Apparatus for transport and analysis of particles using dielectrophoresis

Dielectrophoresis is used to attract particles to an electrode edge then to controllably allow the transport of particles along that edge under a fluid flow to a particular region. The particles may be bacteria which may be maintained in this process in a live state through capture, transport and release.

BACKGROUND OF THE INVENTION

The present invention relates to the manipulation and analysis of particles and, in particular, to a method suitable for manipulating and analyzing live bacterial cells.

The ability to manipulate and analyze nanoscale particles is potentially valuable in the assembly of nanoscale structures, for example, nanorods or nanotubes, into more complex structures. Such techniques could also prove useful in manipulating and analyzing single biological cells such as bacteria.

The manipulation of electrically polarizable particles within a poorly polarizable material (or poorly polarizable particles within a polarizable medium) can be accomplished by placing the particles in a spatially inhomogeneous electric field. In the case of polarizable particles, the field will induce equal and opposite charges on the particle. Unequal field strength will exist on each side of the particle because of the field inhomogeneity, producing a net dielectrophoretic force that pulls the particle toward the greater field concentration.

Such techniques have been used to trap particles and cells at electrodes by drawing the particles and cells to the electrode, or to hold cells within a cage formed of symmetrically balanced electrodes that repel the cell.

While such techniques allow the capture of extremely small particles in a liquid, the ability to precisely control the movement of constrained particles or cells is relatively limited.

BRIEF SUMMARY OF THE INVENTION

The present invention provides controlled movement of particles by attracting the particles to an electrode edge with a reduced force that allows the particles to be conveyed along the edge under the influence of liquid flow. The density and spacing of the particles at the electrode edge may be managed to meter individual or small groupings of particles to a particular location for analysis or treatment and then to release those particles. The invention provides sufficient control of the particles to allow positioning of a single particle between a particle-sized gap between two electrodes for electrical analysis of the particle.

Specifically, the present invention provides a channel for flowing a liquid with suspended particles along a transport axis. A first electrode supported within the channel has an electrode edge extending along the axis. An electrical power source is attached to the electrode for generating a first signal. The first signal provides a dielectrophoretic force on the suspended particles of a strength drawing the particles to the edge while allowing the particles to move along the edge under the force of flowing liquid.

Thus, it is an object of at least one embodiment of the invention to provide for constrained movement of particles along a path defined by an electrode edge. By confining motion of the particles to a single dimension and taking advantage of mutual repulsion of the particles, precise metering and transport of particles may be obtained.

The particles may be bacteria and the electrical power source may provide a signal sufficient to draw the bacteria to the edge while allowing the bacteria to move along the edge under the flow of liquid. The signal may be set not to kill the bacteria.

It is thus another object of at least one embodiment of the invention to provide a transport mechanism suitable for cells and live cells.

The electrode may terminate within the channel at a downstream end adjacent to an analysis area.

Thus it is an object of at least one embodiment of the invention to provide a method of metering particles to an analysis area.

The electrode end may terminate in a sharpened point.

It is thus another object of at least one embodiment of the invention to provide a method of transporting particles to a point isolating the particle and facilitating analysis of one or a small grouping of particles.

The end may be adjacent to a second electrode in an electrical circuit with the power source and the first electrode. The first and second electrodes may be separated substantially by the size of one particle.

Thus it is another object of at least one embodiment of the invention to provide a method of positioning nanoscale particles between electrodes for electronic measurement.

The first signal may include a first component promoting dielectrophoretic force superimposed with a second component allowing independent measurement of the properties of conduction of the particles between the electrodes.

It is thus another object of at least one embodiment of the invention to provide for both transport and analysis of particles by the electrodes. It is another object of the invention to provide a device which may practically direct current through individual particles.

The apparatus may include an impedance measuring circuit communicating with the power source to measure the impedance between the electrodes.

Thus it is another object of at least one embodiment of the invention to provide for electronic detection and analysis of particles.

