Abstract:
A liquid sample sensing device comprises a set of stackable liquid sample base units that are adapted to be stacked together to form multiple sample volumes that are substantially sealed, each with an electroacoustic member disposed therein having a surface adapted for binding to a particular target molecule. Top and bottom units are preferably attached to a closed loop of flexible tubing and a peristaltic pump. Individual electroacoustic members in the stack can each be adapted to bind with different target molecules, allowing for a multiple target assay. Alternately, a plurality of electroacoustic members can be adapted to bind with the same type of target molecule, thereby increasing the sensitivity of the sensing device. The base units are adapted such that sample liquid is caused to flow over and be in close proximity to the sensing surface of each electroacoustic member as the sample liquid flows from one base unit to the next, thereby increasing the efficiency of binding.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention relates to the detection and identification of infectious diseases. Specifically, the present invention relates to a method and apparatus for the detection of disease-causing particles such as bacteria, viruses and other particulate entities in liquid samples with extremely high sensitivity.  
         BACKGROUND OF THE INVENTION  
         [0002]    Quartz crystal microbalances (“QCM”) have been developed as sensitive chemical and biochemical sensing devices and can be used for the detection of disease-related particles such as viruses and bacteria in liquid samples (see e.g. Thompson, M. et al., Analyst Vol. 116, pp. 881-890, 1991; Rickert, J. et al., Biosensors &amp; Bioelectronics Vol. 12, pp. 567-575, 1997; Uttenthaler, E. et al., Biosensors &amp; Bloelectronics 16, 735-743, 2001). In this technology, a binding partner such as an antibody is attached to the surface of a small resonant quartz crystal with a mechanical resonance frequency typically in the 10 to 30 MHz region. If a disease-related particle binds to the antibody, the resonance frequency of the quartz crystal shows a very small shift, whereby such shift in frequency or a correlated phase shift between the electrical excitation and the mechanical vibration is an indication that an antibody-specific binding partner was present in the liquid sample.  
           [0003]    A significant improvement in the detection sensitivity of a QCM biosensor has been achieved by applying the technology of rupture event scanning (“REVS”), (see Dultsev, F. N. et al., Langmuir Vol. 16, 5036-5040, 2000; Cooper, M. A. et al., Nature Biotechnology Vol. 19, 833-837, 2001; WO 01/02857 A1 to Klenerman et al.). In the REVS technology, as in the classic QCM technology, a binding partner such as an antibody is attached to the surface of a small resonant quartz crystal with a mechanical resonance frequency typically in the 10 to 20 MHz region. The liquid sample containing bacteria or viruses is brought into contact with the activated crystal surface so that binding events can take place.  
           [0004]    After a 30-minute incubation period, the resonant quartz crystal is operated as close as possible to the fundamental mechanical resonance frequency, whereby the driving power for the quartz crystal is monotonously increased, until suddenly the binding between the binding partners is broken up. According to the inventors of REVS, the breaking or “rupture” event can be detected due to the emission of noisy sound waves with a preferred frequency spectrum around the third harmonic of the fundamental resonance frequency. The quartz crystal acts as a sensitive microphone, and the generated electrical signal is monitored via an electric resonance circuit tuned to a frequency close to the third harmonic of the fundamental resonance frequency of the crystal. The REVS technology has the potential of detecting the breaking-away of only a few binding partners, thereby enabling extremely sensitive detection.  
           [0005]    As mentioned above, a certain incubation time is required to bring the targets such as bacteria or viruses that are present within the liquid sample into contact with the activated crystal surface, so that binding events can take place. Movement of the targets usually takes place due to diffusion. The time, t, needed for a target to cross over a distance, d, via diffusion is given by the equation t=d 2 /2D, where D is the so-called diffusion coefficient. Assuming a typical diffusion coefficient D=7*10 −7  cm 2 /s for a large molecule, such target would need two hours to cross over a distance of 1 mm. Even larger distances may be required if the sample volume cannot be extremely small, which is often the case for medical samples having a low target concentration.  
