce a fluid independently into each of the m microchannels  36 . 
     In the system  10 , the flow cell  34  is mountable onto the SPR surface in two orientations. One of the two orientations is shown in  FIG. 1A  and is referred to herein as “the probe orientation”. The second orientation, shown in  FIG. 1B  is referred to herein as the “target orientation”. In each of the two orientations, the microchannels are perpendicular to the microchannels in the other orientation. Each microchannel  36  is open on a side of the microchannel facing sensor surface  32  so that fluid flowing in the microchannel, in either orientation, contacts the SPR surface in a rectangular region. In the probe orientation shown in  FIG. 1A , fluid flowing in a microchannel contacts the sensor surface at a respective rectangular region  42  referred to herein as the microchannel&#39;s “probe region” (see  FIG. 1B ). In the target orientation shown in  FIG. 1B , fluid flowing in a microchannel contacts the sensor surface at a respective rectangular region  43  referred to herein as the microchannel&#39;s “target region” (see  FIG. 1A ). The probe regions and the target regions are thus perpendicular to each other. Regions of some microchannels  36  in the system  10  in  FIGS. 1A and 1B  are cut away to show microspots  58  formed at the crossover regions of the probe regions and the target regions. 
       FIG. 2  schematically shows a system  11  for simultaneously carrying out multiple binding assays in accordance with another embodiment of this aspect of the invention. The system  11  includes an SPR device  20  having several components in common with the SPR device  80  shown in  FIGS. 1A and 1B , and similar components are indicated with the same reference numeral in both figures. In particular, the SPR device  80  includes an optical system comprising an array  24  of light sources  26 , a prism  30  having a sensor surface  32 , a lens  46  having an optical axis  48 , and a two dimensional photosurface  54  such as a CCD. A suitable SPR conductor (not shown) is formed on the sensor surface. 
     The system  11  includes a flow cell  34  having m microchannels  36  for flowing liquid across and in contact with the sensor surface  32 . In the device  80 , m=5 microchannels  36   a  to  36   e  are shown. This is by way of example only, and the method of the invention may be carried out using a flow cell having any number m of microchannels. The outer form of flow cell  34  is shown in ghost lines and details of internal features, such as microchannels  36 , of the flow cell are shown in solid lines for clarity of presentation. Each microchannel  36  has at one end an inlet (not visible in the perspectives shown in  FIGS. 1A and 1B ) and, at its other end, an outlet  61  through which fluid flowing in the microchannel exits the microchannel. Each of the inlets is adapted to be independently connected to a suitable pumping apparatus (not shown) in order to introduce a fluid independently into each of the m microchannels  36 . 
     The SPR device  20  has n strip electrodes  33 . The n strip electrodes are used to create n independently activatable regions. While n=5 strip electrodes  33   a  to  33   b  are shown in  FIG. 2 , this is by way of example only and the method of the invention may be carried out with an SPR device having any number of strip electrodes. 
     In the system  11 , the flow cell  34  is mounted onto prism  30  so that the m microchannels are perpendicular to the n strip-electrodes  33 . Each microchannel  36  is open on a side of the microchannel facing sensor surface  32  so that fluid flowing in the microchannel contacts each strip electrode  33  at a microspot  58  located at the crossover region of the microchannel with the strip electrode. In an SPR device having m microchannels and n strip electrodes, a total of m×n microspots are formed at the crossover regions of the m microchannels with the n strip electrodes. Regions of some microchannels  36  in SPR device  20  in  FIG. 2  are cut away to show microspots  58 . 
     Each strip electrode  33  is independently connected to a power supply  60 . Power supply  60  is controllable to independently bring each strip electrode  33  to a voltage relative to a reference electrode  62  connected to the power supply so as to generate an electric field having a component perpendicular to the sensor surface  32 . The electric field passes through the lumen of the microchannels  36  at the crossover region of the microchannels with the strip electrode. 
       FIGS. 3A-3D  schematically shows a method for preparing a probe array on a surface  70  in accordance with one embodiment of the method of the invention. In  FIG. 3A , a first surface region  72   a  on the surface  70  is activated. Activation of a surface region allows probe molecules to be adsorbed to the surface region. One or more probe species  71  are then adsorbed to the activated first surface region  72  ( FIG. 3B ) at distinct microspots in the first surface region  72 .  FIG. 3B  schematically shows the application of 6 probe species  71   a  to  71   f  to the activated first surface region  72   a . This is by way of example only and the method of the invention may be carried out with any number of probe species  71  being adsorbed to the first surface region  72 . This produces the probe array shown in  FIG. 3C , in which each probe species is adsorbed to a different microspot  74 .  FIG. 3C  shows 6 microspots  74   a  to  74   f . The probe species may all be different or some of the probe species may be the same possibly at different concentrations. 
