Patent Application
20130105317
1. Field of the Invention
The invention relates to an apparatus and method for pathogen detection. Specifically, the invention relates to the field of pathogen detection systems and diagnostic devices and their micro-component assembly. More specifically, the invention utilizes an apparatus that includes a dielectrophoretic separator, a dielectrophoretic condenser, a dielectrophoretic trap, microfluidic components, and field effect sensor, such as an ion sensitive sensor, nanowire sensor, or nanoribbon sensor configured as biosensors, to perform a pathogen detection process.
2. Description of Related Art
Bacterial infections cause thousands of diseases in humans and animals every year. Recent deadly outbreaks of E. Coli, Salmonella, and Listeria have highlighted the urgent need for more effective methods of detection, identification, and characterization of pathogens, and their origin and proliferation. Conventional detection methods have proven inadequate because they suffer from long incubation periods, high cost, and require highly trained personnel to operate. There remains a strong need for a reliable, time-efficient apparatus and method for specific detection of bacteria in low concentration.
Conventional methods rely on bacterial culture growth, which require highly qualified personnel and time, both contributing to higher costs for the procedure. The most widely used method for bacterial detection, the standard plate count, takes from 24 to 48 hours due to the time needed for bacteria to grow detectable colonies, and requires a stocked microbiology lab. Although faster methods, such as PCR (Polymerase Chain Reaction) Plates or labeled detection and fluorescent imaging, can reduce the response time to one hour, these require complex sample preparation, highly trained personnel, high cost per test, and have limited portability.
The major challenge in automated sample preparation for detection from blood or other unprocessed liquids using microstructures is efficient separation of the analyte of interest (bacteria, cells, or particles) from large blood components. Red blood cells (RBC) and white blood cells (WBC) range between 6 μm-21 μm in size and constitute over 50% of the whole blood volume. RBC and WBC presence obstructs the detection of bacteria, cells, or particles. The present invention is a miniaturized device for rapid pathogen screening that overcomes these obstacles.
Dielectrophoresis (“DEP”) is a separation method based on size and dielectric properties and has been described in literature as for example in Pohl et al, Science 1966, and Sher Nature 1968, Voldman, Annual Review Of Biomedical Engineering, 2006. The use of DEP to manipulate particles and cells has been previously described, as for example, in H. Pohl, I. Hawk, “Separation Of Living And Dead Cells By Dielectrophoresis,” Science, 152, 3722 (1966); Y. Huang, R. Holzel, R. Pethig, X. Wang, “Differences In The Ac Electrodynamics Of Viable And Non-Viable Yeast Cells Determined Through Combined Dielectrophoresis And Electrorotation Studies,” Phys. Med. Bid., 37, 7 (1992); S. Chang, Y. Cho, “A Continuous Size-Dependent Particle Separator Using A Negative Dielectrophoretic Virtual Pillar Array,” Lab Chip, 8, 1930-1936 (2008); and J. Yang, Y. Huang, X. Wang, F. Becker, P. Gascoyne, “Differential Analysis Of Human Leukocytes By Dielectrophoretic Field-Flow-Fractionation,” Biophysical Journal, 78, 2680-2689 (2000). However, effective methods for cell/pathogen separation on a micro-scale from fluids containing pollutants of comparable size are still unattainable.
High-frequency electric fields when applied to an electrically neutral object cause polarization. A high-frequency non-uniform electric field gives rise to a dielectrophoretic force (DEP) FDEP which acts on the object.
A spherical object of a given electrical permittivity ∈p placed in a medium of a different permittivity ∈m in a spatially varying electric field E(x,ω) is subjected to a dielectrophoretic force, FDEP. The dielectrophoretic force is given by:
F
DEP=2π∈m r3 Re{CM(ω)}·∇E2
where
CM(ω)=(∈{tilde over (p)}−∈{tilde over (m)})/(∈{tilde over (p)}+2∈{tilde over (m)})
{tilde over (∈)}=∈+σ/iω;
Depending on the respective permeability ({tilde over (∈)}) and conductivity (σ) of the object and the medium, the force can be attractive (positive dielectrophoresis (pDEP)), or repulsive (negative dielectrophoresis (nDEP)). If Re{CM(ω)} is positive, then the particle experiences a positive dielectrophoretic force, and if Re{CM(ω)} is negative, then the particle experiences a negative dielectrophoretic force. Different species have different dielectric properties. The dielectric functions ∈m, ∈p depend on the frequency of the external electric field. The permittivity of the medium affects the CM(ω) factor and the value of Re{CM(ω)}. Importantly, if the signs of Re{CM(ω)} for different species are opposite then the species are subject to forces acting in opposite directions and separation occurs.
