The disclosed invention relates to the field of electrochemical sensors. The present invention also relates to the field of microscale devices.
Various scientific and patent publications are referred to herein. Each is incorporated by reference in its entirety.
To monitor the health of patients, identify infectious diseases, screen for cancer, and monitor the safety of water and food supplies, it is often desirable or even necessary to concurrently detect and measure the concentrations of multiple analytes in a given sample. Such analytes can include various chemicals, proteins, viruses, DNA, RNA, cells, bacteria, antibodies, and other biological and non-biological markers.
It is known that accurate and reliable biomarker-based cancer screening requires the concurrent quantification of multiple proteins, and, in certain cases, can entail the establishment of a gene profile of cancer and pre-cancer cells. In the latter case, one takes advantage of the fact that precancerous and cancerous cells exhibit a change in the transcription levels of many genes. Accordingly, the detection of the deviations in mRNA levels provides an informative target for cancer diagnostics. Establishing a gene profile, however, requires the quantification of from about 10 to about 30 genes, which quantification, in turn, is most efficiently accomplished by the use of an array of multiple sensors.
Existing devices for performing such analyses have certain drawbacks, and often require the use of large, complex devices, including cameras, fluorescence meters, optical scanners, and the like. The size and complexity of such devices renders optical detection techniques ill-suited for use in portable devices.
Because of the limitations inherent in multiple analyte detection systems that rely on optical detection, there is a need for devices capable of detecting multiple analytes on a non-optical basis. There is also a related need for methods of fabricating such devices.
The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.
In one aspect, the present invention provides devices, comprising: a capillary, wherein the capillary is capable of being electrically addressed; and at least one particle adjacent to the capillary.
In another aspect, the present invention provides devices, comprising: a substrate, wherein the substrate comprises a surface and a plurality of electrodes, wherein at least one of the electrodes is capable of being individually electrically addressed; and at least one particle comprising at least one functionality, wherein at least one particle comprising at least one functionality is adjacent to at least one electrode capable of being electrically addressed.
Further provided are methods, comprising: contacting a plurality of individually addressable and spatially distributed conductive capture electrodes with a plurality of particles dispersed in a carrier medium, wherein each of the particles comprises at least one sensing functionality, at least one barcode functionality, or any combination thereof; wherein contacting a conductive capture electrode with the plurality of particles dispersed in the carrier medium gives rise to at least one particle being positioned adjacent to at least one conductive capture electrode by action of capillary forces, surface forces, Van der Waals forces, electrokinetic forces, electromagnetic forces, or any combination thereof; and identifying the spatial location of one or more particles positioned adjacent to at least one individually addressable and spatially distributed capture electrode.
Additionally provided are methods, comprising: contacting a first fluid with a plurality of conductive capture electrodes, wherein at least one of the conductive capture electrodes is capable of being individually electrically addressed; flowing a second fluid adjacent to the first fluid, wherein the second fluid resides between the first fluid and a director electrode and comprises a plurality of particles, wherein each of the particles comprise one or more functionalities; and applying an electric potential between the director electrode and the plurality of conductive capture electrodes so as to give rise to at least one particle of the second fluid being positioned adjacent to at least one conductive capture electrode.
The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments of the invention; however, the invention is not limited to the specific methods, compositions, and devices disclosed. In addition, the drawings are not necessarily drawn to scale. In the drawings:
i) depicts a cross-section of a 7-barrel quartz capillary with catalyst-laden inner diameters,
i) is an optical micrograph taken over a time span of about 2.5 seconds of a carbon nanopipette suitable for use in the present invention returning to its original shape after being deformed 90° against a hard surface, and
i) illustrates a particle-tipped probe capped by a conductive particle where the particle acts as an electrode,
i) illustrates an individually-addressed nanoelectrode array for electrochemical detection using a microfabricated planar substrate, and
i) illustrates a particle plugging the tip of a carbon nanoelectrode, and
i) is a schematic illustration of an embodiment of a system of the present invention for electrochemical analysis,
The present invention may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this invention is not limited to the specific devices, methods, applications, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention. Also, as used in the specification including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.
