Abstract:
A nanofluidic biosensor system ( 200 ) comprising a bottom substrate ( 120 ) and a top substrate ( 110 ) between which are defined an input lateral aperture ( 210 ), a nanoslit ( 230 ) which contains at least one functionalized area ( 231 ) and an output lateral aperture ( 220 ) or an internal reservoir ( 221 ), said biosensor system ( 200 ) being adapted to let a solution containing biomolecules ( 320 ) enter the input lateral aperture ( 210 ) and successively pass through said nanoslit ( 230 ) and said output lateral aperture ( 220 ) or internal reservoir ( 221 ); said biosensor system ( 200 ) furthermore comprising a gas evacuation subsystem ( 150 - 155 ) which is located between said nanoslit ( 230 ) and the biosensor external environment.

Description:
FIELD OF INVENTION 
       [0001]    The present invention relates to nanofluidic biosensors with at least one lateral aperture. This kind of biosensor may advantageously be used for accurate rapid quantification of biomedical and biological samples. 
       BACKGROUND OF THE INVENTION 
       [0002]    Nanofluidic biosensors are defined as fluidic systems with nanometer-sized confinements and/or lateral apertures. Applications include quantification of the presence of biomolecules in a solution. A majority of the current nanofluidic biosensor developments are intended for bioengineering and biotechnology applications. In the scope of this invention, biosensors are used to quantify the presence of biomolecules in 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 IB2010/050867 discloses their use with simple optical systems and PCT application IB2012/050527 discloses the method to decrease the incubation time and to increase the sensitivity of the described biosensors. The diffusion of biomolecules in these configurations are slow and require either long waiting times to attain stable measurement conditions or highly concentrated solutions for the observation of the biomolecular interactions. 
         [0004]    Biomarkers, also called biological markers, are substances used as specific indicators for detecting the presence of 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]    Current practices 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 are fluorescence, colorimetry, radioactivity, phosphorescence, bioluminescence and chemiluminescence. Functionalized magnetic beads can also be considered as labeling techniques. Labeled techniques advantages are the sensitivity in comparison to label-free methods and the molecular recognition due to specific labeling. 
         [0007]    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. 
         [0008]    Enzyme linked immunosorbent assay (ELISA) is an important biochemical technique mainly used to detect the presence of soluble biomolecules in serum, 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 is time consuming. 
         [0009]    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 and do not allow high throughput samples testing. 
       OBJECTIVES 
       [0010]    It is an object of this invention to improve the variability of rapid nanofluidic biosensors, which do not require complex manipulations. 
         [0011]    Still another object of the invention is to create crossing-through galleries allowing the evacuation of gas that may be trapped inside the biosensor during its filling by the solution to analyze. 
         [0012]    Still another object of the invention is to enhance the sensitivity of the detection by forcing a higher volume for solution to flow through the biosensor entry (nanoslit). 
       SUMMARY OF THE INVENTION 
       [0013]    This invention is based on the discovery that several air bubbles can appear in a nanofluidic biosensor if the filling front is not perfectly homogenous. In order to evacuate the air trapped, a gas evacuation subsystem allowing the air to exit the biosensor has been invented. 
         [0014]    This invention is also based on the discovery that removing air bubbles is strongly improving the variability inter-biosensors as well as the sensitivity. 
         [0015]    This gas evacuation subsystem according to the invention may be made of porous material. 
         [0016]    Furthermore, this invention highlights the possibility to locally structure one or both of the biosensor substrates in order to define a gas evacuation subsystem. 
         [0017]    In the present text the term “gas evacuation subsystem” has to be understood as any system which may be used for the intended purpose. For instance it may be made of pores, crossing-through holes or slits. 
         [0018]    In the scope of this invention, nanofluidics is used because of its high surface-to-volume ratio, meaning that the surfaces included in the detection volume, maximize the probability of the interactions between biomolecules and immobilized biomarkers on surfaces. It also strongly reduces the background signal of the solution due to the small portion of substrate that is within the detection volume. 
         [0019]    The invention therefore relates to a biosensor as defined in the claims. 
         [0020]    It also relates to an assembly and a method using said biosensor. Some non-limiting examples of the invention are presented in the following chapters. Some of those examples are illustrated. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0021]      FIG. 1 a    is a perspective view of a nanofluidic biosensor system composed of a bottom substrate  120  a spacer layer  130  and a top substrate  110  containing structured or non-structured crossing pores, and a lateral aperture  210 . A solution  300  containing fluorescently-labeled biomolecules is deposited by a pipet system  400  in a way that the solution is entering inside the biosensor from the lateral aperture  210 . An optical system  500  based on a laser beam  510  is typically used for the measurement. 
           [0022]      FIG. 1 b    is a perspective view of a nanofluidic biosensor system composed of a bottom substrate  120  a spacer layer  130  and a top substrate  111  containing structured or not structured crossing pores at defined position  150 , and a lateral aperture  210 . A solution  300  containing fluorescently-labeled biomolecules is deposited by a pipet system  400  in a way that the solution is entering inside the biosensor from the lateral aperture  210 . An optical system  500  based on a laser beam  510  is typically used for the measurement. 