There is a cross-over frequency, ωco, that occurs when the Re{CM(ω)} goes to zero. Critical to separation is that ωco is uniquely different for different cells and bacteria. Separation procedures for stained cells have been described in U.S. Pat. No. 7,153,648 entitled “Dielectrophoretic Separation Of Stained Cells,” where appropriate frequency and amplitude are applied via a function generator, and red blood cells are attracted to electrodes by positive dielectrophoresis force, while stained white blood cells are repelled to the area with weakest electric field by negative dielectrophoresis force. The differential behavior and separation of E. Coli cells from human blood cells on electrodes under applied electric field has been described in patent U.S. Pat. No. 6,989,086 entitled “Channel-less separation of bioparticles on an Electronic Chip by Dielectrophoresis.”
The dielectrophoretic force is affected both by the geometry of the electrodes (gradient of the electric field), the Re{CM(ω)} factor, and depends on the dielectric constant of the medium ∈m.
Using the aforementioned prior art techniques for dielectrophoresis, the separation of bacteria from blood may achieve, at best, an efficiency of approximately 30%.
Bearing in mind the problems and deficiencies of the prior art, it is therefore an object of the present invention to provide a filtration system for pathogen detection that utilizes a plurality of dielectrophoretic modules with distinctive functionality and geometry to obtain separation performance which cannot be obtained using the single module apparatus of the prior art.
It is another object of the present invention to provide a pathogen detection system that includes a capture/release mechanism for solution exchange without cell loss to enhance pathogen detection at low concentrations.
It is yet another object of the present invention to provide a method for separating a low concentration of bacteria (or other pathogens/particles) from a high volume of blood (or other fluids) which is based on the dielectric properties of the products. The species separation being enhanced and promoted by dielectrophoretic forces acting on the test sample in a plurality of microfluidic channels.
It is another aspect of the present invention to provide a filtration system for pathogen detection that can accommodate high and low throughput, capable of processing test sample volumes significantly greater than micro- or pico-liters, yet capable of processing the minute test sample volumes as well.
It is another object of the present invention to provide pathogen detection in liquid media, especially for use with food and agricultural products to improve health standards at the consumer level.
It is another object of the present invention to provide time sensitive pathogen detection for point-of-care diagnostics.
Still other objects and advantages of the invention will in part be obvious and will in part be apparent from the specification.
The above and other objects, which will be apparent to those skilled in the art, are achieved in the present invention which is directed to an apparatus for pathogen detection comprising: a first chamber for storing a test sample including product to be analyzed and microscaled components to be separated from the product to be analyzed; a second chamber for storing a reference solution; a pump for pumping the test sample and the reference solution; a microfluidic separator separating the product to be analyzed from the microscaled components, the microfluidic separator including a plurality of microfluidic channels, each microfluidic channel including: electrodes for producing a dielectrophoretic force on the test sample when the test sample is pumped through each of the microfluidic channels to perform a dielectrophoresis-based separation; and channels for transporting the microscaled components away from the product to be analyzed; a third chamber for storing the microscaled components when separated from the product to be analyzed by the plurality of microfluidic channels; a condenser for capturing the product to be analyzed once the product has passed through the microfluidic channels and is substantially separated from the microscaled components; and a sensor for detecting the product to be analyzed.
The plurality of microfluidic channels may be assembled in an array, each microfluidic channel having electrodes on internal walls for delivering the dielectrophoretic force to the test sample traversing through the microfluidic channel. The electrodes are located on opposing or adjacent internal walls of the microfluidic channels.
