Abstract:
A method and device for multiplexing and calibrating rapid quantification of biomolecules present in a nanofluidic biosensor composed by a nanoslit ( 210 ) is claimed. In particular, the present invention relates to a novel concept defining multiple different functionalized areas containing biomarkers. Functionalized areas can also being structured to decrease the biomarkers density in the nanoslit. The present concept enables the multiplexed quantification biomolecular interactions of interest in the same nanofluidic biosensor.

Description:
FIELD OF INVENTION 
       [0001]    The present invention relates to a method and to a device for the detection of various fluorescently labeled biomolecules in selectively functionalized nanofluidic biosensors, by means of an optical system. The present invention may for example be advantageously used for rapid quantification of biomedical and biological samples. 
       BACKGROUND OF THE INVENTION 
       [0002]    Nanofluidic biosensors are fluidic systems with nanometer-sized confinements and/or lateral apertures, which are used to quantify the presence of biomolecules in a solution. Most nanofluidic biosensor developments are intended for bioengineering and biotechnology applications. In the scope of this invention, the biosensors are used for example to quantify the presence of biomolecules in a solution for in vitro diagnostic applications. 
         [0003]    Swiss patent application CH 01824/09 discloses biosensors with lateral apertures for the detection of biomolecular interactions, PCT application PCT/IB2010/050867 discloses the use of such biosensors with simple optical systems and PCT application PCT/IB2011/050979 discloses a method for avoiding long waiting times to attain stable measurement conditions. However, in all configurations described in these documents, the number of type of biomarker was limited to one per nanofluidic biosensor. 
         [0004]    Biomarkers, also called biological markers, are substances used as specific indicators for detecting the presence of specific biomolecules. It is a characteristic that is objectively measured and evaluated as an indicator of biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. 
         [0005]    Methods for the detection of specific biomolecules can be divided in two categories: (a) the labeled techniques and (b) the label-free techniques. 
         [0006]    Among the labeled techniques, the widely used ones are fluorescence, colorimetry, radioactivity, phosphorescence, bioluminescence and chemiluminescence. Functionalized magnetic beads can also be considered as labeling techniques. Advantages of labeled techniques in comparison to label-free methods are the sensitivity and the molecular recognition due to specific labeling. 
         [0007]    Among the label-free techniques, the widely used ones 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. These technologies however lack molecular specific contrast, sensitivity and reliability. 
         [0008]    Enzyme linked immunosorbent assay (ELISA) is an important biochemical technique mainly used to detect the presence of soluble biomolecules in serum, and is thus widely used as a diagnostic tool in medicine and quality control checks in various industries. ELISA analysis are however expensive, require large amounts of solution and are time consuming. 
         [0009]    The other relevant technologies for biomolecular diagnostics are Western and Northern blots, protein electrophoresis and polymerase chain reaction (PCR). However, these methods require highly concentrated analytes and do not allow high throughput samples testing. 
       Aims of the Present Invention 
       [0010]    An object of the present invention is to provide inexpensive and fast self-calibrated or multiplexed nanofluidic biosensors, which do not require complex manipulations. 
         [0011]    Still another object of the present invention is to geometrically confine the optical measurement volume down to the nanometer scale and to obtain a multiplexed high sensitive biosensor. 
         [0012]    Still another object of the invention is to provide a biosensor able to provide a calibration curve, which can then be used to subtract the biosensor background and to provide quantitative measurements with the concentration value of specific measurements. 
         [0013]    These and other objects of the present invention will become apparent with reference to the following description of preferred embodiments, illustrated by the figures. 
       SUMMARY OF THE INVENTION 
       [0014]    According to embodiments of the present invention, several functionalized areas are differently patterned in order to vary the density of immobilized biomarkers in a nanofluidic biosensor, thereby allowing the extraction of a calibration curve that depends on the biosensor environment and on the solution filling the biosensor. 
         [0015]    Furthermore, according to embodiments of the present invention, various biomarkers are immobilized on various functionalized areas in a same nanofluidic biosensor, thereby allowing quantifying multiple types of biomolecules in the same nanofluidic biosensor. 