It is to be appreciated that certain features of the invention which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. Further, reference to values stated in ranges include each and every value within that range.
As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.
“Adjacent” refers to two or more entities, such as objects, surfaces, or any combination thereof, that reside near, next to, or are in contact with each other and are physically, electrically, or any combination thereof, and may be affected by their proximity to each other. “Directly adjacent” means two or more objects that are in contact with one another.
“Barcode” refers to an aspect or characteristic of an object that is capable of assisting with identifying that object or identifying that object's location.
“Electrical connection” refers to two or more entities, such as objects, surfaces, or any combination thereof, wherein a change in the electrical characteristics of one or more of the entities is capable of affecting or of being detected by one or more of the other entities.
“Electrode” means an object or site capable of passing an electrical current or a magnetic field. The term can also mean an object or site capable of projecting an electrical potential or current or a magnetic field.
In one aspect, the present invention provides devices, comprising: a capillary, wherein the capillary is capable of being electrically addressed; and at least one particle adjacent to the capillary.
Suitable capillaries comprise conducting materials, semiconducting materials, dielectric materials, or any combination thereof. Conductive materials are considered especially suitable.
Suitable capillary materials include carbon, gold, silver, platinum, aluminum, copper, nickel, chromium, indium, tin, or any combination thereof. Other suitable metals will be known to those of ordinary skill in the art. Electrically conductive polymers are also suitable capillary materials.
Capillaries produced by the method of Bau, et al., as described in U.S. patent application Ser. No. 11/231,425, filed on Sep. 21, 2005, are particularly suitable for the present invention. Capillaries made according to that method are produced by direct chemical vapor deposition (“CVD”) inside a pulled quartz or glass capillary and subsequent etching of the quartz or glass tip to expose a predetermined length of carbon capillary.
The technique of Bau, et al., is capable of producing multiple carbon capillaries by CVD of carbon into a multi-barrel quartz pipette structure, as shown in
The resulting capillary structures are biocompatible and display high flexibility. As shown in
Suitable capillaries have lumen diameters in the tens of nanometers up to the micrometer range; lumen diameters can be in the range of from about 10 nm to about 100 micrometers, or from about 20 nm to about 50 micrometers. The wall thickness of the capillaries can be in the range of from about 1 nm to about 1000 nm, and are in the range of from about 100 to about 500 nm, or in the range of from about 10 nm to about 50 nm. Wall thicknesses in the range of about 30 nm are also suitable.
In some embodiments, the capillary and at least one adjacent particle are in electrical connection with one another. It is contemplated that the capillary is capable of detecting a signal related to the binding of one or more functionalities of the particle to one or more analytes to which the particle is contacted.
In other embodiments, the capillary is directly adjacent to a particle. An exemplary illustration of a particle positioned directly adjacent to a capillary is shown in
It is contemplated that the capillaries and particles described herein may be combined so as to form a multiplexed device that comprises multiple, bundled capillaries sand multiple particles. Such a device is illustrated in
Particles suitable for use in the claimed invention include conducting materials, semiconducting materials, insulating materials, and the like. Particles may also comprise a porous material, a nonporous material, or any combination thereof. Functionalized particles are available from, for example, Bangs Laboratories, Inc. (www.bangslabs.com, Fisher, Ind., USA). Suitable particles may be purchased commercially and modified by the user or, alternatively, synthesized by the user. Methods of selecting, synthesizing, and modifying particles will be known to those of ordinary skill in the art.
Suitable particles have a characteristic cross-sectional dimension in the range of from about 20 nm to about 200 microns, or in the range of from about 500 nm to about 50 microns. The optimal particle size for a given application will depend on the needs of the user and will be known to those of ordinary skill in the art. The size of a particle is preferably chosen such that the particle is capable of seating reliably on a capillary or other capture site.
