Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
  • G01N15/147—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/0005—Field flow fractionation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
  • G01N2015/1415—Control of particle position

Definitions

• This invention relates to cell analysis and sorting devices and methods for manipulating cells using these devices. More particularly, the invention relates to a cell analysis and sorting apparatus that can capture and hold single cells at known locations and then selectively release certain of these cells. A method of manipulating the cells using the cell analysis and sorting apparatus is also provided.
• MEMS micro electromechanical systems
• Manipulation of cells is another application of MEMS.
• Sato et al. described in his paper, which is hereby incorporated by reference, Individual and Mass Operation of Biological Cells using Micromechanical Silicon Devices, Sensors and Actuators, 1990, A21-A23.948-953, the use of pressure differentials to hold cells.
• Sato et al. microfabricated hydraulic capture chambers that were used to capture plant cells for use in cell fusion experiments. Pressure differentials were applied so that single cells were sucked down to plug an array of holes. Cells could not be individually released from the array, however, because the pressure differential was applied over the whole array, not to individual holes.
• DEP refers to the action of neutral particles in non-uniform electric fields.
• Neutral polarizable particles experience a force in non-uniform electric fields which propels them toward the electric field maxima or minima, depending on whether the particle is more or less polarizable than the medium it is in.
• an electric field may be produced to stably trap dielectric particles.
• Microfabrication has been utilized to make electrode arrays for cell manipulation since the late 1980s.
• researchers have successfully trapped many different cell types, including mammalian cells, yeast cells, plant cells, and polymeric particles.
• Much work involves manipulating cells by exploiting differences in the dielectric properties of varying cell types to evoke separations, such as separation of viable from non-viable yeast, and enrichment of CD34+ stem cells from bone marrow and peripheral blood stem cells.
• More relevant work on trapping cells in various two- and three-dimensional micro fabricated electrode geometries has been shown by several groups. However, trapping arrays of cells with the intention of releasing selected subpopulations of cells has not yet been widely explored.
• the present invention provides a cell sorting apparatus that is capable of monitoring over time the behavior of each cell in a large population of cells.
• the cell analysis and sorting apparatus contains individually addressable cell locations. Each location is capable of capturing and holding a single cell, and selectively releasing that cell from that particular location.
• the cells are captured and held in wells, and released using vapor bubbles as a means of cell election
• the cells are captured, held and released using electric field traps
• the cell analysis and sorting apparatus has an array of geometric sites for capturing cells traveling along a fluid flow
• the geometric sites are arranged in a defined pattern across a substrate such that individual sites are known and identifiable
• Each geometric site is configured and dimensioned to hold a single cell
• each site contains a release mechanism to selectively release the single cell from that site Because each site is able to hold only one cell, and each site has a unique address, the apparatus allows the user to know the location of any particular cell that has been captured Further, each site is independently controllable so that the user is able to arbitrarily capture cells at select locations, and to release cells at various locations across the array
• the geometric sites are configured as wells As a fluid of cells is flown across the array of specifically sized wells, cells will fall into the wells and become trapped Each well is sized and shaped to capture only a single cell, and is configured such that the cell will not escape into the laminar flow of the fluid above the well
• the single cell can be held inside the well by gravitational forces
• Each well can further be attached via a narrow channel to a chamber located below the well. Within the chamber is a heating element that is able to induce bubble nucleation, the mechanism for releasing the cell from the site.
• the bubble creates volume expansion inside the chamber which, when filled with fluid, will displace a jet of fluid out of the narrow channel and eject the cell out of the well Fluid flow above the well will sweep the ejected cell away to be either collected or discarded
• the geometric sites are formed from a three-dimensional electric field trap.
• Each trap comprises four electrodes arranged m a trapezoidal configuration, where each electrode represents a corner of the trapezoid
• the electric fields of the electrodes create a potential energy well for capturing a single cell within the center of the trap.
• the cell is ejected out of the site and into the fluid flow around the trap. Ejected cells can then be washed out and collected or discarded.
