Abstract:
A method and system for the rapid detection of biomolecular interactions, the system comprising a sensing platform which comprises a primary support structure including recesses designed to be located in front of a detection unit, said recesses containing one or several arrays of biosensors, said system furthermore comprising a reader unit for optical excitation and detection.

Description:
FIELD OF INVENTION 
       [0001]    The invention relates to the detection and the measurement of biomolecular interactions, in particular when several samples have to be quickly handled. 
       BACKGROUND OF THE INVENTION 
       [0002]    Biosensors are defined as fluidic systems with cavities and/or channels, which are used to measure the molecular interactions of diffusing biomolecules with other at the surfaces of the biosensors immobilized molecules. A majority of the current biosensor developments are intended for bioengineering and biotechnology applications. In the scope of this invention, biosensors are used to measure biomolecular interactions for in vitro diagnostic applications. 
         [0003]    Swiss patent application CH 01824/09 discloses biosensors for the detection of biomolecular interactions. The biosensors were described for a use with a confocal microscope. However, confocal microscope reading is difficult to automate, leading to long measurement times. 
         [0004]    Current technologies for the detection of biomolecular interactions can be divided in two categories: (a) the labeled techniques and (b) the label-free techniques. 
         [0005]    Among the labeled techniques, the widely used methods are fluorescence, colorimetry, radioactivity, phosphorescence, bioluminescence and chemiluminescence. Functionalized particles such as nanoparticles or magnetic beads can also be considered as labeling techniques. Their advantages are the sensitivity in comparison to label-free methods and the molecular specificity due to specific labeling. 
         [0006]    Fluorescence microscopy allows to measure the presence and the concentration of biomolecules specifically labeled with a fluorescent molecule called a fluophore. The specimen is illuminated with light of a specific wavelength, which brings it to an excited state, leading to an emission of light at a longer wavelength. The emission is measured by a detector, which allows quantifying the number of fluophores in the measurement volume. 
         [0007]    Fluorescence correlation spectroscopy (FCS), as a known representative of single molecule detection techniques, allows to access, across the fluctuation analysis of fluorescently labeled single biomolecules, static and dynamic molecular parameters, such as the mean number of molecules, their diffusion behavior and kinetic binding constants. This single molecule detection tool enables to measure the specificity of the biomolecule interaction, without being influenced by the presence of the fluorescent molecules outside the detection volume. 
         [0008]    In close relation to FCS several other techniques, known as Photon Counting Histogram (PCH), Fluorescence Intensitiy Distribution Analysis (FIDA) or Fluorescence Lifetime spectroscopy (FLS), use the intrinsic fluorophore mediated properties of single biomolecules for measuring the chemical binding constants, concentration or number of molecules, diffusion properties, etc. All these techniques are substantially compatible with the disclosed invention. 
         [0009]    Nanoparticle-based microscopy is an emerging technique allowing detecting the presence of functionalized nanoparticles that can be attached on biomolecules of interest. This technique has several advantages over fluophores such as chemical stability and no photobleaching. 
         [0010]    Among the label-free techniques, the widely used are electrochemical biosensors, referring to amperometric, capacitive, conductometric or impedimetric sensors, which have the advantage of being rapid and inexpensive. They measure the change in electrical properties of electrode structures as biomolecules become entrapped or immobilized onto or near the electrode. However, all these concepts lack molecular specific contrast, sensitivity and reliability. 
         [0011]    Surface plasmon resonance (SPR) is also a label-free optical technique for monitoring biomolecular interactions occurring in very close vicinity of a transducer gold surface, and has lead to great potential for real-time studying surface-confined affinity interactions without rinsing out unreacted or excess reactants in sample solutions. However, this method is limited to ensemble measurements, meaning that it is not single-molecule sensitive. 
         [0012]    The other important technologies for biomolecular diagnostics are Western and Northern blots, protein electrophoresis and polymerase chain reaction (PCR). However, these methods require highly concentrated analytes. 
       OBJECTIVES 
       [0013]    It is an object of this invention to overcome the limitations of the biosensors use described in Swiss patent application CH 1824/09 by providing a simple handling platform to rapid and automated sensing of multiple different biomolecular interactions. 
         [0014]    Another object of the invention is to use modified compact disc readers to perform the measurement of fluorescence inside the biosensors. 
         [0015]    Another object of the invention is to use modified compact disc readers to precisely control the position of the reading unit by means of rotation and translation in order to scan every biosensors disposed on the platform. 
         [0016]    Still another object of the invention is to use modified compact disc readers to precisely control the position of the reading lens in order to focus the laser beam inside the measurement area of the biosensors disposed on the platform. 
         [0017]    These and other objects of the present invention will be better understood with the following drawings and preferred embodiments. 
       SUMMARY OF THE INVENTION 
       [0018]    This invention is based on the combination of nanofluidic biosensors, a biocompatible sensing platform containing recesses and a reader unit. 