Particles suitable for the present invention also include one or more functionalities. Suitable functionalities include antigens, antibodies, monomers, oligonucleotides, nucleic acids, ligands, enzymes, enzyme substrates, oligomers, dyes, fluorophores, and the like. Particle functionalities can be selected based on the analyte or analytes the device will be used to detect, and can be selected based on being complementary to the analytes. Particles can also be chosen based on size, shape, color, the presence of one or more fluorescent dyes of the particle, electrical resistance, or electrochemical properties. As discussed in further detail elsewhere herein, particle functionalities may be used to bind to target analytes and may also be used to identify a particle or its spatial location.
In one exemplary configuration, the capillary is connected to an electrical device. Suitable electrical devices include a meter capable of detecting electrical signals, a current source, a sensor, a controller, a computer, or any combination thereof. As a non-limiting example, a capillary may be electrically connected to a detector capable of observing changes in the resistance of the capillary when a particle becomes seated on the end of the capillary. Such a detector may be connected in turn to a computer capable of tracking and processing signals from one or more detectors so as to provide information about the positioning of particles across an array of capillaries or capture sites.
One sample configuration is illustrated in
Electrode-particle assemblies can include electrodes adjacently assembled to various particles bearing different functionalities, which assemblies can be combined to form multiplexed devices. Such multiplexed devices are suitable for simultaneously detecting multiple analytes, where the analytes are complementary to the functionalities present on the particles. Such devices are capable of simultaneously detecting the presence of multiple analytes in a given sample. Arranging the capillaries in bundled form is considered especially suitable.
Also provided are devices, comprising: a substrate, wherein the substrate comprises a surface and at least one electrode, wherein at least one electrode is capable of being individually electrically addressed; and at least one particle comprising at least one functionality, wherein at least one particle comprising at least one functionality is adjacent to at least one electrode capable of being individually electrically addressed.
The at least one electrode capable of being electrically addressed is suitably capable of detecting a signal related the binding of one or more functionalities of the adjacent particle to one or more analytes. Such a signal is preferably electrical or magnetic in nature, but optical and radioactive signals are also contemplated.
Substrates comprise one or more materials capable of being shaped or formed. Dielectric materials, conductive materials, semiconductive materials, or any combination thereof are all considered suitable. Ceramics, polymers, alloys, insulated metals, or any combination thereof are suitable substrate materials, as are glass, quartz, silicon, alumina, tungsten, titanium, and the like.
The devices can include at least one electrode capable of being individually electrically addressed, adjacent to an indentation, cup, a bowl, a divot, a cavity, a disk, a stripe, a bump, or any combination thereof. Without being bound to any particular theory of operation, it is believed that positioning the electrode adjacent to a feature capable of accommodating one or more particles will allow for reliable positioning of particles relative to electrodes.
In some embodiments, at least one electrode capable of being individually electrically addressed electrode is characterized as recessed relative to the substrate surface or, in other embodiments, as being even or flush with the substrate surface. In other embodiments, the electrode may protrude above the substrate surface. In some embodiments, at least one electrode capable of being individually electrically addressed is itself characterized as an indentation, a cup, a bowl, a divot, a cavity, a disk, a stripe, a bump, or any combination thereof. Without being bound to any particular mode of operation, it is believed that shaping the electrode in a manner that allows the electrode to accommodate one or more particle can prevent, at least in part, particles from physically separating from adjacent electrodes.
In other embodiments, the electrode is a protuberance, a tube, a projection, and the like. The optimal shape of an electrode will be dictated by the needs of the user, and will be apparent to those of ordinary skill in the art.
Additional configurations include electrodes characterized as being in the form of capillaries. Tubular or straw-shaped electrodes are also suitable.
A schematic view of the disclosed devices is shown in
Electrodes may be capillaries, having lumens in the range of from about 10 nm to about 100 micrometers. Electrodes of other forms—such as pits, depressions, divots, and the like—also suitably have a cross-sectional dimension in the range of from about 10 nm to about 100 micrometers. The dimensions of suitable particles are described elsewhere herein.
At least one electrode capable of being individually electrically addressed can include an electrical connection to a meter capable of detecting electrical signals, a current source, a controller, a computer, and the like.