• an integrated system can be a microfabrication-based dynamic array cytometer ( ⁇ DAC) having as one of its components the cell analysis and sorting apparatus previously described.
• ⁇ DAC microfabrication-based dynamic array cytometer
• the cells can be placed on a cell array chip containing a plurality of cell sites.
• the cells are held in place within the plurality of cell sites in a manner similar to that described above and analyzed, for example, by photometric assay.
• the response of the cells can be measured, with the intensity of the fluorescence reflecting the intensity of the cellular response.
• the cells exhibiting the desired response, or intensity may be selectively released into a cell sorter to be further studied or otherwise selectively processed.
• Such an integrated system would allow researchers to also look at the cell's time response.
• FIGS. 1A, IB, 1C, and ID show the mechanism by which one embodiment of the present invention uses to capture, hold and release a single cell.
• FIGS. 2 A, 2B, and 2C show a process by which another embodiment of the present invention uses to capture, hold and release a single cell.
• FIGS. 3 A and 3B show a top-down view of the cell sorting apparatus of FIG. 2.
• FIG. 4 shows an exploded view of the cell sorting apparatus of FIG 2.
• FIG. 5 shows an exploded view of yet another embodiment of the present invention in which a cell sorting apparatus is integrated into a fluorescence-detecting system.
• FIG. 6 is the thermodynamic pressure-volume diagram for water.
• FIG. 7A shows a top view of a resistor of the present invention.
• FIG. 7B shows a cross-section of the resistor of FIG. 7A.
• FIG. 8 shows thermal resistances as seen by a heater of the present invention.
• FIGS. 9A and 9B show flow lines for flow over rectangular cavities of different aspect ratios.
• FIG. 10 shows a schematic of forces on a particle in a well.
• FIG. 1 1A shows a top view of a heater of the present invention.
• FIG. 1 IB shows a cross-section of the heater of FIG. 1 1A.
• FIG. 12A shows a side view of a cell well of the present invention.
• FIG. 12B shows a top-down view of the cell well of FIG. 12A.
• FIGS. 13A, 13B, and 13C shows a top-down view of a silicon processing mask set for use in the present invention.
• FIG. 14 shows a top-down view of a glass processing mask.
• FIG. 15 shows a diagram of a flow system for testing devices of the present invention.
• FIG. 16A shows a top-down view of a flow chamber of the present invention.
• FIG. 16B shows a side view of the flow chamber of FIG. 16A.
• FIG. 17 is a graph of pressure drop vs. flow rate for the flow chamber of FIGS. 16A and 16B.
• FIG. 18A shows a top-down view of the chamber base of flow chamber of FIG. 16A and l6B.
• FIG. 18B shows a side view of the chamber base of FIG. 18 A.
• FIG. 18C shows a top-down view of the chamber lid of flow chamber of FIG. 16A and 16B.
• FIG. 18D shows a side view of the chamber lid of FIG. 18C.
• FIGS. 19A-19C show a process of fabricating a glass slide of the present invention.
• FIGS. 20A-20H show a process of fabricating a silicon wafer of the present invention.
• FIGS. 21 A-21D show a process of assembling the silicon wafer of FIGS. 20A- 20H onto the glass slide of FIGS. 19A-19C.
• FIG 22 is a graph of temperature v resistance for platinum resistors of the present invention
• FIG 23 shows a configuration for a resistor testing apparatus used in the present invention.
• FIG 24 is a graph of current v voltage for the onset of boiling in platinum line resistors of the present invention.
• FIG 25 is a graph of current v temperature for the platinum line resistors of FIG 24
• FIG. 26 is a graph of temperature v. resistance for a set of annealed platinum line resistors of the present invention
• FIG. 27 is a graph of temperature v. resistance for a set of annealed platinum line resistors which were heated on a hot plate.
