Abstract:
The present invention reports a novel microfluidic analyzer for the high-throughput, label-free measurement of particles suspended in a fluid. 
     The present invention employs the resistive pulse technique (RPT) which affords very high electrical bandwidth for the device, which surpasses that of currently available systems and devices. Further, devices in accordance with the present invention are fabricated with very simple microfabrication technologies, making the present invention more cost efficient and easier to manufacture than currently available devices.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit under 35 U.S.C. Section 119(e) of co-pending and commonly-assigned U.S. Provisional Patent Application Ser. No. 61/437,942, filed on Jan. 31, 2011, by Jean-Luc Fraikin and Andrew N. Cleland, entitled “HIGH THROUGHPUT LABEL FREE NANOPARTICLE DETECTION AND SIZE ASSAY,” which application is incorporated by reference herein. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT 
       [0002]    This invention was made with Government support under Grant/Contract No. U01 HL080718 awarded by the National Institutes of Health. The Government has certain rights in this invention. 
     
    
     BACKGROUND OF THE INVENTION 
       [0003]    1. Field of the Invention 
         [0004]    The present invention relates generally to microfluidics, and specifically to a high-throughput, label-free measurement of nano- and micro-particles suspended in a fluid. 
         [0005]    2. Description of the Related Art 
         [0006]    Counting and measuring particles suspended in a conducting fluid has been taking place in biomedical applications for almost fifty years. A resistive pulse technique has been used to size and count microparticles suspended in conducting fluid since Coulter first patented such in U.S. Pat. No. 2,656,508, which is herein incorporated by reference. Many incremental improvements to this original concept have been used since, and include modifications of the approach in order to achieve nanoparticle (dia.&lt;1 micrometer) detection and measurement. 
         [0007]    Of these improvements, two approaches seem to have provided promising results. The first, set forth in U.S. Pat. No. 7,279,883, which is incorporated by reference herein, uses a four-wire sensing technique, which is inherently limited in bandwidth by the current-sensing capabilities of the electronics. 
         [0008]    The second approach, described in Leif et al. (Clin. Chem. 8: 853 (1973); J. Histo. Chem. 22:626 (1974), J. Histo. Chem. 27:225 (1979)), which is incorporated by reference herein, describe the use of a fluidic voltage divider for the detection of microparticles (dia.&gt;1 micrometer) and cells. These instruments are not nanofabricated, and are instead very complex, three-dimensional large structures which are costly to fabricate and operate. 
         [0009]    It can be seen, then, that there is a need in the art for a non-bandwidth-limited system to measure and detect micro- and nanoparticles in fluid. It can also be seen that there is a need in the art for systems and devices that can measure and detect micro- and nanoparticles, systems and devices that are easier to fabricate and operate than those currently available. 
       SUMMARY OF THE INVENTION 
       [0010]    The present invention describes a novel microfluidic analyzer for the high-throughput, label-free measurement of micro- and nanoparticles suspended in a fluid. 
         [0011]    A device in accordance with one or more embodiments of the present invention comprises a microfluidic channel, which directs the flow of analyte through the electrical sensor and is designed to maximize the measurement bandwidth of the device; and a sensor, which comprises two or more voltage-bias electrodes and one or more readout electrode(s) embedded in the microchannel. 
         [0012]    The present invention employs the resistive pulse technique (RPT) implemented in a manner that affords very high electrical bandwidth for the device, which surpasses that of currently available systems and devices. Further, devices in accordance with the present invention are fabricated with very simple microfabrication technologies, making the present invention more cost efficient and easier to manufacture than currently available devices. 
         [0013]    An apparatus for detecting particles in a fluid in accordance with one or more embodiments of the present invention comprises a first electrode, a sensing electrode, a fluid resistor between the first electrode and the sensing electrode, a second electrode, and a constriction between the sensing electrode and the second electrode, wherein a bias voltage is applied between the first electrode and the second electrode to create a current through the fluid, such that a size and a number of a particle is sensed by a change in voltage sensed at the sensing electrode. 
         [0014]    Such an apparatus further optionally comprises a filter element such that the particles are sensed by the apparatus and undesired particles are blocked by the filter element from being sensed by the apparatus, a bandwidth of the apparatus being greater than 100 kHz, the fluid resistor and the constriction comprise a voltage divider in the apparatus, the sensing electrode being fabricated using lithographic techniques, the bias voltage being a constant voltage, the apparatus sensing different sizes of particles in the fluid, a plurality of constrictions, and an integrated filter element. 