The microfluidic channels preferably comprise a plurality of plates having an electrode configuration, and a plurality of internal channel structures patterned with electrodes, such that each microfluidic channel represents an elongated pathway for the test sample capable of providing a dielectrophoretic force to the test sample as the test sample traverses the microfluidic channel. The plurality of plates and the plurality of internal channel structures are combined to form the array of microfluidic channels.
A collecting electrode is used to attract the product to be analyzed at an inlet of the sensor. The sensor includes a field effect based sensor, nanowire sensor, nanoribbon sensor, or ion sensitive field effect transistor, and is capable of applying a confining dielectrophoretic force, trapping the product to be analyzed.
The pump may be a micro-pump operating in tandem with micro-valves to achieve a fully automated pathogen detection filtration system capable of miniaturization to a chip-scale design.
The apparatus may also include a microfluidic transport module for transporting the product to be analyzed to a location in the vicinity of the sensor.
In a second aspect, the present invention is directed to an apparatus for pathogen detection comprising: a microfluidic assembly including a plurality of microfluidic channels forming an array, each of the microfluidic channels including: electrodes for establishing dielectrophoretic forces on a test sample separating the portions of the test sample into an analyte and a waste product; and adjacent microchannels for receiving the waste product attracted by a dielectrophoretic force, removing the waste product from the analyte; a condenser including an electrode for localizing the analyte for sensing; and a sensor for detecting the analyte.
In a third aspect, the present invention is directed to a method of pathogen detection comprising: providing a test sample including an analyte together with a waste product in a first chamber; providing a reference solution in a second chamber; transporting the test sample through a plurality of microfluidic channels; generating opposing dielectrophoretic forces on the test sample as the test sample is transported through the plurality of microfluidic channels; separating the waste product from the analyte by the opposing dielectrophoretic forces and directing the waste product to a third chamber and the analyte to a condensing area; condensing the analyte in a localized area; flushing the analyte with the reference solution to remove substantially all of remaining waste product from the condensed analyte; detecting low amounts of analyte using a microfluidic sensor.
Dielectrophoretic manipulation of the analyte to the microfluidic sensor is achieved to overcome a diffusion limitation and enable analyte contact with the microfluidic sensor surface for detection.
A pharmaceutical or other substance may be introduced which pierces membranes of an alive analyte component in the reference solution at a predetermined frequency, but not membranes of other analyte components or dead analyte components, and differentiating the alive component from other analyte components and dead analyte components through dielectrophoretic forces.
The method uses the change of a Clausius-Mossotti factor, CM(ω), upon a change of a medium permittivity, ∈m, for analyte capture and release.
The process of capturing the analyte includes: applying an electric field causing a positive dielectrophoretic force to the analyte to facilitate analyte capture; changing the Clausius-Mossotti factor by the flushing; and causing a negative dielectrophoretic force under the electric field acting on the analyte with the changed Clausius-Mossotti factor to facilitate release of the analyte.
The pH or conductivity of the test sample may be adjusted for control of voltage and frequency dependence for the Clausius-Mossotti factor cross-over frequency.
Change of the Clausius-Mossotti factor cross-over frequency may be induced for the analyte by adding or mixing an additional fluid.
The step of separating the waste product from the analyte by the opposing dielectrophoretic forces and directing the waste product to a third chamber and the analyte to a condensing area includes: providing a microfluidic assembly including a plurality of microfluidic channels forming an array, each of the microfluidic channels including: electrodes for establishing dielectrophoretic forces on the test sample separating the portions of the test sample into the analyte and the waste product; and adjacent microchannels for receiving the waste product attracted by a dielectrophoretic force, removing the waste product from the analyte; transporting the analyte to the condenser area; and localizing the analyte at the condenser area by a condenser electrode.
The step of detecting low amounts of analyte using a microfluidic sensor includes using an electric field at predetermined flow conditions, to immobilize the analyte on the surface of a field effect based sensor, nanowire sensor, nanoribbon sensor, or ion sensitive field effect transistor sensor.
Superpositioning or tuning of frequency components, waveform shapes, and waveform tunings, or any combination thereof, may be performed to maximize separation, differentiation, capture, or release of the analyte.