In suitable embodiments, at least one electrode is capable of applying a current, an electric potential, a magnetic field, or any combination thereof. In some configurations, at least one electrode is capable of applying an alternating current, a direct current, a variable current, or any combination thereof.
In other embodiments, an electrode of the device is capable of applying a constant electric potential, an alternating electric potential, a variable electric potential, or any combination thereof. Similarly, electrodes are suitably capable of applying a constant magnetic field, a variable magnetic field, or any combination thereof.
Some embodiments include at least one particle comprising at least one functionality in electrical connection with at least one electrode capable of being electrically addressed. Suitable particles and functionalities are described elsewhere herein. As described elsewhere herein, devices may be assembled, at least in part, by applying a particular field or potential so as to direct particles bearing particular functionalities to particular locations.
Multiple electrode-particle assemblies, like those depicted in
Also disclosed are methods for assembling devices, comprising: contacting a plurality of individually addressable and spatially distributed conductive capture electrodes with a plurality of particles dispersed in a carrier medium, wherein each of the particles comprises at least one sensing functionality, at least one barcode functionality, or any combination thereof; wherein contacting a conductive capture electrode with the plurality of particles dispersed in the carrier medium gives rise to at least one particle being positioned adjacent to at least one conductive capture electrode by action of capillary forces, surface forces, Van der Waals forces, electrokinetic forces, magnetic forces, electromagnetic forces, or any combination thereof; and identifying the spatial location of one or more particles positioned adjacent to at least one individually addressable and spatially distributed capture electrode.
In some embodiments, one or more particles positioned adjacent to at least one conductive capture electrode are in electrical connection with at least one conductive capture electrode.
A conductive capture electrodes can comprise a capillary. Suitable capillaries and their dimensions are described elsewhere herein, and may be fabricated by the methods of Bau, et al. The capillary is in electrical connection with a current source, a current meter, a voltage source, a voltage reader, a controller, a computer, a sensor, or any combination thereof.
The plurality of individually addressable and spatially distributed capture electrodes can comprise a substrate, wherein the substrate comprises one or more electrically conductive and individually electrically addressable sites acting as electrodes. Suitable forms and shapes for such sites are described elsewhere herein. In such embodiments, at least one of the electrically conductive sites is in electrical connection with a current source, a current meter, a voltage source, a voltage meter, a computer, a sensor, a controller, or any combination thereof.
Suitable particles are described elsewhere herein. Sensing functionalities comprise ligands, nucleic acids, enzymes, enzyme substrates, antibodies, oligonucleotides, antigens, oligomers, and the like.
Barcode functionalities can include physical characteristics that distinguish between particles, such as particle size, particle color, intensity of one or more fluorescent dyes of the particle, particle shape, particle electrical resistance, particle dielectric properties, particle magnetic properties, particle electrochemical properties, color of fluorescent dye of the particle, and the like.
Barcode functionalities also include biological functionalities such as ligands, nucleic acids, enzymes, enzyme substrates, antibodies, oligonucleotides, antigens, oligomers, and the like.
Contacting the electrodes with the particles can include the application of a potential or an external field, which include electrical potential, an electrical current, a magnetic potential, a pressure gradient, or any combination thereof.
The frequency of the field or potential is constant or varied. In some embodiments, two or more particles are affected differently by application of the potential. The particles may be affected on the basis of their surface charge, dielectric properties, or any combination thereof.
As a non-limiting example, a mixture of particles having different sensitivities to a potential of a particular frequency is exposed to that frequency. Under such conditions, the particles having a greater sensitivity to that frequency preferentially migrate along the direction of that potential toward capture sites, while the particles having a lesser sensitivity to that frequency remain essentially stationary.
In some embodiments, contacting the capillaries or capture sites with the particles gives rise to a change in a measurable quantity, which quantity is an electrical potential, an electrical current, a magnetic property, a dielectric property, a pressure, and the like. The change in a measurable quantity is suitably monitored and interpreted so as to provide information regarding the status of a particular capillary or capture site or electrode. As an example, a change in the dielectric property of a capture site indicates that one or more particles have become positioned adjacent to that site. Likewise, a change in the electrical resistance or capacitance of a capture site can also indicate that a particle has positioned adjacent to the site.