           [0023]      FIG. 2 a    shows a top view cross section of the substrate  111  composing a nanofluidic biosensor. An input lateral aperture  210 , a nanoslit  230  and an output aperture  220  is composing the fluidic system. The measurement area  231  is defined inside the nanoslit. Once the system has reached its equilibrium, solution may be found in the input lateral aperture  301 , the nanoslit  302  and the output lateral aperture  303 . Gas bubble  350  may be formed if the solution flow front actuated by the liquid-driving component  140  is not perfectly uniform. 
           [0024]      FIG. 2 b    shows a top view cross section of the substrate  111  composing a nanofluidic biosensor. An input lateral aperture  210 , a nanoslit  230  and an output aperture  220  is composing the fluidic system. The measurement area  231  is defined inside the nanoslit  230 , and the substrate crossing-through pores  150  are defined directly inside the output lateral aperture  220 . A liquid-driving component  140  is also present inside the output lateral aperture  220 . Once the system has reached its equilibrium, solution may be found in the input lateral aperture  301 , the biosensor entry (nanoslit)  302  and the output lateral aperture  303 . Gas bubbles are not present as the gas can exhaust through the pores system  150 . 
           [0025]      FIG. 3 a    shows a lateral cross section of the nanofluidic biosensor defined by two substrates  110  and  120 , and composed by an input lateral aperture  210  and an output lateral aperture  220  linked together by a nanoslit  230 . The output lateral aperture  220  may contain a liquid driving system  140 . The substrate  110  is entirely porous with crossing-through pores. 
           [0026]      FIG. 3 b    shows a lateral cross section of the nanofluidic biosensor defined by two substrates  111  and  120 , and composed by an input lateral aperture  210  and an output lateral aperture  220  linked together by a nanoslit  230 . The output lateral aperture  220  may contain a liquid driving system  140 . The substrate  111  may be structured to be locally porous with crossing-through pores  150 . 
           [0027]      FIG. 3 c    shows a lateral cross section of the nanofluidic biosensor defined by two substrates  111  and  120 , and composed by an input lateral aperture  210  and an internal reservoir  221  linked together by a nanoslit  230 . The internal reservoir  221  may contain a liquid driving system  140 . The substrate  111  may be structured to be locally porous with crossing-through pores  150 . 
           [0028]      FIG. 3 d    shows a lateral cross section of the nanofluidic biosensor defined by two substrates  111  and  120 , and composed by an input lateral aperture  210  and an output lateral aperture  220  linked together by a nanoslit  230 . The output lateral aperture  220  may contain a liquid driving system  140 . The substrate  111  may be locally structured with crossing-through holes or slits  151 . 
           [0029]      FIG. 3 e    shows a lateral cross section of the nanofluidic biosensor defined by two substrates  111  and  120 , and composed by an input lateral aperture  210  and an output lateral aperture  220  linked together by a nanoslit  230 . The output lateral aperture  220  may contain a liquid driving system  140 . The substrate  121  may be structured to be locally porous with crossing-through pores  150 . 
           [0030]      FIG. 4  shows a lateral cross section of the nanofluidic biosensor defined by two substrates  111  and  120 , and composed by an input lateral aperture  210  and an output lateral aperture  220  linked together by a nanoslit  230 . Only one of the substrates is locally structured by area  231  that is functionalized by biomarkers  310  and other areas  203  that prevent that functionalization. Reagent solution  300  containing biomolecules enter the nanoslit  230  from the input lateral aperture  210  to the output lateral aperture  220  and is actuated by the internal driving component  140 . When reaching the output lateral aperture  220 , the solution  300  containing the molecules to detected  320  and other molecules  330  is filling the structured cross-through pores  152 . The figure shows fully filled pores  153 , pores that are being filled  154  and pores to be filled  155 . The laser beam  510  monitors the concentration of the immobilized biomolecules  340  in the detection volume  520 . 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0031]    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. 
         [0032]    As used herein, the terms “nanoslit” is intended to be a generic term, which means well-defined microfabricated structure with at least 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 nanoslits with a height lower than few microns, because of the range of the detection volume of the optical system that are typically in the same order of magnitude. 
         [0033]    As used herein, the term “lateral aperture” is intended to be a generic term, which includes for example (but not limited to) input and output channels. 
         [0034]    As used herein, the term “internal reservoir” is intended to be a generic term, which includes for example (but not limited to) spaces that don&#39;t have a direct access to a lateral aperture, but being in contact with the gas evacuation system. 