The power source may alternatively provide a signal drawing the particle to the edge while preventing the particle from moving along the edge under the flow of liquid.

Thus it is another object of at least one embodiment of the invention to provide for independent capture and transport of small particles along the electrode surface.

A controller may operate the power source to cease the electrical signals to release particles from the electrode after the analysis in the analysis area.

Thus it is another object of at least one embodiment of the invention to provide for the capture and release of cells for sequential sampling purposes.

The electrode may be angled with respect to the transport axis.

Thus it is another object of at least one embodiment of the invention to allow multiple electrodes having possibly divergent paths or convergent paths.

The apparatus may include an optical sensor for monitoring the presence of particles near at least one portion of the electrode.

Thus it is another object of at least one embodiment of the invention to allow the manipulation of particles also allowing optical analysis and/or detection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring now toFIG. 1, a particle transport system10per the present invention employs a channel12extending along the longitudinal axis14. The channel12provides generally an inlet16and outlet18opposed along the longitudinal axis14to allow fluid flow56through the channel12along the longitudinal axis14. In one embodiment, the channel12may be three millimeters long along the longitudinal axis14, two millimeters wide along a transverse axis (in and out of the page inFIG. 1) and 1.5 millimeters high. The channel12may be formed out of polydimethysiloxane (PDMS) molded into a channel shaped, for example, by application of liquid PDMS to an etched surface prepared using conventional machining or photolithography/etching techniques.

The fluid in the channel12, in one embodiment, may be water or other liquid holding in suspension nano-sized particles20, for example, nanospheres or nanorods or individual biological cells such as bacteria. A bacterium suitable for use with the present invention isBacillus mycoides, a rod-shaped bacterium approximately one micron wide and five microns long. The bacterium provides a rigid interior coupled with an organic exterior that presents sites that could be used for biomolecular recognition in lieu of bio-functionalized inorganic structures of other nanoparticles. Such bacteria are substantially smaller than protoplasts, yeasts and eukaryotic cells which are typically 10 to 50 microns in diameter. Generally “nanoscale” and nanoparticle as used herein will be particles having a longest dimension of less than 1000 nm, and more typically less than 500 nm or 100 nm.

The channel12provides longitudinally extending PDMS sidewalls closed by a transparent cover slip22on an upper face and a silicon dioxide (SIO2) coated silicon wafer24on a lower face. The latter silicon wafer24may be supported on a polyacrylic base (not shown).

The inner surface of the silicon wafer24facing the cover slip22and exposed to the liquid flowing through the channel12may support at least two longitudinally extending electrodes26and27having a longitudinal gap30therebetween and edges32extending along, but not necessarily parallel with, the longitudinal axis14.

An electrical signal is applied by an electrical power source33across the gap30and between the electrodes26and27. The electrical power source33includes two voltage sources. First, a high-frequency voltage source34provides a sine-wave signal of approximately one megahertz with a controllable amplitude ranging at least between 1.5 volts and 0.5 volts peak-to-peak. This signal will be used to provide dielectrophoresis forces on the particles20. The signal from the high-frequency voltage source34is summed with a signal from a second, low-frequency voltage source36producing a sine-wave signal of from zero to 10 kilohertz at approximately 10 millivolts. This signal will be used as a detection signal and an analysis signal as will be described.

The signals from the high-frequency voltage source34and the low-frequency voltage source36are combined by summing amplifier38and applied to one of the electrodes26. The remaining electrode27is connected through a current-to-voltage converter40which provides a virtual ground for the electrode27and thus a return path to the high-frequency voltage source34and low-frequency voltage source36. The current-to-voltage converter40may provide a sensitivity of 104volts/ampere.

A voltage output42from the current-to-voltage converter40is received by a low pass filter44having a cut off frequency providing passage of the signal from the low-frequency voltage source36but blocking the signal from the high-frequency voltage source34. This filtered signal is provided to a synchronous amplifier46of conventional design also receiving a signal directly from the low-frequency voltage source36to isolate asynchronous current provided by the low-frequency voltage source36. The demodulated output50from the synchronous amplifier46thereby provides a measure of low frequency current conducted between the electrodes26and27largely insensitive to capacitive and inductive effects.