         [0019]    This invention is based on the assembly of the nanofluidic biosensors within recesses of the biocompatible sensing platform. 
         [0020]    Finally, this invention highlights the possibility to modify the detection apparatus of standard compact disc readers in order to perform integrated microscopy for rapid and automated analysis with the above mentioned sensing platform. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0021]      FIG. 1  shows a cross section of the biomolecular diagnostics system composed of a primary structure  110 , containing one or several cavities  111  or capsules, and on which is attached a thin transparent film  120  on the bottom. An array of biosensors  130  may be disposed onto the thin transparent film inside the cavities  111 , or may be inserted inside openings  121 .  FIG. 1A  shows that a solution containing fluorescent biomolecules to analyze  200  may be deposited inside one or several of the cavities  111  or of the capsules  114  in a way that the biosensors  130  are completely immersed.  FIG. 1B  illustrates that a solution containing the fluorescent biomolecules to analyze  200  may be deposited in a way that only a part of the biosensors  130  are immersed. A reading unit  300 , is approached to the thin film  120 , or to the opening  121 , in order to perform the measurement with a laser beam  312  directly inside one or several of the biosensors  130 , from the backside. 
           [0022]      FIG. 2  represents a perspective view of a primary structure  110 , containing several sensing cavities  111  and a central cavity  112  used by the reading unit  300  for the positioning control of the sensing platform  100 . The thin transparent film  120 , also containing a central aperture  121  used for the positioning control, is added on the bottom of the primary structure  110 . Biosensor arrays  130  are assembled inside the cavities  111  directly on the thin transparent film  120 . 
           [0023]      FIG. 3  represents a perspective view of a primary structure  110 , containing several openings  113  and a central cavity  112  used by the reading unit  300  for the positioning control of the sensing platform  100 . Several capsules  114  are disposed in the openings  113  and may be opened before or after insertion. Arrays of biosensors  130  are present in each capsule  114 . 
           [0024]      FIG. 4  is a 3D illustration of the sensing concept. The sensing platform  100  is actuated by the motor  331  in order to place the capsules or the sensing cavities  111  containing the biosensors  130  in the sensing position. The linear motor  332  controls the transversal position of the integrated measurement unit  310 , which is disposed on a rail system  333 , in order to position the measurement volume in the biosensor of interest. The excitation beam  312  is produced by the excitation laser  311  and is deflected on the dichroic minor  313  before to pass through the lens  314 . When focalized in the right position inside one of the biosensors  130 , the excitation beam  312  excites fluorescently labeled biomolecules of the solution  200 , which emit photons that are collected by the lens and finally detected by the detector  315 . 
           [0025]      FIG. 5  represents an illustration of the optical system  310  containing the light source  323  for the positioning of the sensing platform  100  and the biosensor  130 . The positioning beam is directed by the lens  324 , the minor  326 , the dichroic minors  318  and  319 , the mirror  313  and the lens  314  onto the sensing platform where the positioning beam is partly reflected and directed back by the lens  314 , the minor  313 , the dichroic mirror  319 , the emission filter  320  and the lens  322  onto the detector  315 . 
           [0026]    For the fluorescence measurements, the excitation beam  312  produced by the excitation laser  311  is collimated by the lens  316 , cleaned up by the excitation filter  317 , and directed by two dichroic mirrors  318  and  319 , the mirror  313 , and the lens  314  to be focused inside the biosensor  130 . Inside the biosensor  130  fluorescent biomolecules are excited and emit the fluorescent signal  321 , which is directed by the lens  314 , the mirror  313 , the dichroic mirror  319 , the emission filter  320  and the lens  322  onto the detector  315 . The detector  315  can either be a detector surface or an optical fiber guiding the fluorescent signal to a fibered detector. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0027]    As used herein, the term “biomolecules” is intended to be a generic term, which includes for example (but not limited to) polyclonal antibodies, monoclonal antibodies, Fab fragments, recombinant antibodies, globular proteins, amino acids, nucleic acids, enzymes, lipid molecules and polysaccharides. 
         [0028]    As used herein, the term “sensing platform” is intended to be a generic term, which means a device containing one or several arrays of biosensors. It is designed in order to facilitate the reception of the liquid solution to analyze. As used herein, the term “cavities” is intended to be a generic term, which means well-defined wells in the sensing platform, inside which are disposed the biosensors array and that will contain the liquid solution during the measurement. As used herein, the term “capsules” is intended to be a generic term, which means well-defined container disposed in the sensing platform, inside which are disposed the biosensors array and that will contain the liquid solution during the measurement. 
         [0029]    As used herein, the term “compact disc reader” is intended to be a generic term, which means standard reader of compact disc (CD), digital versatile disc (DVD), Laserdisc, Blu-ray or other optical media technologies. 