         [0035]    The present invention aims to enhance the filling of the output lateral aperture  220  or the internal reservoir  221  thanks to a system of gas evacuation that guarantees a low interbiosensor variability of biomolecules concentration measurement. As shown in  FIG. 1 a    and  FIG. 1   b,  a nanofluidic biosensor composed of a substrate  110  or  111  and a substrate  120  sandwiched together with a spacer  130 , and having an input lateral aperture  210 , is immobilized above an optical unit  500 . The substrate  110  may be porous, and the substrate  111  may have locally structured cross-through pores, in order to allow the gas evacuation during the filling of the nanofluidic biosensor. A mix solution  300  containing the biomolecules of interest is disposed at the input lateral aperture  210  by a pipet system  400 . Finally, an optical unit  500  is used to measure the biomolecular interactions inside the biosensors  200  by focusing the laser beam  510  inside the biosensors nanoslit. 
         [0036]      FIG. 2 a    and  FIG. 2 b    illustrate top views of half a nanofluidic biosensor composed of a substrate  111  containing an input lateral aperture  210  and an output lateral aperture  220 , linked together by a nanoslit  230 . In  FIG. 2 a   , the output lateral aperture  220  is not designed with a gas evacuation system whereas in  FIG. 2 b   , the output lateral aperture  220  is structured with a gas evacuation system  150  which can be obtained with a local dry or wet chemical etching process in order to obtain cross-through pores or holes. When a solution containing biomolecules is deposited at the input lateral aperture  210 , the solution will fill firstly the input lateral aperture  301 , fill the nanoslit  302  and then finally fill the output lateral aperture  303 . Despite of an excellent liquid-driving system  140 , the filling of the output lateral aperture  220  is rarely uniform. Typically, the solution may reach uniformly the border of the aperture  220  and after stopping due to surface tensions equilibrium, it can block gas inside the output lateral aperture  220 . This can lead to the apparition of gas bubbles  350  due to the fact that gas cannot exhaust by the lateral aperture  210  or  220 . As depicted in  FIG. 2 b   , gas may exhaust the system through cross-through pores  150 , avoiding the apparition of gas bubbles and guaranteeing the full filling of the output lateral aperture, and thus ensuring low interbiosensor variability. 
         [0037]      FIGS. 3 a , 3 b , 3 c , 3 d  and 3 e    illustrate different configurations of nanofluidic biosensor with lateral apertures and gas evacuation system according to the invention. The system, presented as lateral cross views, is composed of a nanoslit  230  linking an input lateral aperture  210  with either an output lateral aperture  220 , either an internal reservoir  221 . A driving component  140  is structured next or inside the output lateral aperture  220 . In  FIG. 3 a   , the biosensor is composed of a substrate  110  that is entirely porous with cross-through galleries.  FIG. 3 b    presents an alternative where the substrate  111  is locally structured with porous cross-through galleries  150 .  FIG. 3 c    illustrates the case where there is no output lateral aperture as the gas can exhaust through the porous areas  150  locally structured in the substrate  111  as the solution is filling the system.  FIG. 3 d    presents the case where the substrate  111  is locally structured with crossing-through holes  151  with nano-, micro- or millimeter dimensions. Finally  FIG. 3 e    illustrates that the gas evacuation system  150  may be structured on the other substrate  121 , or on both substrates  112  and  121 . 
         [0038]      FIG. 4  illustrates the principle of detection and the cross-section of a biosensor with lateral apertures and gas evacuation system according to the invention. The system presented as a lateral cross view is composed of a nanoslit  230  linking an input lateral aperture  210  with an output lateral output aperture  220 . A liquid-driving component  140  is located next or inside the output lateral aperture  220 . The gas evacuation system  152  is also present in the output lateral aperture  220 . First, biomarkers  310  are immobilized on selectively functionalized nanoslit surfaces of one or both substrates  111  and  120 . The other nanoslit surfaces and the lateral aperture surfaces may be protected by the deposition of a non-functionalized layer  203  in order to prevent non-specificity. Once the solution  300  containing the fluorescently labeled specific biomolecules  320  and non-specific biomolecules  330  is deposited at the input lateral aperture, it fills the system from the input lateral aperture  210  to the output lateral aperture  220  through the nanoslit  230 . After filling the nanoslit  230  and when reaching the liquid-driving component  140 , the solution  300  fills the output lateral aperture  220 . When flowing through the nanoslit  230 , and thanks to Brownian motion, specific biomolecules  320  interact with the biomarkers  310  immobilized inside the nanoslit  230  and form molecular complexes  340 . The non-specific biomolecules  330  will also flow through the nano slit  230  but will not form molecular complexes with the immobilized biomarkers  310  and will continue into the output lateral aperture  220 . When the solution  300  is in contact with the gas evacuation system  152 , the liquid will enter into crossing-through pores  155 , with a transitory filling state  154 , until it has completely filled the pores  153 . Finally, after having reached an equilibrium state, the immobilized fluorescently emitting complexes  340  and the diffusing fluorescently emitting biomolecules  330  diffusing across the optical detection volume are excited by the laser beam  510  and both detected by the optical system. 
         [0039]    According to the present invention, the device offers great improvements in variability and sensitivity for the detection, enumeration, identification and characterization of biomolecules interacting or not with other immobilized biomolecules. Applications of the present invention can cover biomedical, biological or food analysis as well as fundamental studies in analytical and bioanalytical chemistry.