     The first surface region is now deactivated and a second surface region  72   b  is activated. One or more probe species are then adsorbed to distinct microspots on the second surface region  72   b , as explained above for the first surface region  72   a . The process is repeated, each time activating a different one of the surface regions  72  until probe species have been adsorbed to microspots on each of the surface regions  72 . This produces the probe array shown in  FIG. 3D  in which a plurality of probe species is adsorbed to microspots  75 . In  FIG. 3D , 6 surface regions  72   a  to  72   f  are shown. This is by way of example only, and the method of the invention may be carried out with any number of surface regions  72 . In the example shown in  FIG. 3D , on each of the 6 surface regions  72 , 6 probe species were adsorbed. This produces the array of 36 microspots shown in  FIG. 3D . The probe species adsorbed on different surface regions may be different, so that up to 36 different probe species may be adsorbed onto the surface  70 . 
     After the probe array on the surface  70  has been prepared, for each surface region, a target species may presented to the probe species adsorbed to the microspots. 
     The method of preparing a probe array on a surface shown in  FIGS. 3A-3D  will now be demonstrated with reference to the system  10  of  FIGS. 1A and 1B . In this example, m 2  probe species are to be adsorbed to the SPR surface at the m 2  microspots  58  (m 2 =25 in the SPR device  80  of  FIGS. 1A and 1B ) located at the m 2  crossover regions of the m probe regions with the m target regions. To prepare an appropriate microarray of the m 2  probes on the probe regions, the flow cell  34  is first placed in one orientation ( FIG. 1B ) and buffer or water is first pumped through the first microchannels  36  in order to clean and prepare the first surface region  43   a . Flow of buffer or water through the first microchannel  36   a  is then stopped a solution of a chemical surface activator is then made to flow through the first microchannel  36   a  in order to activate the first surface region  43   a . The first surface region is now activated. 
     The flow cell is now rotated 90° to bring it from the target orientation shown in  FIG. 1B  to the probe orientation shown in  FIG. 1A . An appropriate solution comprising a probe species is pumped through each of the m microchannels  36 . The m probe species may all be different, or some may be the same probe species, possibly at different concentrations. As a result of the activation of the first surface region  43   a , each probe species is adsorbed to the first surface region  43   a  and is not adsorbed by the other m−1 surface regions  43   b - 43   e . Each of the probe species is thereby immobilized at a different one of the m microspots  58  located at the m crossover regions of the m probe regions  42  with the first surface region  54   a . The probes are substantially prevented from immobilizing at the m×(m−1) microspots  58  located at the crossover regions of the m probe regions  42  with the m−1 other surface regions  43   b - 43   e.    
     During immobilization of the probes, the process of immobilization and the quantities of probe proteins immobilized at each microspot  58  are monitored by performing an SPR angular scan of the sensor surface  64 , as is known in the art. The signals  57  generated by the CCD  54  responsive to light from each light source  26  reflected at each microspot  58  on the first surface region  43   a  during adsorption of the probes are input to the processor  59 . The processor  59  is configured to analyze the signals so as to determine an SPR parameter for the microspot. The SPR parameter may be, for example, the SPR resonance angle, resonance wavelength, or the reflectance and phase changes that characterize a surface Plasmon resonance. The processor is further configured to analyze the SPR parameter so as to monitor accumulation of the probe immobilized at the microspot. Signals  57  from microspots of the other m−1 surface regions and from regions of the probe surface that are not crossover regions are analyzed by the processor to correct and normalize signals from crossover regions of the first surface region. 
     After termination of the flow of the probe solutions in the microchannels, the flow cell is rotated 90° back to the target orientation ( FIG. 1B ) and a solution containing a surface activator blocker is made to flow through the microchannels  36  to prevent further binding to the first surface region. 
     The above-described process is repeated for each of the other remaining m−1 surface regions  43   b - 43   a  with m probe solutions, until a probe species has been immobilized at each of as many as m 2  different microspots  58  located at the m 2  crossover regions of the m probe regions and the m surface regions. Each surface region  43  may thus be activated individually. As used herein, the term “activatable region” is used to refer to a region that can, when activated, bind one or more probe species. Thus, with the method of the invention, a probe microarray comprising as many as m 2  different probe species may be formed on the SPR surface of the SPR device  80 . 