• FIG 28 is a graph of current v voltage for a set of annealed platinum line resistors of the present invention
• FIG 29 is a graph of current v temperature for the resistors of FIG. 28
• FIG. 30 is a graph of current v voltage for the resistors of FIG 28 under repeated boiling tests
• FIGS. 1A-1D illustrate an exemplary system of the present invention A cell site
• a well 12 sized and shaped to hold a single cell 18 Connected to the bottom of the well 12 is a narrow channel 14 that opens into a chamber 16 situated below the well.
• the well 12 and narrow channel 14 are etched out of a silicon wafer.
• the silicon wafer is attached to a glass slide on which there is a platinum heater 20, and the alignment is such that the heater 20 is sealed mside the chamber 16, which is filled with a fluid such as water
• the well 12 function as a capture and hold mechanism. In operation, fluid containing cells is flown over the top of the apparatus, and then the flow is stopped As shown in FIG.
• FIG IB shows how the well 12 is dimensioned and configured to hold only one cell 18 within the well 12 at a time
• the well 12 is configured such that the cell 18 will not be swept out of the well due to laminar or fluid flow above
• the cells exhibiting the desired characteristics may be selectively released from the wells
• the operator can apply a voltage to the heating element 20 within the chamber 16
• the heating element 20 is then heated to a temperature above the super mit of the fluid contained within the chamber 16 to initiate vapor bubble nucleation at the surface of the heating element 20, as seen in FIG IC
• a microbubble 22 is formed inside the chamber, creating a volume displacement
• the operator can control the size of the microbubble 22
• the volume expansion in the chamber will displace a jet of fluid within the chamber 16 out of the narrow channel 14, ejecting the cell 18 out of the well 12
• the released cell 18 can be swept into the fluid flow outside the well 12, to be later collected or discarded
• the cell site 30 includes electric field traps
• Figures 2A-2C show, in cross-section, two cell sites on a substrate such as a microfab ⁇ cated chip 36
• Each site includes a plurality of electrodes 32
• each cell site 30 contains four electrodes, positioned in a trapezoidal configuration, as seen in Figures 3A and 3B
• the cell site 30 is configured and positioned such that only one cell can be held within the site
• the electrodes 32 create a non-uniform electric field trap within which a single cell 34 can be held and subsequently released
• FIG 4 illustrates how the location and polarity of the electrodes 32 can create an electric field trap for capturing the cell 34
• cells in fluid medium flow over the cell sites 30, as shown in FIG 2A
• a potential energy well can be created within each cell site 30.
• the potential energy well is of sufficient strength to capture a single cell 34 traveling along the fluid flow and to hold the cell 34 within the center of the trap, as seen in FIG. 2B.
• FIG. 2C shows how this in turn removes the potential energy well, releasing the cell 34 back into the fluid flow. The cell 34 can then be collected or discarded.
• the electrodes forming the electric field trap are preferably thin-film poles formed of gold. This creates a three-dimensional electric field trap that is effective in holding a cell against the laminar flow of the fluid surrounding the electrodes.
• the cell sorting apparatus can contain anywhere from a single cell site to an infinite number of cell sites, for sorting mass quantities of cells.
• the embodiments herein are described as holding cells, it is understood that what is meant by cells includes biological cells, cellular fragments, particles, biological molecules, ions, and other biological entities.
• the cell sorting apparatus of the present invention allows the operator to know the location of each cell in the array of cell sites, the operator is able to manipulate the cells and arbitrarily sort the cells based on their characteristic under time-responsive assays.
• One such method contemplates using scanning techniques to observe dynamic responses from cells.
• an integrated cellular analysis system 100 is proposed in which cells are tested using light-emitting assays to determine the cell's response to stimuli over time.
• the integrated system can be a microfabrication-based dynamic array cytometer ( ⁇ DAC).
• ⁇ DAC microfabrication-based dynamic array cytometer
• the tested cells are placed on a cell array chip 110 similar to the cell sorting apparatus above, to be held in place within the plurality of cell sites, such as those described above.
• the response of the cells can be measured, with the intensity of the fluorescence reflecting the intensity of the cellular response.