           [0006]    One could try to resolve this problem by pre-concentrating the target molecules via centrifugation or similar process steps, but this requires significant extra effort and makes the detection more time-consuming and more expensive. One goal of the present invention is, therefore, to avoid the need for a pre-concentration step. Other goals of the invention are to allow for the handling of large sample volumes, and to allow for multiplexed detection within one detection device.  
         SUMMARY OF THE INVENTION  
         [0007]    The present invention is related to diagnostic sensing devices where a liquid sample has to come in contact with a sensing surface, or with a plurality of sensing surfaces. The invention is in particular of interest for sensing devices where the sample liquid contains the target molecules that have to be detected in low concentration, and where, therefore, a large sample volume has to be processed to achieve detection at all. The present invention avoids the need for a pre-concentration step, and also allows for the handling of large sample volumes, and to further increase sensitivity through multiplexed detection within one detection device. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0008]    The invention will be more readily understood with reference to the embodiments illustrated in the attached drawing figures, in which:  
         [0009]    [0009]FIG. 1( a ) shows a top unit that is employed in a detection apparatus according to an embodiment of the present invention;  
         [0010]    [0010]FIG. 1( b ) shows a base unit that is employed in a detection apparatus according to an embodiment of the present invention;  
         [0011]    [0011]FIG. 1( c ) shows a bottom unit that is employed in a detection apparatus according to the present invention;  
         [0012]    [0012]FIG. 2 illustrates a stack of base units, with a top unit at the top, and a bottom unit at the bottom according to an embodiment of the present invention;  
         [0013]    [0013]FIG. 3 shows a stack of base units and a pump, whereby the liquid sample is entered into the detection device through a fill and vent unit, and is re-circulated through the stack of base units by means of a peristaltic pump and a loop of flexible tubing;  
         [0014]    [0014]FIG. 4 illustrates the sample flow within a base unit that provides maximum contact of target molecules with the sensing surface according to an embodiment of the present invention;  
         [0015]    [0015]FIG. 5 illustrates schematically a complete apparatus according to an embodiment of the present invention, whereby the sensing surfaces within the base units are electro-acoustically interrogated according to an embodiment of the present invention;  
         [0016]    [0016]FIG. 6 shows experimental results illustrating the resonance spectrum of a 14.3-MHz piezo-electric quartz crystal under increasing liquid loading conditions;  
         [0017]    [0017]FIG. 7( a ) illustrates experimental data in the form of acoustic spectra with plastic PMMA beads attached to the piezo-electric quartz crystal; and  
         [0018]    [0018]FIG. 7( b ) illustrates experimental data in the form of acoustic spectra with no beads attached. 
     
    
       [0019]    In the drawing figures, it will be understood that like numerals refer to like features and structures.  
       DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0020]    The preferred embodiments of the invention will now be described with reference to the attached drawing figures. An apparatus according to the present invention comprises in its simplest form a base unit as shown in FIG. 1( b ). The base unit contains a housing  100  that has features allowing the stacking of such base units. One of said features has the form of flange  102 , whereby the inner opening of flange  102  has a diameter that is identical to the outer diameter of housing  100  on its opposite end  104 . Due to the presence of flange  102  in a first base unit, a second base unit can be attached to the first base unit with the opposite end  104  of the second base unit. By repeating this operation, a stack of base units  105  as shown in FIG. 2 can be built.  
         [0021]    A stack of base units as shown in FIG. 2 preferably contains identical base units, except for the top unit  107  and the bottom unit  109 . FIG. 1( a ) shows a top unit  107  in more detail, while FIG. 1( c ) shows a bottom unit  109  in more detail. In contrast to a base unit  105 , a top unit  107  has a lid  106  attached to flange  102 . Lid  106  contains an opening  108  for receiving sample fluid, and a second flange  110  allowing attaching a fluid conduit such as flexible tubing. While a base unit  105  comprises a simple through hole  112  in its lower end  104 , a bottom unit  109  contains a through hole  114  and a third flange  116  in its lower end  118 , also allowing attaching a fluid conduit such as flexible tubing.  