     Following preparation of the probe microarray, a solution containing a target species is made to flow in each of the m microchannels  36  in the flow cell with the flow cell in the target orientation. The m target species may all be different, or some of the target species may be the same, possibly at different concentrations. Thus, for each of the m target solutions, the target is presented to each of the m probe species in the m microspots  58  located at the m crossover regions of the target&#39;s target region with the m probe regions. The signals  57  provided by the CCD  54  responsive to light from the light sources reflected from each of the m 2  microspots  58  during flow of the target molecules in the microchannels are input to the processor  59 . The processor  59  is configured to analyze these signals in order to monitor the binding of target to probe at each microspot. A total of as many as m 2  binding reactions can thus be monitored simultaneously involving as many as m 2  different probe species and as many as m different target species. As known in the art, reference surface must be used and be subtracted from any signal obtained from ‘active spot’. In one aspect of this invention, and as a novel outcome of the method, the surface between the spots, termed “inter-spot” is used as a reference surface. 
     The method of preparing a probe array on a surface shown in  FIGS. 3A-3D  will now be demonstrated with reference to the system  11  of  FIG. 2 . In this example, m×n probe species are to be adsorbed to the SPR surface that at the m×n microspots  58  (m×n=25 in the system  11  of  FIG. 2 ) located at the m×n crossover regions of the m microchannels  36  with the n strip electrodes  33 . To prepare an appropriate microarray of the m×n probes on the strip electrodes  33 , buffer or water is first pumped through the microchannels  36  to clean and prepare the strip electrodes for immobilization of the probe molecules at the microspots  58 . Flow of buffer or water through the m microchannels is then stopped and the first strip electrode  33   a  is now activated as explained above. The remaining strip electrodes are all brought to a potential with respect to the reference electrode  62  having a polarity opposite to that of the first electrode. An appropriate solution comprising a probe species is pumped through each of the m microchannels  36 . The m probe species may all be different, or some may be the same probe species, possibly at different concentrations. As a result of the activation of the first strip electrode  33   a  and the charge on the m probe species in the microchannels, each probe species is adsorbed to the first strip electrode  33   a  and is not adsorbed by the other n−1 strip electrodes  33   b - 33   e . Each of the probe species is thereby immobilized at a different one of the m microspots  58  located at the m crossover regions of the m microchannels with the first strip electrode  33   a . The probes are substantially prevented from immobilizing at the m×(n−1) microspots  58  located at the crossover regions of the m microchannels with the n−1 other strip electrodes  33   b - 33   e.    
     During immobilization of the probes, the process of immobilization and the quantities of probe proteins immobilized at each microspot  58  are monitored by performing an SPR angular scan of the sensor surface  64 , as is known in the art. The signals  57  generated by the CCD  54  responsive to light from each light source  26  reflected at each microspot  58  on the first strip electrode  33   a  during adsorption of the probes are input to the processor  59 . The processor  59  is configured to analyze the signals so as to determine an SPR parameter for the microspot. The processor is further configured to analyze the SPR parameter so as to monitor accumulation of the probe immobilized at the microspot. Signals  57  from microspots of the other n−1 strip electrodes  33   b - 33   e  and from regions of the probe surface that are not crossover regions are analyzed by the processor to correct and normalize signals from crossover regions of the first target region. 
     After termination of the flow of the probe solutions in the microchannels, buffer or water is again made to flow through the microchannels  36  to eliminate unbound probe proteins. 
     The above-described process is repeated for each of the other remaining n−1 strip electrodes  33   b - 33   e  with m probe solutions, until a probe species has been immobilized at each of as many as m×n different microspots  58  located at the m×n crossover regions of the m microchannels  36  and the n strip electrodes  33 . Thus, with the method of the invention, a probe microarray comprising as many as m×n different probe species may be formed on the SPR surface of the SPR device  20 . 
