Abstract:
A method and device for the rapid detection of biomolecules ( 320 ) diffusing in a nanometer-confined slit ( 204 ) is claimed. In particular, the present invention relates to a novel concept of fluidic side apertures ( 205 ) that facilitates the filling of the device, the surface coating with biomolecules and that measures the affinity between fluorescently labeled biomolecules in aqueous solution with other biomolecules immobilized on surfaces.

Description:
FIELD OF INVENTION  
       [0001]    The present invention relates to methods and devices for the detection of fluorescently labeled biomolecules in nanofluidic biosensors, using an optical set-up. The present invention may advantageously be used for biomedical and biological analyses. 
       BACKGROUND OF THE INVENTION 
       [0002]    Nanofluidics is defined as fluidic systems with channels in the nanometer range size, and has been applied in microfluidic systems allowing for DNA manipulation, protein separation and sample preconcentration. A majority of the current nanofluidic developments are intended for bioengineering and biotechnology applications. 
         [0003]    Current practices for the detection of specific biomolecules can be divided in two categories: (a) the labeled techniques and (b) the label-free techniques. 
         [0004]    Among the labeled techniques, the widely used are fluorescence, colorimetry, radioactivity, phosphorescence, bioluminescence and chemiluminescence. Functionalized magnetic beads can also be considered as labeling techniques. Their advantages are the sensitivity in comparison to label-free methods and the molecular recognition due to specific labeling. 
         [0005]    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, but all these concepts lack molecular specific contrast, sensitivity and reliability. 
         [0006]    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. 
         [0007]    Enzyme linked immunosorbent assay (ELISA) is an important biochemical technique mainly used to detect the presence of antibodies and antigens, and thus is widely used as diagnostic tool in medicine and quality control check in various industries. ELISA analysis are however expensive, require large amounts of solution and a long time to obtain results. 
       OBJECTIVES  
       [0008]    It is an object of this invention to provide an inexpensive and rapid biosensor based on micro- and nanofluidics, which does not require complex manipulations. 
         [0009]    Still another object of the invention is to geometrically confine the optical measurement volume using nanofluidics and thus to obtain a high sensitivity of the biosensor. 
         [0010]    Still another object of the invention is to simplify the different surface coatings compared to existing biosensors. 
         [0011]    These and other objects of the present invention will become increasingly apparent with reference to the following drawings and preferred embodiments. 
       SUMMARY OF THE INVENTION 
       [0012]    This invention is based on the discovery that apertures can be designed on the sides of micro- and nanofluidics systems, avoiding thereby complex connections between reservoirs of the fluidic systems and external tubing. The device is filled by simple immersion inside a solution containing the biomolecules to assay. It gives the possibility to measure the interaction between diffusing biomolecules and other biomolecules immobilized on surfaces. 
         [0013]    This invention is also based on the discovery that the immersion water usually used with water immersion objectives may also be replaced by a solution containing a small concentration of fluorescent biomolecules to assay if necessary. 
         [0014]    Finally, this invention highlights the possibility to functionalize every single die with different biomolecules and to dispose these dies in an array configuration in order to perform rapid multiplexed tests. 
         [0015]    In the scope of this invention, nanofluidics is used because of its high surface-to-volume ratio, meaning that the surfaces are included in the detection volume, maximizing the detection of the interactions between diffusing biomolecules and other immobilized biomolecules on surfaces. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]      FIG. 1A  is a schematic of the system composed of a support  100  on which is attached an array of nanofluidic biosensors  200 . A solution containing the fluorescent biomolecules  300  is deposited on the devices and a optical system  500 , covered with a contamination filter  400 , is used for the measurement. 
           [0017]      FIG. 1B  is a schematic of an alternative of the biosensing system composed of a support  100  on which is attached an array of nanofluidic devices  200 . A solution containing the fluorescent biomolecules  300  is deposited on the devices and a optical system  500 , is used in epi-detection. 
           [0018]      FIG. 2  shows a cross section of the nanoslit defined by two substrates  201  and  202 . The intersection between the optical volume  510  and the nanoslit  204  delimits the zone of detection of fluorescent biomolecules  331 . Diffusing fluorescent biomolecules  320  may interact with immobilized biomolecules on surface  310 , and thus create molecular complexes  330  when a specific binding exists. 