In a fourth aspect, the present invention may be directed to a filtration system for pathogen detection comprising: microchannels for fluid transport; a dielectrophoretic separator for separating the fluid into constituent components; a dielectrophoretic condenser for condensing at least one constituent component of the fluid; a dielectrophoretic transport module; and a field effect based sensor, nanowire sensor, nanoribbon sensor, or ion sensitive field effect transistor, or any combination thereof, for detecting the at least one constituent component of the fluid.
The dielectrophoretic separator includes a parallel multichannel structure having a plurality of microfluidic channels in multiple stacked layers, and branched with waste channels.
The features of the invention believed to be novel and the elements characteristic of the invention are set forth with particularity in the appended claims. The figures are for illustration purposes only and are not drawn to scale. The invention itself, however, both as to organization and method of operation, may best be understood by reference to the detailed description which follows taken in conjunction with the accompanying drawings in which:
In describing the preferred embodiment of the present invention, reference will be made herein to
The filtration system of the present invention performs pathogen detection using a plurality of dielectrophoretic modules of microfluidic channels with distinctive functionality and geometry to obtain separation performance which cannot be obtained in the prior art. Additionally, the present invention integrates a nano-scaled sensor with the filtration system. Advantageously, all components of the filtration system may be embedded, forming an integrated electronic-microfluidic circuit.
The assembled filtration system automatically transports, separates, condenses, and detects low amounts of particles, cells, and bacteria, or the like, from liquids in a portable configuration that minimizes false positives and negatives.
The present invention defines a robust method for separating bacteria from blood components based on their dielectric properties, localizing the bacteria, flushing substantially all remaining by-products from the bacteria, which generally are on the order of micro-sized or microscaled components, and detecting low concentrations of the bacteria. The separation is fast and reliable as species movement is caused by a module array imparting dielectrophoretic forces, which may be opposing forces for enhancing separation. The module array includes a plurality of microfluidic channels with connecting microfluidic waste channels for directing undesired material away from bacteria. The process enables separation of low concentration of bacteria or other pathogens or particles from blood or other fluids, which then enables detection of the low concentration of these species. This is of great importance for medical diagnostics and determining food safety.
In a preferred embodiment, the invention includes an electronic device capable of detecting of a low number of bacteria or other pathogens or particles from milliliter or larger volumes of different liquid media on a minute time scale by integrating a plurality of modules of microfluidic channels capable of performing a dielectrophoresis-based separation, and incorporating a unique capture system using field-effect-transistor based biosensors.
Bacteria present in a sample even of different types will be subject to a dielectrophoretic force in one direction, whereas all large blood components red blood cells and white blood cells will be subject to a dielectrophoretic force in another direction. Effective separation improves the detection of pathogens. Without separation, blood components which constitute a vast majority of micro-sized particles in blood, would clog active sensor sites and prevent detection of low concentrations of bacteria present in the same sample.
Furthermore, many types of sensors cannot operate in high ionic solutions such as blood plasma. Consequently, the solution containing the sample to be sensed, most likely bacteria, has to be changed to a more suitable reference solution, such as a buffer. Often a pre-concentration step is required. The capture/release mechanism presented by the present invention provides an excellent method for solution exchange without cell loss.
Electric field cell capture, release, and separation based on forces acting in opposite directions allow precise control of cell separation without risk of cell loss or contamination. Advantageously, the present invention may be applied for separating any species of comparable size in any liquid medium; however, bacteria separation from blood components, white blood cells and red blood cells, is illustrated for exemplary purposes, and represents a predominate utilization of the present invention.
Unlike the prior art, the microfluidic separator 16 of the present invention includes a plurality of channels that apply dielectrophoretic forces that are exerted on the particles, cells, bacteria, and/or micro-scale components as they flow through the channels. The dielectrophoretic field is carefully chosen such that the components of interest that flow through will experience an opposite force as compared to the rest of the components or waste-products that are desired to be separated out. The waste-products are drained throughout the process from the plurality of microfluidic channels to waste chamber 22 through multiple microfluidic channel outlets.