     Following preparation of the probe microarray, a solution containing a target species is made to flow in each of the m microchannels  36 . The m target species may all be different, or some of the target species may be the same, possibly at different concentrations. Thus, for each of the m target solutions, the target is presented to each of the n probe species in the n microspots  58  located at the n crossover regions of the target&#39;s microchannel with the n strip electrodes. The signals  57  provided by the CCD  54  responsive to light from the light sources reflected from each of the m 2  microspots  58  during flow of the target molecules in the microchannels are input to the processor  59 . The processor  59  is configured to analyze these signals in order to monitor the binding of target to probe at each microspot. A total of as many as m×n binding reactions can thus be monitored simultaneously involving as many as m×n different probe species and as many as m different target species. Ina preferred embodiment, a region of the surface, referred to as “an interspot” is used as a reference surface to provide a reference signal. 
       FIGS. 4A and 4B  shows a method for preparing a probe array on a surface  80  in accordance with another embodiment of the method of the invention (termed “OSK” or “one-shot kinetics”). This embodiment may be used when it is desired to perform a binding assay involving one probe species and one target species at different combinations of probe and target concentrations. In this embodiment, m probe regions  82  are simultaneously activated.  6  probe regions  82   a  to  82   f  are shown in  FIG. 4A . This is by way of example only, and the method may be carried out with any number of probe regions. The m probe regions  82  are activated and the probe is adsorbed onto the probe regions  82 . A different probe concentration is adsorbed onto each probe region. One of the probe regions  82   f  may be used as a reference region upon which no probe is adsorbed. 
     As depicted in  FIG. 4B , n concentrations of the target are then presented to the probe array (designated analytes  1 - 6 ). Each concentration is presented to a different microspot on each of the probe regions. The reaction assay thus consists of all of the m×n combinations of the probe and target concentrations. 
     The method of performing a binding assay shown in  FIGS. 4A and 4B  will now be demonstrated with reference to the system  10  of  FIGS. 1A and 1B . This embodiment is used when it is desired to perform a binding assay involving one probe species and one target species at different combinations of probe and target concentrations. The probe species is applied to each of the m probe regions at a different concentration, and the target is applied to each of the m target regions at a different concentration. To prepare this microarray, the flow cell  34  is first placed in the probe orientation ( FIG. 1A ) and buffer or water is first pumped through the m microchannels  36  in order to clean and prepare the m probe regions  42 . Flow of buffer or water through the m microchannels  36  is then stopped and any residual buffer or water in the flow system is drained away. A solution of a chemical surface activator is then made to flow through the m microchannels  36  in order to activate the m probe regions  42 . The surface activator may be, for example, EDC/NHS. The m probe regions are now activated. 
     An appropriate solution comprising the probe is pumped through each of the m microchannels  36 . In this embodiment, the probe is present in each of the different microchannels at a different concentration. As a result of the activation of the target regions  42 , probe molecules in each microchannel are adsorbed to the n microspots  58  in contact with the microchannel. 
     During immobilization of the probes, the process of immobilization and the quantities of probe proteins immobilized at each microspot  58  are monitored by performing an SPR angular scan of the sensor surface  64 , as is known in the art. The signals  57  generated by the CCD  54  responsive to light from each light source  26  reflected at each probe region  42  during adsorption of the probe are input to the processor  59 . The processor  59  is configured to analyze the signals so as to determine an SPR parameter for each probe region  42 . The processor is further configured to analyze the SPR parameter so as to monitor accumulation of the probe immobilized on each probe region  42 . Signals  57  regions of the SPR surface not in a probe region are analyzed by the processor to correct and normalize signals from the probe regions. 
     After termination of the flow of the probe solutions in the microchannels, a solution containing a surface activator blocker is made to flow through the microchannels  36  to prevent further binding of molecules to the SPR surface. The surface activator blocker may be, for example, ethanolamine. 