         [0016]    In the present text the term “patterned functionalized areas” has to be understood as surfaces that are structured in a way that the total surface where biomarkers are immobilized, is limited according to a determined pattern. The patterning can be realized by the geometry of one or more functionalized surfaces or by varying the concentration of the biomarkers within a specific area. 
         [0017]    In the scope of this invention, nanofluidics is used because of its high surface-to-volume ratio, which means that the surfaces comprised in the detection volume maximize the probability of interaction between biomolecules and the biomarkers immobilized on said surfaces. Nanofluidics also strongly reduces the background signal of the solution due to the small portion of substrate that is within the detection volume. 
         [0018]    The invention relates to a biosensor as defined in the claims. 
         [0019]    It also relates to an assembly and a method using said biosensor. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]      FIG. 1  is a perspective schematic view of a system  100  comprising nanofluidic biosensors  200  with lateral apertures. A solution  300  containing fluorescently-labeled biomolecules is deposited in the system  100  by a pipet system  400 . An optical system  500  including a probing laser  510  is used for the measurement. 
           [0021]      FIG. 2  is a perspective view of an embodiment of a nanofluidic biosensor  200  with a lateral input aperture  221  and a lateral output aperture  222 . A nanometer-sized channel  223  is defined by the height of a structured material  230  sandwiched between a first substrate  210  and a second substrate  220 . Several functionalized areas  240  are present in the nanometer sized slit  223 . 
           [0022]      FIG. 3  is a schematic representation of a partial cross section of the nanofluidic biosensor of  FIG. 2 , comprising two substrates  210  and  220  and locally structured areas  240  that are each functionalized by different biomarkers  331 ,  332 ,  333 ,  334 ,  335 ,  336  and other areas  250  that prevent that functionalization. The solution  300  containing biomolecules  311 ,  312 ,  313 ,  314 ,  315 ,  320  enters the nanoslit  223  from the lateral input aperture of the biosensor  221  to the lateral output aperture  222 , with a flow front  301 . Molecules  311 ,  312 ,  313 ,  314 ,  315 ,  320  may interact with the different immobilized biomarkers  331 ,  332 ,  333 ,  334 ,  335 ,  336  and may form complexes  341 ,  343 ,  344 ,  345 ,  346  if specific to the biomarkers. The laser beam  510  monitors the concentration of the immobilized biomolecules on their specific biomarkers in the detection volume  520 . Molecules  320  that are not specific to any functionalized biomarkers won&#39;t be measured by the system. Finally, negative control areas  335  and positive control areas  336  may for example be used to calibrate the biosensor. 
           [0023]      FIGS. 4 a , 4 b  and 4 c    are a partial top view cross sections of biosensors according to different embodiments of the invention, where the nanofluidic biosensor comprises a nanoslit  223  with a lateral input aperture  221 , a lateral output aperture  222 , and sides  223 . The solution  300  containing biomolecules enters the nanoslit  223  with a flow front  301 . Several functionalized areas  240 ,  241 ,  242  and not functionalized areas  250  are present in the nanoslit  223 . Functionalized areas  240 ,  241 ,  242  may have various shapes such as lines  240 , small polygons  241  or rounded areas  242 . 
           [0024]      FIG. 5  is a partial top view cross sections of a biosensor according to still another embodiment of the invention, with a first functionalized area  240  for immobilizing one type of biomarker (specific detection), and other areas that are functionalized for the calibration of the test. For example, functionalized area  243  is the 100% positive control, functionalized area  244  is 75% positive control, functionalized area  245  is 50% positive control, functionalized area  246  is 25% positive control and functionalized area  247  the negative control. The laser beam  510  monitors the concentration of the immobilized biomolecules on their specific biomarkers in the detection volume  520  for each different functionalized area. 
           [0025]      FIG. 6  illustrates the various sequences for the detection using a biosensor of  FIG. 5 . First the specific detection is measured in the functionalized area A), then the positive control B), several intermediate controls C), D), E), and finally the negative control F). 