         [0030]    As used herein, the term “reading unit” is intended to be a generic term, which means the device containing the measurement system, including the compact disc reader. 
         [0031]    The present invention aims to provide a simple method for detecting biomolecular interactions by combining microfluidic and nanofluidic biosensors described in the patent [1], a biocompatible sensing platform containing cavities or capsules, and a reader unit. 
         [0032]    As shown in  FIG. 1 , the sensing platform is composed of a primary support structure  110  containing cavities  111  or openings  113 . This primary structure may be a single component or may be composed of a primary structure, on which is attached a transparent biocompatible thin film  120 . An array of biosensors  130  may be disposed in the capsules  114  or on the thin film  120  within the cavities of the primary structure  110 . The solution  200  containing the fluorescent biomolecules to detect is deposited directly in one of the cavities  111  or capsules  114  in order to fill the biosensors  130  by capillarity, The solution  200  can also be disposed in a way that only a part of the biosensor  130  is immersed. A reading unit  300  is approached by the opposite side of the thin film  120 . Its laser beam  312  is focused inside the biosensors  130 , such as the measurement volume is always right-positioned in the detection area during every measurement. 
         [0033]    The biomolecules contained in the solution  200  diffuse in every biosensor, interact with those preliminary fixed on the biosensors surfaces, and may create a molecular complex (depending on the specificity). The immobilized biomolecules and those freely diffusing across the optical detection volume are both detected by the reading unit  300  that is inserted or connected to a computer or an analyzing unit. Finally, the measurements are directly presented to the user who will interpret their meaning. 
         [0034]    A possible principle of assembly of the sensing platform  100  is illustrated in  FIG. 2 . First, the primary support structure  110  containing the cavities  111  and a central aperture  112  is used to place the assembly in the measurement position. The transparent biocompatible thin film  120 , also containing a central aperture  121  that is larger than the one of the primary structure  110 , is added. Biosensor arrays  130  are assembled on the thin film  120  within the cavities  111  of the primary structure  110 . 
         [0035]    Another possible principle of assembly of the sensing platform  100  is illustrated in  FIG. 3 . First, the primary support structure  110  containing the openings  113  and a central aperture  112  is used to place the assembly in the measurement position. Capsules  114 , which contain biosensors array  130 , are disposed in the openings  113 . 
         [0036]    The sensing principle is presented in  FIG. 4 . The sensing platform  100  is positioned by the motor  331  in order to place sensing cavity  111  and especially one of the biosensors  130  in the sensing position. The linear motor  332  controls the transversal position of the integrated measurement unit  310 , which is disposed on a rail system  333 , in order to position precisely the measurement volume inside the biosensor of interest. The excitation beam  312  is produced by the laser  311  and is deflected on the dichroic mirror  313  before passing through the lens  314 . When focused in the right height position inside the biosensor, the laser beam  312  excites fluorescently labeled biomolecules, which emit photons that are collected by the lens  314  and finally detected by the detector  315 . The detector  315  is controlled by an electronic interface, which is connected to a computer or an analyzing unit that will present the measurements to the user. 
         [0037]    The optical system  310  is presented in  FIG. 5 . By means of the light source  323  the biosensors  130  on the sensing platform  100  is correctly positioned for the fluorescence measurement. The positioning beam  325  is collimated by the collimating lens  324 , deflected by the minor  326 , transmitted through the dichroic mirror  318 , partly deflected by the dichroic minor  319 , deflected by the mirror  313  and then focused onto the sensing platform  100  by the lens  314 . Part of the positioning beam  325  is reflected by the sensing platform  100  and the biosensor  130 , collected by the lens  314 , deflected by the mirror  313 , partly transmitted through the dichroic mirror  319  and the emission filter  320 , and focused by the lens  322  onto the detector  315 . The signal from the positioning beam  325  is then analyzed for the correct positioning of the biosensors  130  in preparation of the fluorescent measurement. 
         [0038]    The excitation beam  312  is produced by the excitation laser  311  and collimated by the collimating lens  316 , cleaned up by the excitation filter  317 , deflected by two dichroic minors  318  and  319 , and the minor  313 , in order to be focused on the sensing platform  100  and inside the biosensor  130  by the lens  314 . Inside the biosensor  130  fluorescent biomolecules are excited, which then emit the fluorescent signal  321  being collected by the lens  314 , deflected by the minor  313 , transmitted through the dichroic minor  319  and the emission filter  320 , and focused by the lens  322  onto the detector  315 . The detector  315  can either be a detector surface or an optical fiber guiding the fluorescent signal to a fibered detector. 
         [0039]    The method of measurement presented in this invention shows great promise for the detection, enumeration, identification and characterization of biomolecular interactions. Applications of the present invention can cover biomedical, biological and food analysis as well as fundamental studies in analytical and bioanalytical chemistry.