In some configurations, identifying the location of one or more particles adjacent to one or more conductive capture electrodes comprises: contacting the one or more particles positioned adjacent to at least one capture electrode with at least one reduction-oxidation effector species, wherein the reduction-oxidation effector species has a known reduction-oxidation potential; and monitoring one or more evolved threshold potentials necessary for reduction and oxidation reactions to take place. In these configurations, the threshold potential of the one or more particles positioned adjacent to at least one capture electrode is known and is compared to the evolved threshold potential affected by contacting the particles with the reduction-oxidation effector species. By comparing the evolved potential of electrode-adjacent particles of different known threshold potentials to the evolved potential at given electrode-adjacent particle, it is then determined which particle is adjacent to which electrode. By ensuring particles made of different materials also bear different functionalities, identifying the location of particular particles at particular electrodes in turn identifies which functionalities are present at those electrodes.
Identifying the spatial location of the one or more particles positioned adjacent to one or more conductive capture electrodes can include: contacting the one or more particles with a plurality of identifier agents, wherein at least one of the identifier agents is capable of binding uniquely to a particular type of barcode functionality; and monitoring one or more signals arising from the binding of at least one identifier agent to at least one barcode functionality. Suitable identifier agents comprise ligands, nucleic acids, enzymes, enzyme substrates, antibodies, antigens, oligomers, dyes, flurophores, oligonucleotides, or any combination thereof. Identifier agents are complementary to particle barcode functionalities, described elsewhere herein. The signals provided by these embodiments include an electrical signal, an optical signal, a magnetic signal, an electrochemical signal, a radioactive signal, or any combination thereof.
Identifying the spatial location of the particles can further comprise monitoring the voltage, current, impedence, resistance, or any combination thereof of the one or more particles adjacent to one or more conductive capture electrodes before, during, or after the binding of the identifier agent to the barcode functionality, so as to identify the spatial location of the one or more particles positioned adjacent to one or more conductive capture electrodes.
In other configurations, various types of identifier molecules, one type at a time, are brought into contact with particles adjacent to the conductive capture electrodes. By monitoring, on an electrode-by-electrode basis, the signals evolved from the interactions of identifier agents and the barcode functionalities of particles adjacent to electrodes, the locations of particular particles bearing particular barcode functionalities and particular sensing functionalities are determined. By knowing a priori which sensing functionalities and which barcode functionalities are present on particular particles, the spatial locations of particles bearing particular sensing functionalities may then be determined.
Where barcode functionalities comprise one or more physical characteristics of a particle, including particle size, particle color, the intensity of one or more fluorescent dyes of the particle, particle shape, particle electrical resistance, and particle electrochemical properties, the spatial locations of particles are determined by monitoring, on an electrode-by-electrode basis, electrical or optical signals evolved from the particles adjacent to electrodes. By a priori knowing the sensing functionalities of particular particles of particular barcode physical characteristics, the spatial locations of particles bearing particular sensing functionalities is then determined.
As one example, beads that are complementary to analyte A might bear a red fluorescent molecules, whereas beads that are complementary to analyte B might bear a green fluorescent molecules. In such an embodiment, once the beads are attached or proximate to capillaries or capture sites, the entire device may be fluorescently interrogated so as to determine the locations of those beads—and associated capture sites—that are complementary to analyte A (i.e., those beads that display a red fluorescence) and those that are complementary to analyte B. (i.e., those beads that display a green fluorescence). Once such a map of bead locations is generated, the device may then be contacted to an analyte-containing sample, and the presence of particular analytes may be determined by monitoring particular capture sites known to be associated with beads complementary to the analytes.
As will be apparent to those of ordinary skill in the art, there are innumerable permutations of barcode markers and functionalities that may be used on beads. By careful design, the user may assemble a broad library of particles bearing identifying functionalities, thus enabling the assembly of detector devices that may be customized to detect any number of targets or analytes.