In this manner, the filtration system of the present invention is composed of modules/segments each tuned such that the class of objects under study (i.e., the analyte) has the same response. For example, all bacterial have an nDEP, which is in the middle of the response spectrum. An additional “filter” is then applied for increased accuracy of targeting the analyte. The assembly of independent modules for this application is comprised of multiple, but not necessarily continuous, wires. Microchannel outlets, waste channels, break the continuous wire configuration. Using this geometry, surface electrode configurations may be employed, moving away from continuous conducting wire of the prior art.
In this example, isolated bacteria flows into a condenser chamber 24, which has a collecting electrode to attract the bacteria to the inlet of a microfluidic sensor 26 containing sensor arrays. Movement of bacteria to the field-effect-transistor based sensor is enhanced using the electric field and the dielectrophoretic force to overcome the diffusion limitation of the motion. Furthermore, the present invention is capable of tuning the electric field such that predominantly only the particle of interest is isolated, the remaining product is substantially eliminated. Thus, the analyte may be detected without any labeling or marking process steps, since the analyte is substantially isolated from other related product. This makes the detection essentially label-free, it does not require sensor functionalization with specific antibodies or other known tags for labeling.
The method is based on dielectrophoretic separation followed by dielectrophoretic concentration, and replacement or partial replacement or dilution of the original liquid with at least one reference solution, preferably a conducive solution. The next step is dielectrophoretic manipulation of bacteria to the sensor surface to overcome the diffusion limitation and enable bacteria contact with the sensor surface for detection.
The device operation and automated sample preparation is described in some detail below. First, a test sample injection is distributed into the system. This is performed by a pump that causes the automated distribution of the test sample, placing the test sample in a plurality microfluidic channels (microchannels) via capillary forces (porous media) and pump-pressure driven flow. Each of the plurality of microfluidic channels are lined with electrode geometry capable of establishing an electric field and a dielectrophoretic force on the test sample. Separation within the microfluidic channels is then performed by the dielectrophoretic force. In order to achieve adequate and efficient separation, waveform tuning of the electric field is selected with the intention that two types of species are subjected to forces acting in opposite directions. The separation occurs within microfluidic separator 16. The unwanted micro-scaled components and blood cells (waste-product) separated from the analyte (bacteria/cells/particles) are collected in waste chamber 22. The separated analyte is then collected on condenser 20. An electrode immobilizes the desired analyte material.
In order to remove the waste-product, extraneous serum and other unwanted blood products, the remaining analyte is exposed to a reference solution while held by the condenser electrode. In this manner the unwanted blood products are flushed away, replaced or at least partially replaced, and/or diluted by the reference solution. The remaining analyte is localized to a sensor surface. Dielectrophoretic manipulation of bacteria is used at the sensor surface to overcome the diffusion limitation and enable bacteria contact with the sensor surface for detection.
Preferably the sensor is of nanowire or nanoribben technology, which enables the filtration system of the present invention to be integrated on a semiconductor chipset. Once the final analyte is interrogated, the output may be digitized for automated data processing and readout.
In a preferred embodiment, a multi-step approach to filtration for pathogen detection is achieved using a plurality of dielectrophoretic modules including a plurality of microfluidic channels in an array fashion. The microfluidic separator 16 separates the test sample components of interest (e.g., bacteria) from pollutants (e.g., blood cells and blood serum). In a subsequent process step, the surrounding medium is then exchanged or diluted with a reference solution more suitable for comprehensive electronic detection applications.
The process introduces condensation of the analyte onto a concentrating electrode using a dielectrophoretic (pDEP) force. Once all remaining analyte from the sample is collected on the condensing electrode and the remaining waste-product has been exchanged with the reference solution, the frequency of the applied electric field is then changed (generally from high to low) so that the dielectrophoretic force changes sign and becomes repulsive, and the analyte is then released into a small (˜1 μl) volume of the reference solution. Next, the analyte is transported to the sensor chamber and restricted in the vicinity of the sensor. Detection is then performed by sensor arrays, selectively functionalized for the target analyte (bacteria) of interest.
Microfluidic separator 16 is comprised of a high throughput system of multiple microchannels, preferably an array 10×100 microchannels although any number of microchannels may be utilized with varying degrees of efficiency.