         [0015]    A method of detecting particles in a fluid in accordance with one or more embodiments of the present invention comprises applying a bias voltage between a first electrode and a second electrode, filtering the fluid prior to entering any smaller constrictions, restricting the fluid in a constriction between the first electrode and the second electrode, and sensing a change in voltage at a point between the constriction and the second electrode, wherein the change in voltage is proportional to a volume filling fraction of a particle in the constriction. 
         [0016]    Such a method further optionally includes a bandwidth of the apparatus being greater than 100 kHz, creating a voltage divider in the apparatus using the constriction, the sensing electrode being fabricated using lithographic techniques, the bias voltage being a constant voltage, sensing different sizes of particles in the fluid, and constricting the fluid in a plurality of constrictions. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]    Referring now to the drawings in which like reference numbers represent corresponding parts throughout: 
           [0018]      FIGS. 1A-1C  illustrate device schematics, detector response, and operation of one or more embodiments of the present invention. 
           [0019]      FIG. 1D  illustrates a filtration system in accordance with one or more embodiments of the present invention. 
           [0020]      FIG. 2  illustrates a process chart in accordance with one or more embodiments of the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0021]    In the following description of the preferred embodiment, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. 
       Overview 
       [0022]      FIGS. 1A-1C  illustrate device schematics, detector response, and operation of an embodiment of the present invention. 
         [0023]      FIG. 1A  illustrates system  100 , with electrode H  102 , electrode L  104 , and sensing electrode  106 . Between electrode H  102  and sensing electrode  106  is a constriction  108 , which can be fabricated with cross-sectional dimensions ranging from nanometers to micrometers, and between sensing electrode  106  and electrode L  104  is a fluid resistor  110 . Particles  112  suspended in a pressure driven solution  114 , typically a saline solution, are driven along the channel between electrode H  102  and electrode L  104  through the constriction  108 , and possibly although not necessarily through the fluid resistor  110 . Electrodes H  102  and L  104  are voltage-biased, and changes in the electrical potential of the fluid  114  adjacent to the constriction  108  are detected by the sensing electrode  106 . The passage of particles through the constriction  108  changes the effective electrical resistance of the fluid  114  contained in the constriction  108 , which combined with the voltage bias provided by electrodes H  102  and L  104 , changes the electrical potential of the sensing electrode  106 . The optional passage of particles through the fluid resistor  110  does not appreciably change the corresponding fluidic resistance, so that no corresponding signal is generated in sensing electrode  106 , in contrast to passage of the particles through the constriction  108 . The sensing electrode is typically, though not necessarily, lithographically manufactured. Although shown in the center between electrodes H  102  and L  104 , sensing electrode  106  can be placed anywhere in the region between electrodes H  102  and L  104 , so long as the changes in the effective electrical resistance of the fluid  114  contained in the constriction  108  can be properly and/or accurately detected by sensing electrode  106 . Further, fluid resistor  110  and constriction  108  can be placed anywhere in the system  100 . 
         [0024]      FIG. 1B  illustrates an electrical equivalent circuit to the system  100  shown in  FIG. 1A . Typically, a constant bias voltage Vh  116  is applied to electrode H  102 , and a constant bias voltage Vl  118  is applied to electrode L  104 , although varying bias voltages can be applied if desired. The application of a bias voltage difference between the electrodes  102  and  104  causes an ionic current to flow in the fluid  114 . Resistors Ra  120  and Rb  122  represent the electrical resistance of the fluid in the constriction  108  and the fluidic resistor  110  respectively. The sensing electrode  106  is capacitively-coupled to the fluid through the electric double layer capacitance Cdl  124 . The circuit elements Ra  120 , Rb  122 , and Cdl  124  are measurable directly. The output voltage Vout  126  is measured as the output from sensing electrode  106 . 
         [0025]    The ionic current between electrodes  102  and  104  creates voltage drops across the constriction  108  in the fluid channel and the fluid resistor  110  which typically presents an approximately equal fluidic electrical resistance but has a larger volume. The sensing electrode  106  is embedded in the fluid between the constriction  108  and the fluid resistor  110 . When a particle enters the constriction  108 , it alters the ionic electrical current and, because of the voltage division between the two fluidic resistances, changes the electrical potential of the sensing electrode  106 . The time-varying nature of the particle passage through the constriction makes the electrical potential at the sensing electrode  106  time-varying as well, which allows this signal to be electrically coupled through the electrical double layer capacitance Cdl  124 . The signal  126  can be amplified with an operational amplifier or other type of amplifier as desired to properly measure the output voltage  126 . 