     The flow cell is now rotated 90° from the probe orientation to the target orientation ( FIG. 1B ). A solution containing the target species is made to flow in each of the m microchannels  36  of the flow cell. In this embodiment, the target is present in the different microchannels at a different concentration. Thus, for each of the m target solutions, the target is presented to each of the m probe concentrations species in the m microspots  58  located at the m crossover regions of the target solution with the m probe regions. The signals  57  provided by the CCD  54  responsive to light from the light sources reflected from each of the m 2  microspots  58  during flow of the target molecules in the microchannels are input to the processor  59 . The processor  59  is configured to analyze these signals in order to monitor the binding of target to probe at each microspot. In this embodiment, a total of m 2  binding reactions are thus monitored simultaneously involving as many as m 2  different combinations of probe concentration and target concentration. This allows the collection of kinetic data on the binding of the target to the probe for kinetic analysis in a single binding assay, without the need to regenerate the surface at any time. This is in contrast to prior art methods in which reactions are performed sequentially, each time with a different combination of probe and target concentrations which requires regeneration of the surface between successive binding reactions. 
     The method of performing a binding assay shown in  FIGS. 4A and 4B  will now be demonstrated with reference to the system  11  of  FIG. 2 . The probe species is applied to each of the m probe regions at a different concentration, and the target is applied to each of the m target regions at a different concentration. To prepare this microarray, the flow cell  34  is positioned as shown in  FIG. 2  with the m microchannels  36  perpendicular to the n strip electrodes  33 . Buffer or water is first pumped through the microchannels  36  to clean and prepare the SPR surface in contact with the microchannels Flow of buffer or water through the m microchannels is then stopped and the n strip electrodes  33  are now activated as explained above. An appropriate solution comprising the probe is pumped through each of the m microchannels  36 . In this embodiment, the probe is present in each of the different microchannels at a different concentration. As a result of the activation of the strip electrodes  33  and the charge on the probe in the microchannels, probe molecules are adsorbed to the strip electrodes  33 . Probe molecules are thereby immobilized at a different one of the n microspots  58  located at the n crossover regions of the microchannel with the n strip electrodes  33 . 
     During immobilization of the probes, the process of immobilization and the quantities of probe proteins immobilized at each microspot  58  are monitored by performing an SPR angular scan of the sensor surface  64 , as is known in the art. The signals  57  generated by the CCD  54  responsive to light from each light source  26  reflected at each microspot  58  on the first strip electrode  33   a  during adsorption of the probes are input to the processor  59 . The processor  59  is configured to analyze the signals so as to determine an SPR parameter for the microspot. The processor is further configured to analyze the SPR parameter so as to monitor accumulation of the probe immobilized at each microspot. Signals  57  from regions of the probe surface that are not microspots are analyzed by the processor to correct and normalize signals from of the microspots. 
     After termination of the flow of the probe solutions in the microchannels, buffer or water is again made to flow through the microchannels  36  to eliminate unbound probe proteins. 
     The flow cell  34  now removed from the SPR surface and a second flow cell (not shown) having n microchannels is positioned on the SPR surface with a microchannel overlying each of the n strip electrodes  33 . In the case that m=n, the flow cell  34  may also be used as the second flow cells by rotting it 90° from the orientation shown in  FIG. 2  to an orientation (not shown) in which the microchannels  36  overlay the strip electrodes  33 . A solution containing the target is made to flow in each of the n microchannels. In this embodiment, the target is present in the different microchannels at a different concentration. Thus, for each of the n target solutions, the target is presented to each of the m probe concentrations in the m microspots  58  located at the m crossover regions of the targets microchannel with the m probe regions. The signals  57  provided by the CCD  54  responsive to light from the light sources reflected from each of the n×m microspots  58  during flow of the target molecules in the microchannels are input to the processor  59 . The processor  59  is configured to analyze these signals in order to monitor the binding of target to probe at each microspot. This allows collection of kinetic data on the binding of the target to the probe for kinetic analysis in a single binding assay, without the need to regenerate the surface at any time. This is in contrast to prior art methods in which reactions are performed sequentially, each time with a different combination of probe and target concentrations which requires regeneration of the surface between successive binding reactions. 
     EXAMPLES 
     Example 1 
     A binding assay was carried out using the system  10  shown in  FIGS. 1A and 1B . Anti-IL-4 antibody (αIL-4) was used as a probe in this experiment and was localized on the SPR surface in each of six rectangular probe regions  42  (see  FIGS. 1A and 1B ), as explained above in the description of the system  10 . The probe regions were labeled (a) to (f). The density of the antibody, in “response units” (RU), in each of the 6 probe regions is given in Table 1. 
     