It is envisioned that the plurality of electrodes is suitable for use as a multiplexed device capable of simultaneously detecting multiple analytes, wherein the analytes are complementary to the sensing functionalities present on particles in known spatial locations.
The disclosed inventions also include devices made according to the disclosed methods. Such a device is used as a probe, a sensor, a detector for a single analyte, a detector for multiple analytes, or any combination thereof. Such devices also include electronic, optoelectronic, or electromechanical devices, and the like.
Further provided are methods for assembling devices, comprising: contacting a first fluid with a plurality of conductive capture electrodes, wherein at least one of the conductive capture electrodes is capable of being individually electrically addressed; flowing a second fluid adjacent to the first fluid, wherein the second fluid resides between the first fluid and a director electrode and comprises a plurality of particles, wherein each of the particles comprise one or more functionalities; and applying an electric potential between the director electrode and the plurality of conductive capture electrodes so as to give rise to at least one particle of the second fluid being positioned adjacent to at least one conductive capture electrode.
It is contemplated that one or more particles of the second fluid positioned adjacent to at least one conductive capture electrode induce one or more electrical signals in at least one conductive capture electrode. Without being bound by any particular theory of operation, it is believed that the one or more particles positioned adjacent to at least one conductive capture electrode are maintained in position by capillary forces, surface forces, Van der Waals forces, electrostatic forces, electromagnetic forces, magnetic forces, chemical bonds, or any combination thereof.
Suitable conductive capture electrodes include capillaries. Suitable capillaries are described elsewhere herein.
In other embodiments, at least one conductive capture electrode is adjacent to an indentation, a bowl, a cup, a disk, a ring, a cylinder, a disk, a strip, or any combination thereof, or, in still other embodiments, at least one conductive capture electrode is directly adjacent to an indentation, a bowl, a cup, a disk, a ring, a cylinder, a disk, a strip, or any combination thereof.
At least one conductive capture electrode is in electrical connection with a current source, a meter capable of detecting an electrical signal, a computer, a controller, a sensor, or any combination thereof.
The method contemplates that the first fluid, second fluid, or both, flow relative to the plurality of conductive capture electrodes. The first and second fluid flows can be laminar, and are preferably characterized by low particle-based Reynolds numbers; such particle-based Reynolds numbers are less than about 1, or even less than about 0.1.
The first and second fluid layers can be characterized as having a thickness in the range of from about 50 microns to about 200 microns, or in the range of from about 100 to about 150 microns.
Suitable potentials include a DC electric field, an alternating electric field, a magnetic field, a pressure gradient, and the like.
The electrical potential is suitably less than about 5 volts, but other magnitudes may be suitable, depending on the user's needs and design constraints. In some embodiments, the electrical potential is periodic, irregular, or both. It is envisioned that the frequency of a potential ranges from about 1 to about 10 MHz.
Suitable particles and functionalities are described elsewhere herein.
The parameters of the first and second fluid flows and the sizes of the particles are each selected such that the positioning of the particles is governed by the electric potential and not governed by particle diffusion.
The method also includes monitoring one or more signals of the capture electrode while the electrode is in contact with the first and second fluids. These signals vary according to the position of the one or more particles relative to the conductive capture electrode. In some configurations, the signal may be a signal present only when a particle is adjacent to an electrode. Suitable signals include electrical signals, optical signals, mechanical signals, or any combination thereof.
The method further comprises terminating the potential to an individual electrode when the signal indicates at least one particle is positioned adjacent to at least one conductive capture electrode. The signal can include a change in resistance, in impedance, in optical intensity, in optical wavelength, or any combination thereof. The potential can be terminated so as to avoid the formation of a chain or particles.
Potentials may also include DC electric potential, an AC electric potential, a magnetic field, pressure field, or any combination thereof. The frequency of the potential may be varied.
The methods contemplate that one or more particles may be motivated on the basis of the electric charge of the particles. Particles are also motivated, in some embodiments, on the basis of a dielectric property of one or more of the particles, or even on the basis of a magnetic property of the one or more particles.