• the cells exhibiting the desired response, or intensity may be selectively released, to be collected or later discarded.
• Any light-emitting assay in which the cell's response may vary in time is suited for study using this proposed system. It is ideally suited for finding phenotype inhomogeneities in a nominally homogeneous cell population. Such a system could be used to investigate time-based cellular responses for which practical assays do not currently exist.
• the researcher can look at its time response. Furthermore, the researcher can gain information about a statistically significant number of cells without the potential of masking important differences as might occur in a bulk experiment. Specific applications may include the study of molecular interactions such as receptor- ligand binding or protein-protein interactions. Signal transduction pathways, such as those involving intracellular calcium, can also be investigated.
• An advantage of the proposed integrated system is that the full time-response of all the cells can be accumulated and then sorting can be performed. This is contrasted with flow cytometry, where each cell is only analyzed at one time-point and sorting must happen concurrently with acquisition. Geneticists can look at gene expression, such as with immediate-early genes, either in response to environmental stimuli or for cell-cycle analysis. Another large application area is drug discovery using reporter-gene based assays. The integrated system can also be used to investigate fundamental biological issues dealing with the kinetics of drug interactions with cells, sorting and analyzing cells that display interesting pharmacodynamic responses. Another application is looking at heterogeneity in gene expression to investigate stochastic processes in cell regulation.
• the integrated system can be used in a clinical setting to diagnose disease and monitor treatment by looking for abnormal time responses in patients' cells.
• One objective of the present invention is to provide a cell analysis and sorting apparatus which uses hydraulic forces to capture individual cells into addressable locations, and can utilize microbubble actuation to release these individual cells from their locations.
• bubble nucleation There are two modes of bubble nucleation: homogeneous and heterogeneous. Homogeneous nucleation occurs in a pure liquid, whereas heterogeneous nucleation occurs on a heated surface. In a pure liquid containing no foreign objects, bubbles are nucleated by high- energy molecular groups. According to kinetic theory, pure liquids have local fluctuations in density, or vapor clusters. These are groups of highly energized molecules which have energies significantly higher than the average energy of molecules in the liquid. These molecules are called activated molecules and their excess energy is called the energy of activation. The nucleation process occurs by a stepwise collision process that is reversible, whereby molecules may increase or decrease their energy. When a cluster of activated molecules reaches a critical size, then bubble nucleation can occur.
• FIG. 6 is the thermodynamic pressure-volume diagram for water, which shows a region of stable liquid to the far left, stable vapor to the far right, metastable regions, and an unstable region in the center of the dashed curve.
• the dashed line is called the spinodal, and to the left of the critical point represents the upper limit to the existence of a superheated liquid.
• Equation ( 1-1 ) holds true, and within the spinodal, Equation ( 1-2 ) applies.
• thermodynamic superheat limit of water was computed. The results are shown below in Table 1.
• a kinetic limit of superheat may also be computed using the kinetic theory of the activated molecular clusters.
• the kinetic limit of superheat for water is about 300°C.
• T w is the surface temperature
• T ia is the saturation temperature ( 100°C for water)
• ⁇ is the surface tension
• h tg is the latent heat of vaporization
• p v is the vapor density
• ⁇ is the cavity radius
• the surface temperature necessary to nucleate bubbles in water with a surface that has a l ⁇ m cavity radius is about 133°C
• the temperature to nucleate a bubble is about 432°C, well above the highest thermodynamic water superheat limit of 322°C.
• agglomeration rate is insignificant at anneal temperatures below 700°C
• the onset of agglomeration can cause small voids in the platinum with radii of up to about 0.5 ⁇ m.
• heterogeneous nucleation would be possible at a temperature of about 166°C.
• L is the characteristic length for conduction and a is the thermal diffusivity of the material.
• a is the thermal diffusivity of the material.
• the Biot number measures the ratio of internal conduction resistance to external convection resistance. Since the Biot number was much less than unity, the lumped body approximation was used and an assumption was made that the entire resistor was at a uniform temperature.