         [0026]    The present invention optionally includes filtration systems  130  manufactured in a size commensurate with the constriction  108  to avoid clogging by large particles and to allow for longer uninterrupted run times. The filtration systems can be scaled in size and can be manufactured using existing fabrication techniques currently used elsewhere in the device. Although shown near fluidic resistor  110 , filtration system  130  can be placed anywhere in the system  100  without departing from the scope of the present invention. Further, the system  100  can be optimized in the channel design to allow automation of the channel filling process. Such optimization can include specialized purpose-built electronics, possibly in miniaturized electronic formats, and microfluidic features that can be manufactured using the techniques shown in the present invention. 
         [0027]    The system  100  of the present invention can also comprise a sorting mechanism, which can be a part of fluidic resistor  110  or optional filtration system  130 , or a separate sorting mechanism if desired, so that particles meeting selection criteria may be separated from a fluid  114  mixture. 
         [0028]    The present invention can be manufactured in an array geometry, allowing scaling to large numbers of parallel detection channels (e.g., parallel constrictions  108  and/or parallel fluid resistors  110  and/or parallel filtration systems) which can be employed in the present invention to increase the range of particle sizes that can be detected with the sensor electrode  106  (or parallel sensor electrodes  106 ). Such approaches can also be used to develop size-based antibody assays, and to size and measure the concentration of particles in solution, and to determine other properties of the particles, such as electrical charge. Related art approaches currently do not provide the size resolution and direct measurement of particle concentration as is possible through the present invention. 
         [0029]      FIG. 1C  illustrates a measurement of Vout  126  as a function of time as a single particle of nominal diameter 117 nm traverses the constriction  108 , showing excellent time resolution and signal-to-noise ratio. The peak voltage change ΔVout is proportional to the bias voltage difference (Vh  116 -Vl  118 ) and to the volume filling fraction of the particle in the constriction  108 . 
         [0030]      FIG. 1D  illustrates a filtration system in accordance with one or more embodiments of the present invention. 
         [0031]    Filtration system  130  is shown, with individual elements  132  of the optional filter shown in detail section  134 . Other optional filtration systems are possible within the scope of the present invention as needed for a specific application; some applications may not need or desire any filtration system  130  within system  100 . 
       Analyzer Features 
       [0032]    The analyzer of the present invention has several advantages over analyzers of the related art. First, the analyzer of the present invention is fabricated using a completely scalable process, allowing parallel lithographic or other manufacture of large numbers of identical or similar devices. The fluidic components of the device can be made of molded poly-(dimethylsiloxane) (PDMS), using well-established molding or nano-imprint lithography. The sensing electrode can be made of gold, patterned on a glass substrate using single-layer optical lithography. Other materials can be used without departing from the scope of the present invention. The scalable fabrication process of the present invention allows for very low-cost fabrication of analyzer chips, to the point where the chip is disposable. 
         [0033]    Another characteristic of the device of the present invention is a high-throughput detection capability, which results from the type of signal transduction. Rather than cumbersome four-wire techniques, or large devices that are complex to build and operate, the fluidic network of the present invention acts as a balanced voltage divider, so a voltage output signal is produced using a voltage bias. 
         [0034]    The analyzer of the present invention also has excellent size resolution. Rather than particle size distributions being extracted from bulk measurements such as dynamic light scattering or disk centrifugation, which methods are inherently ensemble-averaging and limited in size resolution and unable to detect rare particles, the present invention has better size and particle counting resolution through the use of the constriction and simple electronic design, with the further ability to perform sophisticated population analysis using the individual-particle detection capability. 
         [0035]    Related art approaches are also inherently limited in bandwidth to audio frequencies (less than a few kilohertz) by the requirement of current-sourcing, or current-sensing electronics. These limitations prevent the adoption of RPT methods as practical analytic tools for high-throughput detection of particles smaller than approximately 1 micrometer (um) in diameter. The present invention allows for significantly higher detection bandwidth (approximately 1000 times greater at 650 kHz bandwidths) allowing for a much greater throughput, sufficient to detect particles with diameter&lt;100 nm at rates exceeding 500,000 particles per second, so that large sample numbers and significant population statistics can be achieved in much shorter sampling times. The present invention is capable of bandwidths greater than 100 kHz, and has been experimentally shown at 650 kHz, which is far in excess of the related art techniques. 