           [0026]      FIG. 7  represents a typical calibration curve, established from functionalized areas  243 ,  244 ,  245 ,  246  and  247 , allowing calibrating the specific detection value (CA) in function of the biosensor environment (background, maximum value, etc.) and of the characteristics of the solution  300  (viscosity, density of biomolecules, etc.). 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0027]    As used herein, the term “system” is intended to be a generic term, which includes for example (but not limited to) a capsule, a surface, a disc or any environment that can immobilize nanofluidic biosensors. 
         [0028]    As used herein, the term “biomolecules” is intended to be a generic term, which includes for example (but not limited to) proteins such as antibodies or cytokines, peptides, nucleic acids, lipid molecules, polysaccharides and virus. 
         [0029]    As used herein, the term “nanoslit” is intended to be a generic term, which means well-defined microfabricated structure with one nanometer-sized dimension. The nanometer-sized dimension of the nanoslit is defined to be higher than 2 nm because of the size of the smallest biomolecules to be detected that have to enter into the slit and that are in the same order of magnitude. The present invention is limited to nanoslit with only one dimension lower than one micron, because of the range of the detection volume of the optical system that are typically in the same order of magnitude. 
         [0030]    An object of the present invention, according to embodiments, is to allow the detection of several substances, in particular of several biomolecules in a single nanofluidic biosensor. 
         [0031]    As illustrated in  FIG. 1 , according to embodiments of the invention, several nanofluidic biosensors  200  are immobilized in a system  100 . A mixed solution  300  containing fluorescently-labeled biomolecules of interest is deposited inside the system  100  for example with a pipet system  400 . An optical unit  500  is used to measure the biomolecular interactions inside the biosensors  200  by focusing the laser beam  510  inside the biosensors&#39; nanoslit. 
         [0032]      FIG. 2  illustrates the structure of a nanofluidic biosensor according to an embodiment of the invention. A layer  230  of a thickness ranging for example from 2 to 1,000 nm is deposited on a lower substrate  220  and structured using standard photolithography techniques in order to define the geometry of a nanoslit  223 . An upper substrate  210  is added, or stacked, onto the lower substrate  220 . At least one of the lower or upper substrates has to be compatible with the microscope objective in terms of transparency and optical aberrations. The nanofluidic biosensor also comprises an input lateral aperture  221  and an output lateral aperture  222 . The nanoslit  223  is in fluidic communication with said lateral apertures  222 ,  223  and links the input lateral aperture  221  with the output lateral aperture  222 . The nanoslit&#39;s height is determined by the spacing between the upper and the lower substrates  210 ,  220 , i.e. by the thickness of layer  230 . The layer  230  thus acts as a spacer to define the height of the nanoslit  223 . Several areas  240  inside the nanoslit  223  are functionalized for the detection of biomolecules. 
         [0033]      FIG. 3  illustrates the principle of detection and the cross-section of a nanoslit  223  of a nanofluidic biosensor according to an embodiment of the invention. The biosensor comprises a nanoslit  223  extending from a lateral input aperture  221  to a lateral output aperture  222 , thereby creating a fluidic connection between said apertures. A fluidic solution  300  containing fluorescently labeled specific biomolecules ( 311 ,  312 ,  313 ,  314 ,  315 ) and non-specific biomolecules  320  is filled into the biosensor, entering the biosensor from the input lateral aperture  221 , flowing through the nanoslit  223  and to the output lateral aperture  222  with a flow front  301 . In embodiments, various biomarkers ( 331 ,  332 ,  333 ,  334 ,  335 ,  336 ) are immobilized on various selectively functionalized structured surfaces  240 . Remaining surfaces  250  of the nanofluidic biosensor are for example treated to avoid unwanted functionalization of biomarkers or non-specific binding of biomolecules. During the filling of the output lateral aperture and thanks to Brownian motion, biomolecules ( 311 ,  312 ,  313 ,  314 ,  315 ,  320 ) interact with the biomarkers ( 331 ,  332 ,  333 ,  334 ,  335 ,  336 ) immobilized inside the nanochannel  223  and may create molecular complexes ( 341 ,  342 ,  343 ,  344 ,  346 ). The non-specific biomolecules  320  will also diffuse in the nanoslit  222  but will not form molecular complexes with the immobilized biomarkers ( 331 ,  332 ,  333 ,  334 ,  335 ,  336 ). 