The disclosed methods further include changing the position of one or more positioned particles, which is accomplished by applying an electrical, magnetic, electromagnetic, or mechanical potential to the one or more positioned particles. Hence, in some embodiments, particles that have previously been positioned atop a capillary or adjacent to a capture site are then dislodged from that location by application of a potential. This enables the re-use of the capillaries or captures sites with a different set of beads, in turn enabling the construction of a broad range of detection devices, limited only by the functionalities contained within a given user's library of beads.
It is contemplated that the positioning of a particle adjacent to at least one conductive capture electrode gives rise to a change in a measurable quantity, which measurable quantity includes and electrical potential, an electrical current, a magnetic property, a dielectric property, a pressure, or any combination thereof. As discussed elsewhere herein, the change in a measurable quantity is suitably monitored and interpreted so as to provide information regarding the status of a particular capillary or capture site or electrode.
Also provided are devices made according to the method, wherein the devices are used as probes, sensors, detectors, or any combination thereof. Such devices can be electronic, optoelectronic, or electromechanical devices, or any combination thereof.
A depiction of the disclosed methods is shown in
A carbon nano-capillary was brought into a contact with a dilute solution of suspended nanoparticles with diameters slightly larger than the inner diameter of the carbon capillary. The solution penetrated the carbon capillary by capillary suction and a particle locked into place by Van der Waals forces, as depicted in
One method of fabricating the sensing devices of the instant invention is the directed assembly method. This method permits the positioning of particular particles adjacent or directly adjacent to particular electrodes, and eliminates the need for post-attachment analysis to determine which particles are adjacent to which electrodes.
The directed assembly can be performed using dielectrophoresis. To perform directed assembly, an electrode probe, such as a nano-scale capillary, is brought into contact with a dilute suspension of particles. A director electrode is then positioned opposite the electrode probe, and an alternating (AC) potential difference is applied between the nano-scale capillary and the director electrode. In the presence of the resulting, non-uniform electric field, one or more particles polarize and migrate towards the location of the maximum field intensity, at the opening of the nano-scale capillary. After the particle migrates to the nano-scale capillary, the particle locks into position by surface forces, as depicted in
Measures can be taken to prevent particles from reaching non-active electrodes by diffusion. To prevent diffusion-effected transport of particles, a microfluidic device, as shown in
The particle's attachment or positioning adjacent to an electrode is in turn sensed as an electrical signal. This signal opens the circuit that actuates the particular capillary to which the particle has attached. Deactivating the capillary in turn prevents one attached particle from acting as an electrode that attracts additional particles, and eliminates the possibility of chain formation. This method allows direction of a particle of known functionalization to a target capillary electrode and eliminates the need for subsequent registration (identification) of the particles.
After the particle attaches to the capillary, the electrochemical characteristics of the capillary electrode-particle assembly are examined with a standard bench-top electrochemical analyzer, such as the potentiostat and associated electronics of the Epsilon benchtop electrochemical analyzer (BASi, Co.). See
Identifying the particle through its electrochemical characteristics is also feasible. In one case, the particle's material is used as a barcode to identify a particle attached to an electrode. To this end, a particle-electrode assembly is inserted into an electrolyte solution containing reduction-oxidation species. The threshold potentials needed for reduction and oxidization reactions are measured and then correlated with the potentials known for the particle's material, thus identifying which particle has attached to which electrode in an array of electrodes. Particles made from various alloys and pure materials can be used.
To characterize the electrochemical behavior of the particle-electrode sensors, the contact resistance between the functionalized particle and the carbon nanopipe and the overall resistance of the particle-tipped electrode is measured. The using the probe for electrochemical detection is tested by using a sandwich assay to detect various concentrations of avidin in solution. The microfluidic cell of Example 2 is exposed to flow of avidin solution at various dilutions. Subsequently, the sensor is then washed and the solution is replaced with biotinated enzyme. The biotin-enzyme complex then binds to the immobilized avidin. After a rigorous wash, a substrate to be processed by the enzyme is flowed across the bound enzyme complex, and the resulting electric current is detected. Such experiments establish the correlation between signal amplitude and analyte concentration. The experiments also provide an estimate of the sensitivity and detection limits of the sensor.