       
         
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   
                 Target (αIl-4) 
               
               
                   
                   
                 concentration 
               
               
                   
                 Probe region 
                 (pg/mm 2 ) (RU) 
               
               
                   
                   
               
             
             
               
                   
                 (a) 
                 530 
               
               
                   
                 (b) 
                 315 
               
               
                   
                 (c) 
                 346 
               
               
                   
                 (d) 
                 360 
               
               
                   
                 (e) 
                 334 
               
               
                   
                 (f) 
                 355 
               
               
                   
                   
               
             
          
         
       
     
     IL-4 was used as the target in this experiment was presented to the αIL-4 in each of five target regions  43  (see  FIGS. 1A and 1B ), as explained above. The target regions were numbered  1  to  5 . The concentration of IL-4 in each target region is given in Table 2. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Probe 
                 Target (IL-4) 
               
               
                   
                 Localization 
                 concentration 
               
               
                   
                 Site 
                 (nM) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 (1) 
                 26.7 
               
               
                   
                 (2) 
                 8.89 
               
               
                   
                 (3) 
                 2.96 
               
               
                   
                 (4) 
                 0.98 
               
               
                   
                 (5) 
                 0.33 
               
               
                   
                   
               
             
          
         
       
     
     The binding assay thus involved 30 binding reactions that were performed simultaneously. Binding of IL-4 to αIL-4 in the 30 microspots was monitored simultaneously as described above. The results of the binding are shown in  FIGS. 5A-5F . Each graph in  FIGS. 5A-5F  shows binding of IL-4 to αIL-4 in the probe region indicated in the graph. Each of the 5 curves in the graph shows the results of the binding of IL-4 to αIL-4 in the microspot located at the intersection of the probe region of the graph and the target region specified for each curve. At the times indicated by the arrow in each graph, unbound IL-4 was rinsed away, and the dissociation of bound IL-4 from αIl-4 in the 30 microspots was monitored simultaneously by the method of the invention. The processor  63  was configured to analyze the curves in each graph to obtain the association constant (Ka) and the dissociation constant (Kd) of the binding of Il-4 to αIL-4 at the antibody concentration of the graph. The Ka and Kd of each graph are shown in each of the graphs in  FIGS. 5A-5F . From these, the affinity constant (KD) can be derived, as is known in the art. 
     Example 2 
     Binding between 6 antibody probes (αIgG1, αIgG2b, αIgA, αIgG2a and αIgG3) to 5 antigen targets (IgG1, IgG1, IgG2a, IgG2b and IgG3) was studied using the system  10  of  FIGS. 1A and 1B . The concentrations used of the probes and targets are given in Tables 3 and 4, respectively. The binding curves obtained are shown in  FIGS. 6A-6E  and the binding response of each of the 30 binding reactions is shown in Table 5. 
     