• FIG. 8 shows the thermal resistances between the resistor and the ambient temperature. For the purpose of this order of magnitude estimate of the heat transfer mechanisms, steady state conditions were used in determining thermal resistances. First, the thermal resistance due to convection through the water was computed. For this case it was assumed there was natural convection since the water above the heater was stagnant, and boiling was not occurring. The thermal resistance due to convection was calculated below.
• L is the length through which heat conducts
• A is the cross-sectional area.
• the length through which heat conducts was very long (12mm) and the cross-sectional area was very small, resulting in a high thermal resistance:
• t is the platinum film thickness (0. l ⁇ m)
• Lp is the length through which heat conducts ( 12mm)
• w is the width of the resistor (3 ⁇ m)
• k Pt is the conductivity of platinum (71.5W/mK).
• L g is the length of glass through which heat conducts ( 1mm)
• k g is the conductivity of glass (0.81 W/mK)
• L is the length of the resistor (lOOO ⁇ m)
• w is the width of the resistor (3 ⁇ m)
• L w is the length of water through which heat conducts (450 ⁇ m)
• k w is the conductivity of water (0.67 W/mK).
• K is the thermal conductivity (0.61 W/mK for water and 0.88 W/mK for glass)
• q is the heat flux
• the subscript ' 1 ' denotes water
• '2' denotes glass.
• T 0 is the initial temperature of the body.
• the solution was also used to check the semi-infinite body assumption. For times equal to or less than 1 ms, and a reasonable heat flux such as 2.5x10 W/m , the heat penetration depths into the glass and water were less than lOO ⁇ m. The total thickness of the water was 450 ⁇ m and of the glass was 1mm, so the semi-infinite body assumption held true.
• the one-dimensional model was sufficient for determining the temperature of the resistor at small times.
• the heat flux necessary to heat the resistor to 305°C in 1ms was computed from ( 1-19 ) to be 1.32x10 W/m " .
• the necessary power was about 120mW.
• FIG. 9 shows the flow pattern for laminar flow over a rectangular cavity for two different width to height aspect ratios. From these flow patterns it was seen that there was a separating flow line which penetrates slightly into the cavity. Below this line there were one or two vortices, depending on the aspect ratio of the cavities. A particle below the separating flow line would not be swept out of the cavity by a slow flow in the laminar range, though the vortex may agitate the particle.
• FIG. 10 A diagram of a particle in a well with flow over the top is shown in FIG. 10.
• the force of gravity acting on the particle was dependent on the difference in density between the particle and the water, ⁇ p.
• the density of water is approximately 1000kg/m 3
• the density of the polystyrene beads used in the experiments was given by the manufacturer as 1060kg m 3 .
• the density of cells ranges from 1050-1 100kg/m 3 . Accordingly, the force of gravity, F g was computed as shown:
• the viscous shear force acting on the particle was computed by assuming the top of the particle was at the top of the well, and that the flow profile was parabolic.
• the shear stress at the wall was:
• ⁇ is the viscosity of water (1x10 " kg/ms) and u(y) is the velocity profile as a function ⁇ y, the distance from the wall.
• the flow profile was calculated for a known chamber height and volume flow rate.
• V Q_ wh ( 1-23 )
• V is the average flow velocity
• w is the chamber width
• h is the chamber height.
• the viscous shear force on the cell was estimated as the wall shear stress multiplied by the area being effected, approximately ⁇ a " .
• a is the sphere radius (5 ⁇ m for a polystyrene bead)
• p s is the density of the bead (about 1060kg/m )
• p is the density of water (lOOOkg/m )
• ⁇ is the viscosity of water.
• the Reynolds number is the ratio of inertial effects to viscous forces. For this case, only the highly viscous regime applied and inertial effects were negligible.
• Peclet number This is the ratio of sedimentation to diffusion. For the particles to settle, the Peclet number must be sufficiently high, otherwise the particles will diffuse throughout the liquid.