To examine the multiplexing methods developed to perform multiple electrochemical measurements in a solution, nanoscale carbon multi-electrodes are formed by depositing carbon inside multi-barrel quartz micropipette substrates so as to form multiple carbon capillary electrodes. See
After undergoing a CVD process, the quartz is removed by wet-etching to expose the carbon tubes formed on their quartz tubes' inner diameters. Seven independent wires are attached with silver conductive epoxy to the tail end of each pipette probe to make electrical connections for electrochemical analysis.
After the carbon tubes are exposed, they are contacted with a dilute suspension of differently functionalized nanoparticles. The particles are positioned in place using the directed assembly methods described in Example 1 to position selected particles at the desired electrodes.
Following assembly of particles to electrodes, the particle-tipped electrodes are submerged in an aqueous solution containing anti-A, anti-B, and anti-C antibodies as shown in
A bundle of capillaries is contacted with a fluid comprising two different types of beads, each kind of bead (1) bearing a particular functionality that is uniquely complementary to a specific target and (2) each kind of being comprised of a material with a different set of dielectric properties that render that bead uniquely sensitive to a particular frequency of electricity.
By applying an electric field or current of a particular frequency, only those beads that react to such a frequency will migrate across the fluid to particular capillaries, after which migration the location of the migrated beads is catalogued. A second, different frequency is then applied to the fluid, which in turn causes migration of the other type of beads to unoccupied capillaries, and the location of these newly-migrated beads is then catalogued. Alternatively, different capillaries or capture sites may apply different electrical frequencies to the fluid, such that only a bead sensitive to a particular frequency will migrate to the capillary or capture site that applies that frequency. In such a configuration, successive application of different frequencies enables successive assembly of differently-functionalized beads onto specific capillaries. In this way, specifically-functionalized beads may be directed to specific capillaries or capture sites.
The user is then left with a device having beads in known locations that are specifically complementary to specific targets. Such a device is then useful for detecting the presence of known targets in a particular sample. Cups, electrodes, pits, protruberances, or other shapes and forms may be used in addition to or in place of capillaries.
A bundle of capillaries is contacted to a fluid comprising magnetizable particles. A magnetic potential is effected across the fluid to the capillaries, such that one or more particles migrate to the capillaries and remain positioned on the capillaries by capillary forces or by effect of the magnetic field. In one embodiment, one or more capillaries is at least partially filled with a ferrofluid so as to effect attraction between the capillary and one or more particles.
A device comprising magnetizable functionalized beads proximate to capillaries is assembled according to the disclosed methods. The user applies a magnetic potential oriented so as to dislodge the beads from the capillaries to separate the beads from the capillaries. The dislodged beads are then flushed away from the capillaries. The capillaries are the brought into contact with a new, different population of beads bearing particular functionalities, and a new detection device is assembled according to the methods of the present invention. In other configurations, the beads are dislodged by electric potential, or even by vibration.
In such configurations, a device may be assembled to detect target molecule A and B and is then used to detect—simultaneously—molecules A and B in a sample. After the detection of A and B is performed, the detector beads are dislodged and flushed away from the capillaries or electrodes, and the capillaries are then contacted with beads capable of detecting targets C and D, and a new device is then assembled, that is capable of detecting C and D. In such a way, the same set of capillaries may be used as the basis for a number of customizable, reusable detector devices by using the capillaries in conjunction with a library of beads that are complementary to any number of targets or analytes.
This application is the National Stage of International Application No. PCT/US2008/001554, filed Feb. 4, 2008, which claims the benefit of U.S. Provisional Application No. 60/888,375, filed Feb. 4, 2007, the disclosures of which are incorporated herein by reference in their entirety.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US2008/001554 | 2/4/2008 | WO | 00 | 9/16/2010 |
Publishing Document | Publishing Date | Country | Kind |
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WO2008/130463 | 10/30/2008 | WO | A |
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Number | Date | Country | |
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20110027913 A1 | Feb 2011 | US |
Number | Date | Country | |
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60888375 | Feb 2007 | US |