         [0022]    As indicated in FIGS.  1 ( a ),  1 ( b ), and  1 ( c ), base units  105 , top units  107 , and bottom units  109  each contain a body  120  with a sensing surface, whereby the sensing surface has preferably been prepared to allow for specific binding of target molecules. In the example of FIG. 1, body  120  represents a piezo-acoustic crystal that can be brought into mechanical vibration by means of an electrical RF excitation. For that purpose, crystal  120  contains electrodes  122  and  124 , and crystal  120  is mounted within a unit by means of two support wires  126  and  128  that serve also as electrical conductors for bringing the RF field to the crystal, and picking up an electrical detection signal from the crystal. Support wires  126  and  128  are preferably connected to feed-throughs  130  and  132  that extend through a unit&#39;s housing  100  to the outside of the unit.  
         [0023]    It should be understood by those of ordinary skill in the art, that body  120  is presently contemplated to embody the best mode of the invention as an electro-acoustic body such as a piezo-electric device. However, a broad range of sensing bodies are considered to be within the scope of the present invention, including bodies with surfaces prepared for binding to specific target molecules, as well as bodies with surface not intended for binding. Furthermore, sensing bodies that generate a sensor output signal based on changes in the bodies&#39; electrical conductivity, and electro-chemical sensing bodies are specifically considered to be within the scope of the present invention. Other types of sensing bodies should be considered within the scope of the invention as well.  
         [0024]    No matter if one base unit  105  is used, or if a number of base units  105  is connected together to build a stack of such units as shown in FIG. 2, the liquid sample is guided into the unit(s) via tubing  134 , which is illustrated in FIG. 3. A closed loop of such tubing  134  is provided to allow for continuous re-circulation of sample fluid through the unit(s)  136 . The fluid circulation is preferably achieved by utilizing flexible tubing, and by mounting a peristaltic pump  138  onto the closed tubing loop. A fill-and-vent unit  140  with a removable lid  142  allows bringing the liquid sample into the system. As indicated by arrows  144  in FIG. 3, the liquid sample enters the stack of base units at flange  110 . After passing through all units, the liquid sample leaves the stack of units at flange  116 , passes peristaltic pump  138 , and re-enters the stack of units again at flange  110 .  
         [0025]    Due to the narrow entrance at through hole  108  and exit  112  of the base unit as shown in FIG. 4, the liquid sample containing the target molecules in low concentration is guided across the sensing surface or surfaces of crystal(s)  120 , which is indicated by arrows  146  and  148 . This more readily brings the few target molecules in close proximity to the sensing surface, resulting in more efficient specific binding during the continuous re-circulation of the liquid sample. Therefore, no pre-concentration step such as centrifugation is required.  
         [0026]    [0026]FIG. 5 illustrates schematically a complete apparatus according to the present invention, whereby the sensing surfaces within the stack of base units  136  are interrogated by applying principles of electro-acoustics.  
         [0027]    The liquid sample containing the target molecules of interest in low concentration is introduced into the apparatus at fill-and-vent unit  140 , which has a removable lid  142 . A closed loop of tubing  134  connects fill-and-vent unit  140  with the stack of units  136 , and with a peristaltic pump  138 . Preferably, tubing  134  is made out of flexible material. A computer  150  is connected with an electronic signal source  152 , which is in turn connected with a connector and directional coupler  154  at the feed-throughs described and/or shown in FIGS.  1  to  4 . Connector and directional coupler  154  guides an RF signal from signal source  152  towards piezo-acoustic crystals  120  within the stack of units  136 . Connector and directional coupler  154  is also connected with the input of an RF receiver  156 , and the output of RF receiver  156  is connected to computer  150 . Finally, computer  150  is connected to a pump driver  158  that in turn is connected to peristaltic pump  138 . Arrows  144  in FIG. 5 show how the liquid sample circulates within tubing  134 .  
         [0028]    In operation, after the liquid sample has been introduced into the system, computer  150  triggers pump driver  158  and, consequently, peristaltic pump  138 . Due to this action, the liquid sample starts circulating within the system and, as shown in FIG. 4, passes the sensing surfaces of piezo-electric crystals  120  within stack  136  in close proximity, which enhances the probability for specific binding on said surfaces. Preferably, computer  150  and pump driver  158  are programmed to periodically reverse the flow or liquid to further enhance binding efficiency and to reduce dead-zones in the liquid, such as might develop in corners of housing  100 . After a predetermined incubation time period (e.g. 30 minutes) computer  150  activates signal source  152 , which results in an oscillation of said sensing surfaces on crystals  120 .  