In a preferred embodiment, array 40 is composed of multiple plates 36 sandwiching internal channel structures 38 such that when stacked they form an array of 10 (horizontal)×100 (vertical) microfluidic channels. The 11 layers are aligned to the edges and thermally bonded.
In an assembled microfluidic separator 16, laminar flow conditions are provided for separation. Under preferred operating conditions, flow velocity, v, is approximately 100 μm/s, channel length, L, is about 1 cm, the total flow time through a single channel, tflow, is on average about 100 s, and the flow rate per channel is about 1 nL/s. Thus, in total 1 cc is pumped very quickly through 1000 microchannels.
The test sample throughput may be “tuned” by increasing or decreasing the number of parallel microchannels, increasing or decreasing the parallel stacked microchannel assemblies 30, and changing the flow velocity by setting the pumping speed.
The invention utilizes the frequency dependence of the sign of the CM factor between different contaminant/blood species for separation in a series of custom designed dielectrophoretic modules.
For the purposes of the present invention, a coefficient α will be defined as follows:
α=2π∈m r3 Re{CM(ω)}; and
F
DEP
=α·∇E
2;
where CM(ω) is as defined previously. The α coefficient accounts for the particle size r and the dielectric properties of the particle itself ∈p and the surrounding medium ∈m.
Thus, the invention provides for separation of species based on the different signs of the Re{CM(ω)} factor and which follows the different signs of the α coefficient at a chosen operating frequency. The separation method of two select groups of the components of interest, i.e., pathogens/cells/bacteria/particles (group 1) and blood cells (group 2), is based on tuning the electric field frequency such that the Re{CM(ω)} factor is positive for one group and negative for the other group. This causes component movements in different directions, which leads to separation. Unlike the prior art, the microfluidic separator is uniquely designed to complete separation, and the subsequent condensing and flushing process steps result in pure isolation of the bacteria, cells, or particles of interest.
A description for bacteria in blood is provided below for the separation, capture, and release mechanisms, and for flow conditions inside a microfluidic channel. Figures of force fields are generated from program runs using COMSOL Multiphysics software.
An electric field gradient is generated by an electrical waveform applied to sets of electrodes on the plurality of microchannel walls.
As an illustrative example, the values of a are selected for E. Coli bacteria, red blood cells, and white blood cells based on pre-determined permittivity data (real and imaginary permittivity ∈p, ∈m, particle radius r, and conductivity σ).
The dielectrophoretic force acting on E. Coli bacteria, red blood cells (RBC), and white blood cells (WBC) is generated by applying a voltage, which in the preferred embodiment is approximately 20V, on the electrodes at different operating frequencies. Selected analytical values for the coefficient α for RBC, WBC, and bacteria at 10 MHz in blood serum are shown in Table I identified in
In Table I (
In this example, α is negative for bacteria and positive for the blood components, thus effecting separation under dielectrophoretic force. At an electric field frequency of 10 MHz, and using blood serum as a surrounding medium, bacteria (E. Coli and Micrococcus), experience a negative dielectrophoretic force, while at the same operating conditions the blood components, WBC and RBC experience a positive dielectrophoretic force. Bacteria and red blood cell trajectories upon applied dielectrophoretic force in the microchannel are depicted in
The dielectrophoretic force acting on red blood cells is directed towards the field maximum, where the waste collecting electrode is placed. The dielectrophoretic force confines bacteria within a certain “safe” region of the microchannel as shown in
In the current example, utilizing the preferred array geometry for the microfluidic separator array with lateral and vertical DEP electrodes, the provided separation efficiency of E. Coli from RBC and WBC components was nearly 95% in about 15 seconds, and 100% for an approximately 100 micron channel length in a timeframe of approximately one minute. The microfluidic separator comprising an array of microfluidic channels, each acting to separate the test sample and direct waste-product towards a waste chamber.