The demodulated output50is then provided to an analog-to-digital converter (not shown) forming an input to a control computer52. The control computer52also incorporates to a digital-to-analog converter (not shown) applying a voltage control signal51to the high-frequency voltage source34controlling its amplitude as will be described. The control computer52may optionally receive a video signal53from a camera70viewing the electrodes26and27through the cover slip22as will be described further below

The control computer52is programmable to execute a stored program to control the voltage of the high-frequency voltage source34for various operating modes as will be described below and to output a graphical representation of data collected from the demodulated output50and video signal53using a human machine interface54such as display terminal, keyboard mouse and the like.

Referring now toFIGS. 2 and 3, an exemplary use of the particle transport system10ofFIG. 1provides a gentle liquid flow56of a liquid along the longitudinal axis14past electrodes26and27. For example, the liquid may be a 90 percent water, 10 percent glycerol mixture suspending bacteria as particles20, the liquid moving at a linear velocity of approximately 0.1 millimeter per second.

As indicated by process block60ofFIG. 3, the control computer52may first apply capture voltage from the high-frequency voltage source34across the electrodes26and27. This capture voltage, for example, a signal having 1.5 Volts peak to peak, causes some of the particles20to be drawn against the edge32of electrode26by virtue of the high electrical field gradient at the edge of the electrode26. Lower voltages such as 200 mV may also be used. While the capture voltage is applied, the captured particles20do not move significantly under the influence of the liquid flow56; however, if another particle20is captured, the adjacent particles will readjust their positions slightly. While Applicant does not wish to be bound by a particular theory, this readjustment may be a result of mutual electrostatic repulsion between the particles20caused by their induced charge.

The amplitude and frequency of the capture voltage can be used to discriminate between live and dead bacteria, and in addition is should be possible to discriminate between different species.

Referring to process block62after a predetermined period of time at which a desired number of particles20have been captured by the edge32, the control computer52may change the voltage of the high-frequency voltage source34to a transport voltage, for example, 0.5 volts peak-to-peak. Under this voltage, the particles20are transported downward along the edge32under the influence of the flow56of liquid while retained at the edge32.

In the example ofFIG. 2, the electrodes26and27provide for an opposed T-bar configuration with longitudinally extending electrode trunks57terminating in opposition at transversely extending T-bars59perpendicular to the longitudinally extending electrode trunks57. The T-bars59are separated by a gap30approximately equal to the longest dimension of the particles20.

While the control computer52continues to apply the transport voltage, particles20will continue to move in the direction of the flow56either passing around the T-bar59or across its top under the influence of the flow56. When at least one particle20is within the gap30, it is held against further movement by the force of two the transverse edges of the T-bars59of the electrodes26and27and thus may resist further movement with the flow56.

If the transport voltage is retained, then particles20will continue to accumulate within the gap30after moving conveyor-like along the edge32.

The gap30may be at an analysis area whereby analysis or treatment of individual particles20may be performed. This analysis, which may include detection, may be performed by the signal (for example 20 millivolts peak to peak) from the low-frequency voltage source36passing through the particle20from electrode26to electrode27, as will be described, but may alternatively be optical analysis using a camera70including but not limited to analysis with visual frequencies of light or fluorescence measurement using visible or ultraviolet light frequencies. The analysis may further include treatment of the individual particles20with reagents or other substances introduced near the gap30.

Referring toFIG. 3, once sufficient particles20have accumulated in the analysis area of the gap30, the capture voltage of process block60may be restored preventing additional particles from moving along the edge32into the gap30.

Upon completion of the analysis of the particular particles20in the gap30, the control computer52may change the voltage on the high-frequency voltage source34to a release voltage indicated by a process block64, for example 10 millivolts, allowing release of the particles within the gap30to continue with the flow56.