       
         
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                   
                 Probe 
                 Probe concentrations 
               
               
                   
                 Probe 
                 region 
                 (pg/mm 2 ) (RU) 
               
               
                   
                   
               
             
             
               
                   
                 Anti mouse IgG2a 
                 a 
                 3410 
               
               
                   
                 Anti mouse IgG2b 
                 b 
                 4170 
               
               
                   
                 Anti mouse IgG1 
                 c 
                 3970 
               
               
                   
                 Anti mouse IgG3 
                 d 
                 3500 
               
               
                   
                 Anti mouse IgA 
                 e 
                 3770 
               
               
                   
                 Reference surface 
                 f 
                 — 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                   
                 Target 
               
               
                   
                 Target 
                 concentrations (μg/ml) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 IgG1 (anti IL-2) 
                 2.5 
               
               
                   
                 IgG1 (anti IL-4) 
                 2.5 
               
               
                   
                 IgG2a 
                 5 
               
               
                   
                 IgG2b 
                 5 
               
               
                   
                 IgG3 
                 5 
               
               
                   
                 Mouse IgG 
                 5 
               
               
                   
                   
               
             
          
         
       
     
     The Binding Responses to Different Antibody Subclasses (in Response Units) 
     
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                   
                 Anti 
                 Anti 
                 Anti 
                 Anti 
                 Anti 
               
               
                 Ligand 
                 mouse 
                 mouse 
                 mouse 
                 mouse 
                 mouse 
               
               
                 Analyte 
                 IgG2a 
                 IgG2b 
                 IgG1 
                 IgG3 
                 IgA 
               
               
                   
               
             
             
               
                 IgG1 
                 21 
                 42 
                 44 
                 — 
                 — 
               
               
                 (anti IL-2) 
                   
                   
                   
                   
                   
               
               
                 IgG1 
                 23 
                 — 
                 45 
                 — 
                 — 
               
               
                 (anti IL-4) 
                   
                   
                   
                   
                   
               
               
                 IgG2a 
                 90 
                 — 
                 — 
                 — 
                 — 
               
               
                 IgG2b 
                 — 
                 241  
                 — 
                 — 
                 — 
               
               
                 IgG3 
                 — 
                 — 
                 — 
                 97 
                 — 
               
               
                 IgG polyclonal 
                 122  
                 67 
                 44 
                 30 
                 — 
               
               
                   
               
             
          
         
       
     
     Example 3 
     The binding of five Cytochrome-P450 (CYP) enzyme probes (3A4, 2C19, 1A2, 2C9 and 2D6) with 6 different targets (Ketoconazole, Nifedipine, Dextromethorphan, Diclofenac, Dulfaphenazole and Propranolol) was carried out using the system  10  of  FIGS. 1A and 1B . The targets were presented at concentrations of 1,000, 500, 250, 125, 62.5, 31.25, 15.5, and 7.8 μM. The affinity constant, KD was determined for each reaction. The results are shown in  FIGS. 7A-7F  and Table 6. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Affinity constants (KD in [M]) determined for binding of 
               
               
                 various compounds to five CYP enzymes. 
               
             
          
           
               
                   
                 CYP-P450 
               
             
          
           
               
                   
                 3A4 
                 2C19 
                 1A2 
                 2C9 
                 2D6 
               
               
                   
               
               
                 Ketoconazole 
                 2.59E−05 
                 5.21E−05 
                 1.33E−03 
                 7.65E−05 
                 2.10E−04 
               
               
                 Nifedipine 
                 1.84E−03 
                 2.24E−03 
                 5.81E−02 
                 1.42E−03 
                 4.37E−03 
               
               
                 Dexo- 
                 1.26E−02 
                 7.90E−03 
                 — 
                 2.83E−02 
                 6.04E−02 
               
               
                 methorphan 
                   
                   
                   
                   
                   
               
               
                 Diclofenac 
                 4.47E−04 
                 7.17E−04 
                 1.42E−02 
                 1.66E−04 
                 6.81E−04 
               
               
                 Sulfaphenazole 
                 1.65E−01 
                 1.14E−02 
                 1.15E−03 
                 2.06E−03 
                 7.11E−02 
               
               
                 Propranolol 
                 7.53E−02 
                 6.59E−03 
                 8.73E−04 
                 5.13E−03 
                 5.22E−03 
               
               
                   
               
             
          
         
       
     
     Example 4 
     Table 7 shows immobilization of Rabbit IgG and Goat IgG probes on 36 independent microspots prepared by the method shown in  FIGS. 3A-3D , using the system  10  of  FIGS. 1A and 1B . Each probe region was sequentially activated and six alternate probes of Rabbit IgG and Goat IgG were adsorbed onto the activated probe region. This resulted in the immobilization of 36 alternate probes in the 36 microspots (6 in each surface region), as shown in Table 7. 
     