Unique to the present invention, a branched microfluidic design allows for separated components to be discarded as waste, while the target of interest, for example E. Coli, is transferred to a condenser, flushed, and then localized for pathogen detection by an electronic sensor. The invention is not dependent upon a single critical dimension fabrication or alignment, and the waveform frequencies may be tuned to change the differential sign of the Re{CM(ω)} factor for different components to be separated. The cross-over frequency varies for different particles, bacteria, and/or cells in different media.
The values of the α coefficient for bacteria E. Coli and Micrococcus in buffer solution and blood serum at frequencies 10 MHz and 400 Mhz are provided in Table I of
Continuing with the example above, the α coefficient for E. Coli and Micrococcus is negative and has a different magnitude in blood serum at 10 MHz, which for E. Coli α=−0.0044(10−24) J(m/V)2, and for Micrococcus α=−0.0027(10−24) J(m/V)2, while the α coefficient is positive and has a similar magnitude in blood serum at 400 MHz (E. Coli α=0.0044(10−24) J(m/V)2, Micrococcus α=0.0043(10−24) J(m/V)2). Micrococcus and E. Coli will experience a very similar force in blood serum at 400 MHz, while they will experience a very different (opposite) force in the same medium, blood serum, at a frequency of 10 MHz.
The α coefficient for T. Lymphocytes is positive (α=0.0136(10−24) J(m/V)2) in blood serum at 10 MHz. Thus, the DEP force (negative DEP) exerted on bacteria in blood serum at 10 MHz has an opposite sign then the DEP force (positive DEP) exerted on T. Lymphocytes in blood serum at 10 MHz.
Consequently, a waveform applied to the electronic device of the present invention, containing only a frequency component at 400 MHz will result in a very similar behavior of both E. Coli and Micrococcus, causing similar motion of both products. A waveform applied to the electronic device containing only a frequency component at 10 MHz will result in a similar motion of both E. Coli and Micrococcus, and this motion will be in the opposite direction of T. Lymphocytes.
A waveform applied to the device containing both frequency components 10 MHz and 400 MHz will result in a motion of Micrococcus while the force will cancel for E. Coli, resulting in a lack of motion of E. Coli.
A choice of a waveform in the same medium allows differentiating and fingerprinting different species. Unique to the present invention, a sequence of an array of modules with tuned waveforms would allow selecting species based on their unique dielectric function.
After passing through the segments of microfluidic separator 16, the first component of the filtration system, the targets of interest (e.g., bacteria) are separated from pollutants (e.g., blood cells), at which point, the targets of interest are then condensed by condenser 20.
In a preferred embodiment, condenser 20 uses the change of the Re{CM(ω)} factor upon the change of the medium permittivity (∈m) for species capture on a capturing electrode, to reduce the volume of the sample and condense the species bacteria, cells, and/or particles in a significantly lower volume. A collecting electrode attracts the bacteria to the inlet of a microfluidic sensor 26 containing sensor arrays. Movement of bacteria to a field-effect-transistor based sensor is enhanced using the electric field and the dielectrophoretic force to overcome the diffusion limitation of the motion.
At a matching frequency, the Re{CM(ω)} in the medium surrounding the species is positive, which results in a positive dielectrophoretic force directed towards a capturing electrode. The set of electrodes and their geometry in the microchannel cross-section is shown in
To enhance separation, overcome the limitation caused by high ionic strength of the solution, and obtain functional analyte (bacteria) response, the initial medium (e.g., blood serum) is diluted and partially replaced by the buffer solution. As a result, the dielectric constant of the medium ∈m changes and the Re{CM(ω)} factor changes resulting in a change of the magnitude and potentially direction of the force.
The change of the Re{CM(ω)} factor upon the change of the particle/cell/bacteria permittivity (∈p) is used to obtain a differential functional response.
This form of α-screen testing allows for a portable platform for rapid multiplexed analyte detection, such as bacteria, from blood samples of ill patients at a point-of-care application. Doctors would be able to diagnose the bacteria of infection, and accurately prescribe only the necessary antibiotic, resulting in a more efficient disease treatment, and limiting antibiotic-resistance formation.
Using the apparatus of the present invention, this a-screen testing does not require additional laboratory space, and is low in energy consumption. It may be used with a sensor network integrated with food processing lines in food processing plants for continuous food product quality monitoring, or used in food storage and transport. It may be integrated in a hand-held unit for rapid Vibrio cholera and E. Coli bacteria detection from water samples to determine water safety.