• ⁇ is the particle volume fraction (about 0.01 for this case). Accordingly, the time necessary for all the particles to settle to the bottom of the flow chamber was calculated using the hindered velocity and the chamber height, the maximum distance to be traveled.
• O is the volume flow rate
• ⁇ P is the pressure drop
• c is the aperture radius (-2.5 or 4 ⁇ m)
• ⁇ is the water viscosity
• the volume flow rate out of the chamber was estimated in a different way. Because water is incompressible, it was assumed in the model that the bubble formation as a volume injection into the chamber resulted in the same volume being ejected from the chamber over the characteristic bubble formation time. For instance, if it took 1ms to form a lO ⁇ m diameter bubble, then the resulting volume flow rate out of the chamber was calculated as follows.
• ⁇ p is the difference in densities between the water and the polystyrene beads (60 kg/m 3 ). It was seen that as the particle radius increased, the effect of gravity increased. For typical cells, the radius ranges from 5 ⁇ m (red blood cells) to 20 ⁇ m (most other cells) to lOO ⁇ m (embryos and eggs). This device will most likely be used for cells on the order of 5-10 ⁇ m in radius so the above calculation was representative of the expected applications.
• resistive heaters were used.
• the heaters were made of thin-film platinum on standard glass slides.
• the design constraint for this step was the need to keep the current density below the electromigration limit of platinum, while retaining an adequate degree of ohmic heating.
• the electromigration limit is the maximum current density which platinum can endure before the atoms begin to migrate leaving the resistor inoperable.
• the resistance of a line heater is calculated as follows.
• R is the resistance ( ⁇ )
• L is the length of the resistor (m)
• t is the film thickness (m)
• w is the width of the resistor (m)
• p is resistivity of platinum ( ⁇ m).
• the power output of a resistor is a function of the current and resistance, as shown below.
• FIG. 1 1A is a top view of a heater configuration, while FIG. 1 IB shows a cross-sectional view of the heater and its dimensions.
• Table 2 A table of resistor dimensions, maximum currents, and maximum power outputs is also shown in Table 2.
• the lines connecting the contact pads to the heaters were designed to have a far lower resistance than the heaters. This was done to ensure that the lines did not heat up, and that they remained approximately at the ambient temperature.
• the connector line widths were chosen to be 1500 ⁇ m with lengths of 12mm. The total resistance of each line was about 7.7 ⁇ .
• Photomasks for use in the device fabrication were created using standard mask layout software.
• the mask set for the silicon processing are shown in FIGS. 13A-13C and the glass mask set is shown in FIG. 14A and 14B.
• FIG. 13 A Three masks were designed for the silicon portion of the device processing.
• One mask was created for the cell wells (FIG. 13 A), one for the narrow channels within the wells (FIG. 13B), and one for the large wells (FIG. 13C) etched from the backside of the wafer to enclose the heaters.
• Two masks were made for the fabrication of the platinum heaters on the glass slides.
• One mask (FIG. 14) was designed to pattern the metal.
• a fluidic system as illustrated in FIG. 15 was designed and assembled.
• a syringe pump 150 was used as the flow source for the bulk fluid, and flow rates ranging from 1 to 100 ⁇ L/min were specified. Beads, cells, or cell stimuli were injected through the sample injection valve 152.
• a pressure sensor 154 was located before the flow chamber 156 so that the pressure drop across the chamber could be monitored. All fluid was outlet into a waste beaker 158 which could be reused if desired.
• FIGS. 16A and 16B A schematic of the flow chamber 156 is shown in FIGS. 16A and 16B.
• the flow chamber was machined from plexiglass so that it was clear and a microscope was used to observe cell behavior from above the chamber.
• HPLC high-performance liquid chromatography
• fittings were used with tube dimensions of 1/16 inch outer diameter and 0.020 inch inner diameter.
• the gasket between the slide and the top cover were made from PDMS (poly dimethyl siloxane), a flexible polymer.
• a seal was formed by screwing the top plate down onto the bottom plate.