When the release voltage is applied, the particles20attached to the edge32are also released but because their natural trajectory is along the edge32they may be reattached to the edge32when the transport voltage of process block62is restored.

The application of capture, transport, and release voltage may be flexibly controlled and timed to manipulate the particles20into and out of the region of the gap30.

Applicant has determined that bacterial samples captured with this device using the described voltages may be released without damage to the bacteria. At larger voltages greater than 2 volts peak to peak, however, the bacteria are irreversibly immobilized possibly because of perforation of the cell walls.

Referring now toFIG. 4, an alternative electrode design provides a “teardrop” end to the electrodes26and27in which no surface of the ends is perpendicular to the flow56. Again the ends of the electrodes26and27are separated across a gap30substantially equal to the dimension of the particles20; however the gap30provides for opposed sharpened points66suitable for concentrating and locating a single particle20both longitudinally and transversely in a particular location. The gap30is approximately 3.5 microns for these electrodes. A “pearl-chain” structure, in which bacteria are aligned end-to-end, can be created using an electrode structure with a larger gap. In this process, one particle is captured and directed to the gap, and then another particle applied, etc, to create a controlled sequence of particles that is electrically verifiable.

The edge32of the electrodes26and27in this example are also not perfectly aligned with the longitudinal axis14. This ability to cant the electrode edges32allows diverging and converging electrodes that may be useful for sorting or separating bacterial or nanoparticle samples.

Referring again toFIG. 1, the location of a particle20within the gap30may be confirmed by means of the camera70coupled to a microscope objective focusing through the cover slip22to the gap30. Alternatively or in addition the present invention contemplates that the particles20arriving in the gap30may be detected electronically by monitoring the current attributable to the signal from low-frequency voltage source36. This current may be used to deduce the impedance across the gap using the known voltage of the low-frequency voltage source36(for example 20 mVpp) in Ohm's law and may be calculated by the control computer52.

A larger voltages may be used to provide a semi-permanent “fixation” of cells between electrode gap30. In this way, the cells may be adhered to particular locations and receptors on their surface as a scaffold for building more complex nano-structures. A voltage on the order of 2 V is appears to be sufficient to “glue” the bacteria in place to that a continued voltage is no longer required to hold them to the electrode.

Referring now toFIG. 5, a measurement of that current with time shows changes in current flow and thus impedance across the gap caused by the capture and release of bacterium at points labeled R for release and C for capture. As can be seen, the capture of bacteria particles20lowers the impedance across the gap30whereas the release provides for an abrupt increase in that impedance. A combination of video monitoring and impedance monitoring may be performed. The changes in current are not instantaneous but occur slowly over the period of about twenty seconds. While the Applicants do not wish to be bound by a particular theory, it is believed that in some cases bacteria do not bridge perfectly and make and break the electrical contact several times. It is possible that slow changes in the polysaccharide layer occur over the time span of twenty seconds to improve electrical contact. Over the course of several minutes, there is a steady increase in background current which is believed to be the result of ions that leak from the bacteria over time increasing solution conductivity. Controlled experiments using a solution lacking bacteria show no such increase.

Other types of electrical analysis of the particles20may be performed using this technique, including, for example, a frequency response, by sweeping the frequency of the sine wave signal from low-frequency voltage source36and monitoring impedance as a function of frequency. No notable differences in frequency response were observed between individual bacterium by the inventors; however, frequency response may help to distinguish other forms of nanoparticles including other types bacterium or man-made nanoparticles incidentally or by design having particular frequency response characteristics.

One benefit of the use of bacterial cells, as opposed to manmade nanoscale objects such as nanotubes and nanowires, is that the external surfaces of the bacteria may be engineered or selected to express specific proteins and thus may be further manipulated with secondary biological interaction such as antibody binding to create more complex nanoscale structures.

Generally, the ability to manipulate particles20by transporting them controllably along a defined edge32may be used in a variety of applications including the sorting of particular cells.