By introducing to the medium a reference solution, such as a buffer, and additional pharmaceuticals, the dielectric constant of the medium ∈m changes, the dielectric constants of the particles/bacteria/cells ∈p change, and Re{CM(ω)} change for different species, resulting in a change in FDEP allowing to distinguish between the analyte components.
Using the previous values as an illustrative example, the α coefficient for E. Coli and Micrococcus is negative and has a different magnitude in blood serum at 10 MHz (E. Coli α=−0.0044 (10−24) J(mN)2, Micrococcus α=−0.0027 (10−24) J(mN)2), while a is positive in a PBS buffer solution at 10 MHz (E. Coli α=0.0055 (10−24) J(mN)2, Micrococcus α=0.0106 (10−24) J(mN)2}. The force FDEP on Micrococcus in serum will have a lower magnitude than on E. Coli; however, in a buffer solution (such as PBS) the force on Micrococcus will be stronger than on E. Coli. Introducing a pharmaceutical or a substance (antibiotic) which pierces only the membrane of alive Micrococcus at 10 MHz in PBS, but not the membrane of E. Coli or dead Micrococcus in buffer will allow differentiating alive from dead Micrococcus and E. Coli, since the dielectrophoretic force depends on the size of the particle/bacteria/cell, where FDEP is proportional to r3.
Thus, a tuned chemical modification of the medium allows differentiating and fingerprinting different species. A sequence of modules with tuned chemical modifications will allow species selection.
In the preferred embodiment, the invention applies an electrical waveform and a dielectrophoretic force for enclosing the separated bacteria in a small volume around a sensor to significantly decrease diffusion time to the sensor. Bacteria trapping on a nanowire or nanoribbon sensor is a resultant of the dielectrophoretic trapping mechanism and surface modification of the sensor for capture. In this manner, a dielectrophoretic force is used as a confining force for trapping micro-sized blood components (RBC, WBC, bacteria, and the like).
The DEP capture mechanism for bacteria decreases the volume of diffusion of a product of interest (particle, bacteria, and/or cell) in the sensor chamber, and decreases the time for the product of interest to diffuse towards the sensor surface, which is necessary for detection.
The electronic device that implements this separation may be miniaturized to an integrated circuit, and does not require trained personnel—the user only introduces a sample (such as blood or water) into the inlet chamber, and an automated process performs sampling, separation, condensation, transport, and detection. Using dielectrophoresis, the device automatically separates any present bacteria from the rest of the sample—for example, with blood, the large blood components (e.g., red and white blood cells). The separated bacteria are concentrated by a second dielectrophoretic region, and finally detected using label-free nanosensors which may be functionalized with bacteria specific antibodies for selectivity.
The levels of integration of the present invention on an integrated circuit chip are generally depicted by the fabrication steps of
Step B of
Step C adds insulator layer 76 with openings for the separator, the condenser chamber, and a microfluidic channel connecting chambers, e.g. the condenser with the sensor chamber 77.
Step D depicts the addition of the microfluidic separator module 78.
Step E adds an insulator layer forming the walls of the condenser 79, an electrode 80, and outlet 81 from the sensor chamber.
Step F adds insulator layer 82 forming the walls of the test sample chamber 83, buffer/reference liquid chamber 84, waste chamber 85, and the insulator layer 86 forming the walls of the separator.
Step G adds lid 87 with inlets to the chambers for sample, liquid storing, inlets 88 to the separator, and outlet 89 from the sensor chamber and waste chamber.
In one embodiment the electrical connections and microfluidic connections between components are provided in embedded layers as shown in
While the present invention has been particularly described, in conjunction with a specific preferred embodiment, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art in light of the foregoing description. It is therefore contemplated that the appended claims will embrace any such alternatives, modifications and variations as falling within the true scope and spirit of the present invention.
Thus, having described the invention,
| Number | Date | Country | |
|---|---|---|---|
| 61553413 | Oct 2011 | US | |
| 61557654 | Nov 2011 | US |