• Aluminum molds were machined in order to create PDMS gaskets of the proper dimensions. Gaskets were compressed until a hard stop was reached. The stop was provided by the spacers, made of metal shim stock, in order to accurately specify the channel height.
• the aspect ratio of the channel's width to height was greater than 10, allowing the assumption of a parabolic velocity profile- plane Poiseuille flow.
• the height of the flow chamber was 790 ⁇ m (determined by thickness of metal spacer). Flow rates ranged from 1 to 100 ⁇ L/min and corresponded to Reynolds numbers of 0.001-0.1. In this creeping flow regime, the entrance length for fully developed flow was calculated to be negligible. These calculations are shown below.
• V mm is the minimum average velocity
• Q mm is the minimum volume flow rate ( 1 ⁇ L/min)
• V mm is the maximum average velocity
• Q mdK is the maximum volume flow rate ( lOO ⁇ L/min)
• Re is the Reynolds number
• v is the kinematic viscosity of water ( l xl 0 '6 m 2 /s)
• X e is the entrance length for fully-developed flow.
• the pressure drop through the tubing was calculated using the following equation.
• ⁇ is the viscosity of water ( lxl0 "3 kg/ms)
• r is the tube radius (0.254mm)
• zlx is the tube length (m).
• the pressure drop through the chamber was calculated to be negligible in comparison.
• the flow chamber schematic with dimensions is shown in FIGS. 18A-18D.
• the platinum heaters were fabricated on standard lx3in glass slides using a lift- off process.
• the process flow is shown in FIGS. 19A- 19C.
• photoresist was spun onto the glass slide, exposed using mask 4, and developed.
• lOOA of titanium and lOOOA of platinum were evaporated onto the slide, as seen in FIG. 19B.
• the titanium served as an adhesion layer between the glass and the platinum.
• the slide was submerged in acetone to dissolve the photoresist and lift away the metal which was deposited on top of the photoresist, as depicted in FIG. 19C. Only the platinum resistors were left on the glass slide.
• DSP four inch diameter silicon wafers were used.
• FIG. 20A l ⁇ m of thermal oxide was grown on the wafer.
• the oxide was patterned using mask 1, FIG. 20B.
• Resist was spun on top of the oxide and patterned using mask 2.
• FIG. 20C The resulting configuration was called a nested mask, shown as FIG. 20C.
• the photoresist mask was used to etch the narrow 5 ⁇ m trenches, then the oxide mask was used to etch the cell wells, as shown in FIGS. 20D and 20E.
• FIGS. 20D and 20E the wafer was turned over and photoresist was deposited and patterned on the back side using mask 3 (FIG. 20F).
• a deep silicon etch was then performed to etch through the wafer and intersect the narrow trenches etched previously (FIG. 20G) to obtain a finished wafer (FIG. 20H).
• a complete device consisted of a silicon chip attached to a glass slide by photoresist, as shown in FIGS. 21C and D.
• the resist provided a water-tight seal so that volume expansion in the bubble wells resulted in a burst of fluid being pushed through the narrow channel and ejecting a cell.
• alignment marks were fabricated on the glass slide and matching holes were etched in the silicon chip.
• the alignment tolerances were sufficiently large (about 2mm) that the chip could be aligned to the slide by hand using just the naked eye, while still positioning the bubble wells over the platinum heaters.
• Photoresist was painted onto the silicon chip around the bubble wells using a toothpick. Drops of water were deposited into each well using a pipette, then the glass slide was visually aligned from above and stuck down onto the chip. The drops of water served to fill the bubble wells and get pushed through the narrow channel to fill it with water. The device was now ready to be tested in the flow chamber. Next, the resistance of the platinum resistors were studied. The film thickness was first measured using a profilometer.
• the platinum thickness measurements ranged from about 800-900A, so the average value of 85 ⁇ A was used in the subsequent calculations.
• the resistance along metal lines wide enough not to be strongly affected by variation of a few microns was measured using a multimeter.