         [0029]    If binding events have taken place during said incubation time period, target molecules will break away from said sensing surfaces due to the surfaces&#39; oscillation, which results in an acousto-electric signal that is being detected by RF receiver  156 .  
         [0030]    [0030]FIG. 6 shows experimental results illustrating the resonance spectrum of a 14.3-MHz piezo-acoustic quartz crystal under increasing liquid loading conditions. FIG. 6 illustrates the fact that if only a small percentage of the sensing surface comes into contact with the liquid sample (0.3 microliter water load), then the resonance curve is very narrow. If the crystal is completely immersed in liquid, as is the case in an apparatus according to the present invention, then the resonance curve becomes wider, but it is still narrow enough to allow for acousto-electric detection of binding events.  
         [0031]    [0031]FIG. 7( a ) illustrates experimental data in the form of acoustic spectra with plastic PMMA beads attached to the above-mentioned piezo-acoustic quartz crystal. FIG. 7( b ) illustrates experimental data in the form of acoustic spectra with no beads attached. As illustrated in FIG. 7( a ), if targets such as small beads had been bound to the sensing surface, peaks  160  with amplitudes larger than noise peaks are observed. FIG. 7( b ) illustrates lower amplitude noise peaks  162 .  
         [0032]    In an apparatus according to the present invention, the stack of base units  136  may comprise base units having identical sensing surfaces, or may comprise base units having sensing surfaces that have been activated for the binding of different target molecules on different crystals. Including sensing surfaces activated for binding to different target molecules achieves multiplexed analyte detection. Including identical sensing surfaces in the stack  136  results in an increased overall size of sensing surface, and, consequently, in an improved detection limit for low analyte concentration.  
         [0033]    The base units for an apparatus according to an embodiment of the present invention are easily manufactured, and advantageously can be produced in large numbers at low cost. The sensing surfaces are easily prepared for single base units, before more than one base unit are connected together. Due to the open flange design, before connecting them, the interior of a base unit is readily accessible, which also makes the mounting of crystals  120  very easy. After preparing groups of base units, where each group has been activated for a particular target molecule of interest, members from multiple groups can be connected together to form stacks of base units  136  that represent specific multi-analyte assays.  
         [0034]    The re-circulation of the sample liquid can be performed over an extended incubation time period to allow for binding of all target molecules that are present within the sample volume. It is possible and advantageous to reverse the flow direction for the liquid sample periodically to avoid trapping of target molecules in corners or other dead zones of the base units.  
         [0035]    In a modification of the invention, the fill-and-vent unit  140  shown in FIGS. 3 and 5 can become part of lid  106  in a top unit  107  shown in FIGS.  1  to  4 .  
         [0036]    A typical electro-acoustic crystal  120  according to a preferred embodiment of the invention has the shape of a disc with a thickness of approximately 0.1 mm, and a diameter between 5 and 10 mm. Based on this, the volume of a base unit can be as low as 100 microliters. This would result in an overall sample volume around 1 ml for a stacked detection device  136  containing ten base units.  
         [0037]    The closed loop tubing  134  is preferably made out of a flexible plastic material, but of course it will be readily understood by those of skill in the art that a stiff tubing material or any other suitable material could be used without departing from the spirit of the invention. Those of skill in the art will also recognize that peristaltic pump  138  can be replaced with any other kind of suitable pumping device.  
         [0038]    It will be appreciated that an apparatus according to the present invention is not limited to the use of piezo-acoustic crystals. It would still be within the scope of the invention to use piezo-acoustic plastic materials, piezo-acoustic foils, piezo-acoustic microstructures on solid substrates, or the like.  
         [0039]    While the invention herein disclosed has been described by means of specific embodiments and applications thereof, numerous modifications and variations can be made thereto by those skilled in the art without departing from the scope of the invention as set forth in the claims.