         [0036]    Other voltage divider techniques of the related art are very complex, three-dimensional large structures, which limit the commercial use and application of such devices. The simple, scalable fabrication techniques of the present invention reduce costs and make such devices more available to a wider application in industry and scientific research. 
         [0037]    Although individual particles can be studied by electron microscopy, this approach is costly, slow, and not practical for collecting large population statistics. Because the present invention measures each particle individually as it passes through the sensing volume, it has inherently finer size resolution than bulk measurement techniques and can detect rare particles. The present invention has experimentally distinguished subcomponents of a polydisperse particle mixture with diameters larger than about 30-40 nm, a task that dynamic light scattering, the most commonly used technique for sizing nanoparticles below 1 um, cannot accomplish. 
         [0038]    The present invention allows for analyzers to be used in several applications, e.g., size distribution analysis and concentration measurements of synthetic and biological particle mixtures, particle surface chemistry analysis using a change in size resulting from particle agglomeration, a change in size caused directly by molecular binding of a specific reporter molecule to the particles, biomolecular detection using a change in size resulting from particle agglomeration, a change in size caused directly by molecular binding of the target to the particles, virus or other particle-targeting antibody development, and analysis of other particle properties that lead to differences in the change in effective resistance of the constriction caused by the particles, such as surface charge. 
       Process Chart 
       [0039]      FIG. 2  illustrates a process chart in accordance with one or more embodiments of the present invention. 
         [0040]    Box  200  illustrates applying a bias voltage between a first electrode and a second electrode. 
         [0041]    Box  202  illustrates constricting the fluid in a constriction between the first electrode and the second electrode. 
         [0042]    Box  204  illustrates sensing a change in voltage at a point between the constriction and the second electrode, wherein the change in voltage is proportional to a volume filling fraction of a particle in the constriction. 
       Device Applications 
       [0043]    Although the present invention is described in terms of a device, it is also contemplated within the scope of the present invention that the device of the present invention is used in conjunction with other devices in various applications. For example, and not by way of limitation, the device of the present invention can be used to measure particles and/or nanoparticles in a variety of applications, such as blood plasma sampling devices, measuring particles and/or nanoparticles in protein agglomeration, measuring particle distributions in products such as cosmetics, industrial products, ink, paint, personal care products, etc. Such applications of the present invention can be used, for example, for quality control of products and chemical mechanical processes, as a feedback mechanism for industrial machinery, as a measuring device for real-time or delayed sampling, or other applications where particle measurement, sampling, and volume filling fractions are desired or required. 
       CONCLUSION 
       [0044]    This concludes the description of the preferred embodiment of the present invention. 
         [0045]    In conclusion, an apparatus for detecting particles in a fluid in accordance with one or more embodiments of the present invention comprises a first electrode, a sensing electrode, a fluid resistor between the first electrode and the sensing electrode, a second electrode, and a constriction between the sensing electrode and the second electrode, wherein a bias voltage is applied between the first electrode and the second electrode to create a current through the fluid, such that a size and a number of a particle is sensed by a change in voltage sensed at the sensing electrode. 
         [0046]    Such an apparatus further optionally comprises a filter element such that the particles are sensed by the apparatus and undesired particles are blocked by the filter element from being sensed by the apparatus, a bandwidth of the apparatus being greater than 100 kHz, the fluid resistor and the constriction comprise a voltage divider in the apparatus, the sensing electrode being fabricated using lithographic techniques, the bias voltage being a constant voltage, the apparatus sensing different sizes of particles in the fluid, a plurality of constrictions, and an integrated filter element. 
         [0047]    A method of detecting particles in a fluid in accordance with one or more embodiments of the present invention comprises applying a bias voltage between a first electrode and a second electrode, filtering the fluid prior to entering any smaller constrictions, restricting the fluid in a constriction between the first electrode and the second electrode, and sensing a change in voltage at a point between the constriction and the second electrode, wherein the change in voltage is proportional to a volume filling fraction of a particle in the constriction. 
         [0048]    Such a method further optionally includes a bandwidth of the apparatus being greater than 100 kHz, creating a voltage divider in the apparatus using the constriction, the sensing electrode being fabricated using lithographic techniques, the bias voltage being a constant voltage, sensing different sizes of particles in the fluid, and constricting the fluid in a plurality of constrictions. 
         [0049]    The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto and the full range of equivalents to the claims appended hereto.