• the lines used for this measurement were measured in an optical microscope to be about 1510 ⁇ m wide.
• the length of the lines was about 8mm. Knowing the width, thickness, and length of these lines, as well as the measured resistance, the resistivity of the thin film platinum at room temperature was determined. The measured resistance was 15 ⁇ , and the computed resistivity was calculated below.
• the main objective for the resistors was that they be able to reach high enough temperatures to boil water.
• the resistors were tested on a probe station using an
• FIG. 23 is a schematic of this configuration.
• FIG. 24 An I-V curve for the onset of boiling on a line resistor is shown in FIG. 24.
• this I-V curve it is shown that for the first run when no bubbles were present on the line, there is a sharp jump in current at the onset of boiling.
• the second run residual bubbles were left on the heater and served as nucleation sites for boiling resulting in a smooth I-V curve with boiling beginning at a lower temperature. The two curves are very close after the boiling begins for run 1.
• the jump in the I-V curve occurred during each heating cycle for the resistors, since there were no residual air bubbles left when the power was turned off.
• the boiling points for the 5 resistors tested ranged from 250°C to 308°C.
• the lowest calculated value for the superheat limit of water was found to be 273°C, so these measured boiling points suggest that the bubble nucleation occurs either in the homogeneous regime, or by a weak heterogeneous mechanism.
• new resistor slides were annealed at 600°C for 1 hour as the last step in their process. This temperature is higher than operating temperatures are likely to reach, but not so high that major agglomeration will result. Once the anneal was complete, the new resistors were characterized as described above for the first generation resistors.
• the resistivity of the platinum at room temperature was found to be 2.056x10 '7 ⁇ m, less than the unannealed resistors that were 2.41xl0 "7 ⁇ m.
• the resistances were measured using a multimeter, and the line widths were computed as before, as shown in Table 6.
• the temperature-resistance characteristic or the resistors was then measured on a hotplate as described above, and is shown in FIG. 27.
• a resulting I-V curve is shown in Figure 28, and the corresponding temperature curve is shown in Figure 29. From the curve we can see that the onset of boiling occurred at about 200°C, a much lower temperature than for the first generation resistors, and well below the superheat limit of water. For the 8 second generation resistors tested, boiling points ranged from 128°C-200°C, with the majority of the temperatures above 180°C. This suggests that the boiling is in the heterogeneous nucleation regime as discussed earlier. The cavity radii corresponding to these boiling inception temperatures are calculated from Equation ( 1-59 ).
• bubbles were nucleated in radii ranging from 0.3- 1.2 ⁇ m. As discussed previously, these cavities were most likely formed during the 600°C anneal, during which the grooves at the grain boundaries widened creating cavities.
• the second generation resistors were also tested for the repeatability of their boiling temperatures. I-V curves were measured as in the previous section, and then remeasured for the same conditions several times. Between measurements, time was given for the vapor bubbles to dissipate so that the characteristic jump in the I-V curve at boiling could be observed with each measurement. The boiling point was found to be very repeatable, and an example of the results is shown in Figure 30. This result demonstrated the potential of a control system based on a jump in the I-V curve at the onset of boiling, since the boiling point remained fixed. Another interesting result from this testing is that for a particular resistor, the bubbles tended to nucleate in the same locations on the resistor each time. This strengthens the hypothesis that the bubbles are nucleating in the heterogeneous regime, in cavities created by thermal grooving caused by the annealing.
• the cell chip was attached to the glass resistor slide as described earlier, and then tested in two ways. First tests were done with stagnant fluid on the device. Then the device was put into the flow chamber for testing. The results of these tests are described below. For these tests, several drops of bulk solution were placed on top of the cell chip, and contained by the PDMS gasket. A drop of the polystyrene bead solution was then added to the bulk fluid and allowed to settle. The bulk solution was a 0.05% solution of Triton x-100 surfactant in deionized water. The bead solution was about 1% beads diluted in the same bulk solution. Some of the beads settled into wells, as shown in Figure 42.

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