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
               
               
                   
                  543 RU 
                 1640 RU 
                  963 RU 
                 1950 RU 
                 1050 RU 
                 1420 RU 
               
               
                 2 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
               
               
                   
                 1620 RU 
                 1060 RU 
                 1800 RU 
                 1020 RU 
                 1320 RU 
                 1060 RU 
               
               
                 3 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
               
               
                   
                  525 RU 
                 1870 RU 
                 1200 RU 
                 1960 RU 
                 1300 RU 
                 1430 RU 
               
               
                 4 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
               
               
                   
                 1730 RU 
                 1300 RU 
                 2070 RU 
                 1240 RU 
                 1540 RU 
                 1360 RU 
               
               
                 5 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
               
               
                   
                  608 RU 
                 1660 RU 
                 1200 RU 
                 2160 RU 
                 1340 RU 
                 1730 RU 
               
               
                 6 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
                 Rabbit IgG 
                 Goat IgG 
               
               
                   
                 1680 RU 
                 1080 RU 
                 1910 RU 
                 1120 RU 
                 1530 RU 
                 1110 RU 
               
               
                   
               
             
          
         
       
     
     Mouse anti-rabbit and mouse anti-goat antibody targets were then presented to the probe array. Table 8 shows the target binding responses. Each of the 36 independently selected probes in the probe array reacts with its corresponding target allowing 36 different and independent interactions to be performed and monitored simultaneously (in a “checker board” pattern). 
     
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
               
               
                   
               
             
             
               
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
               
               
                 (0 RU) 
                 (348 RU) 
                 (2 RU) 
                 (431 RU) 
                 (1 RU) 
                 (291 RU) 
               
               
                 Anti Goat 
                 Anti Goat 
                 Anti Goat 
                 Anti Goat 
                 (Anti Goat 
                 Anti Goat 
               
               
                 (308 RU) 
                 (5 RU) 
                 (553 RU) 
                 (0 RU) 
                 (584 RU) 
                 (0 RU) 
               
               
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
               
               
                 (354 RU) 
                 (0 RU) 
                 (415 RU) 
                 (0 RU) 
                 (262 RU) 
                 (0 RU) 
               
               
                 Anti Goat 
                 Anti Goat 
                 Anti Goat 
                 Anti Goat 
                 (Anti Goat 
                 Anti Goat 
               
               
                 (0 RU) 
                 (573 RU) 
                 (0 RU) 
                 (571 RU) 
                 (1 RU) 
                 (579 RU) 
               
               
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
               
               
                 (4 RU) 
                 (402 RU) 
                 (1 RU) 
                 (435 RU) 
                 (0 RU) 
                 (291 RU) 
               
               
                 Anti Goat 
                 Anti Goat 
                 Anti Goat 
                 Anti Goat 
                 (Anti Goat 
                 Anti Goat 
               
               
                 (299 RU) 
                 (4 RU) 
                 (650 RU) 
                 (1 RU) 
                 (687 RU) 
                 (1 RU) 
               
               
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
               
               
                 (362 RU) 
                 (0 RU) 
                 (480 RU) 
                 (0 RU) 
                 (309 RU) 
                 (0 RU) 
               
               
                 Anti Goat 
                 Anti Goat 
                 Anti Goat 
                 Anti Goat 
                 (Anti Goat 
                 Anti Goat 
               
               
                 (1 RU) 
                 (674 RU) 
                 (0 RU) 
                 (660 RU) 
                 (1 RU) 
                 (704 RU) 
               
               
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
                 Anti Rabbit 
               
               
                 (0 RU) 
                 (355 RU) 
                 (3 RU) 
                 (475 RU) 
                 (1 RU) 
                 (360 RU) 
               
               
                 Anti Goat 
                 Anti Goat 
                 Anti Goat 
                 Anti Goat 
                 (Anti Goat 
                 Anti Goat 
               
               
                 (353 RU) 
                 (0 RU) 
                 (642 RU) 
                 (0 RU) 
                 (708 RU) 
                 (0 RU)