           [0019]      FIG. 3  is a perspective view of an array of microfabricated device composed of a bottom structure  202 , on which is deposited a layer of amorphous silicon  203  defining one or several nanoslits  204 . A substrate cover  201  is affixed by anodic bonding. The liquid enters in the device from the lateral apertures on the sides  205 . 
           [0020]      FIG. 4  represents a top view of a support  100 , on which is fixed an array of nanofluidic devices  200 . (A) illustrates a rectangular array showing for example a 2-die arrangement  210  and a 3-die arrangement  220 , and (B) illustrates an hexagonal array showing for example a 3-die arrangement  230  or a 4-die arrangement  240 . 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0021]    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, polysaccharides and virus. 
         [0022]    As used herein, the term “nanoslit” is intended to be a generic term, which means well-defined microfabricated structure with a nanometer-sized height, of which the width and the length are larger. The nanometer-sized height of the nanoslit is defined to be higher than 2 nm because of the size of the smallest proteins to detect, that have to enter into the slit and are in the same order of magnitude. The present invention is limited to nanoslits with a height lower than the micron, because of the range of the detection volume of the optical system that are typically in the same order of magnitude. 
         [0023]    As used herein, the term “nanochannel” is intended to be a generic term, which means well-defined microfabricated structure with a nanometer-sized height and width, of which the length is larger. 
         [0024]    The present invention aims to simplify the measurement of the presence and of the interaction of specific diffusing biomolecules with surfaces, or with other biomolecules immobilized on surfaces. As shown in  FIG. 1 , an array of nanofluidic devices  200  is fixed on a support  100 , such as standard microscope cover glass or plastic capsule for example. An aqueous solution containing fluorescently labeled biomolecules is disposed on the support, so that at least one of the lateral apertures  205  of the nanofluidic device is included in the solution  300 , which results in filling its channels. If necessary and as highlighted in  FIG. 1A , a water-immersion microscope objective  500 , on which a contamination filter  400  is previously fixed, can be put in contact with the solution. Otherwise, as depicted in  FIG. 1B , the optical system is used in epi-detection. 
         [0025]      FIG. 2  illustrates the principle of detection and  FIG. 3  illustrates the structure of an embodiment of a biosensor according to the invention. First, biomolecules  310  are fixed on surfaces of substrates  201  and  202 . The detection volume  510  has to be focused inside a nanoslit  204  in a way that the intersection volume defined by the volume of the nanoslit  204  and the detection volume  510  is maximal. Then, the solution  300  containing the fluorescently labeled biomolecules  320  is filled into the system by capillarity. The biomolecules  320  diffuse and interact with those  310  fixed inside the nanoslit  204  and may create a molecular complex  330 ,  331 . The immobilized fluorescently emitting complexes  331  and the diffusing fluorescently emitting biomolecules  320  diffusing across the optical detection volume are both detected by the optical system. 
         [0026]    The present invention is distinguishable from biosensors currently being used to detect molecules interactions. The unique design of side apertures allows the liquid solution to directly enter the fluidic system. This is different from current biosensors based on micro- and nanofluidics reservoirs, which have to be mechanically connected with flexible tubes. Those solutions require injecting the solution containing the biomolecules to analyze, and require driving them through micro- or nanochannels, increasing the manipulation complexity of the system. 
         [0027]    The biosensor illustrated on  FIG. 3  may be manufactured as follows: First, the lateral apertures of a wafer  202  are etched by wet or dry etching. Then, an amorphous silicon layer  203  of thickness from 2 to 1,000 nm is deposited and structured using standard photolithography techniques, allowing definition of the nanoslit  204  geometry. A second wafer  201  is anodically bonded onto the first wafer  202 . The height of this second wafer  201  has to be compatible with the microscope objective. Afterwards, the wafers  201 ,  202  are diced into individual dies. The nanoslit  204  is linking the two side apertures  205  and is defined by the spacing between the two wafers  201 ,  202 . The amorphous silicon layer  203  is acting as spacer to define the nano slit  204  height. 
         [0028]      FIG. 4  shows an array of biosensors  200  that are fixed onto the microscope-mountable support  100 . The disposition of the biosensors  200  may be (A) rectangular or (B) hexagonal, but any other form can be contemplated. 
         [0029]    The handling of the device according to the present invention shows great promise for the detection, enumeration, identification and characterization of the biomolecules interacting or not with other immobilized biomolecules. Applications of the present invention can cover biomedical, biological and food analysis as well as fundamental studies in analytical and bioanalytical chemistry.