         [0034]    In order to optimize the detection of a particular biomolecule, a detection volume  520  is focused inside the nanoslit  223  such that the intersection volume defined by the volume of the nanoslit  223  and the detection volume  520  is maximal, and located in one of the functionalized areas  240 . When excited by a laser beam, the immobilized fluorescently emitting complexes  341 ,  342 ,  343 ,  344 ,  346 , respectively, and the diffusing fluorescently emitting biomolecules  320  diffusing across the optical detection volume  520  are both detected by the optical system. 
         [0035]    The present invention thus allows detecting and/or measuring different biomarkers or different concentrations of the same biomarker in a single nanofluidic biosensor. This allows benefiting from the potential of multiplexing detections or of performing calibration. 
         [0036]      FIGS. 4 a  to 4 c    show a top-view cross section of a nanofluidic biosensor according to embodiments of the invention. A nanoslit  223  with a height defined by the thickness of a layer  230  is linked to an input lateral aperture  221  and to an output lateral aperture  222 . A solution  300  containing biomolecules is filling the biosensor with a flow front  301 . The nanoslit  223  comprises functionalized areas  240 ,  241 ,  242  and non-functionalized areas  250 . As depicted in  FIG. 4 a   , the functionalized areas may be lines or rectangles  240 , small polygon structures  241  as illustrated in  FIG. 4 b    or round or oval structures  242  as proposed in  FIG. 4 c   . Any other shape is however possible within the scope of the invention. 
         [0037]      FIG. 5  illustrates an example of another embodiment of the present invention. According to the illustrated example, a first functionalized area  240  is prepared with a first type of biomarker, and the other functionalized areas  243 ,  244 ,  245 ,  246 ,  247  with another type of biomarker and are used to calibrate the result obtained with the first functionalized area  240 . For example, a functionalized area  243  is 100% covered with a positive control biomarker and a functionalized area  247  is 100% covered with a negative control biomarker. Functionalized areas  244 ,  245  and  246  are only partially covered with a positive control biomarker. The partial coverage is for example performed by applying different patterns during the microfabrication of the functionalized areas  244 ,  245 ,  246  or by varying the concentration of the biomarker during the immobilization process. The laser spot  520  measuring the concentration of biomolecules is illustrated in the negative control functionalized area  247  in  FIG. 5 . 
         [0038]      FIG. 6  schematically illustrates the method of calibrating a nanofluidic biosensor according to embodiments of the invention. Firstly, the laser beam measures the specific detection in the first functionalized area (A). Then, it measures a positive control (B), several intermediate control n 1  (C), n 2  (D), nx (E) and finally a negative control (F). Microfabricated patterns of the functionalized areas (in black) are also represented for each step. 
         [0039]    In order to calibrate the nanofluidic biosensor and thus to obtain an accurate quantitative specific detection, a calibration curve is measured for different concentration of control biomarkers.  FIG. 7  illustrates a calibration curve. From measurements realized in the functionalized areas  243 ,  244 ,  245 ,  246  and  247 , the specific detection value (CA) is for example adjusted in function of the biosensor environment (background, maximum value, etc.) and of the characteristics of the solution  300 , such as for example the solution&#39;s viscosity, the density of biomolecules, etc. 
         [0040]    According to the present invention, the definition of precise functionalization areas in a nanoslit provides for great improvements to the multiplexed detection and calibration of biomolecules interacting or not with other immobilized biomolecules. Applications of the present invention for example include biomedical, biological or food analysis as well as fundamental studies in analytical and bioanalytical chemistry.