Patent Application: US-73950908-A

Abstract:
microsystem for monitoring cell growth . a microfluidic structure is designed to allow cells to circulate therethrough and the microfluidic structure includes modules to monitor mass , mass density and fluorescence of the cell .

Description:
with reference first to fig1 , a microsystem 10 includes a microfluidic rotary structure 12 through which cells 14 travel in single file . the cells are circulated by means of a pump 16 and analyte delivery module 18 delivers nutrients and analytes into the rotary structure 12 . as shown in fig1 , the cells 14 pass several modules that detect mass , mass density , fluorescence , and other properties of the cells . still referring to fig1 , a mass detector 20 is a suspended microchannel resonator 22 shown in fig2 [ 1 ]. the resonance frequency of a suspended microchannel resonator ( smr ) is highly sensitive to the presence of cells whose mass density differs from that of the solution . cells in solution flow through the resonator 22 and its resulting frequency shift depends on their mass and position within the channel . for dilute suspensions , this measurement yields a series of well - separated peaks whose heights are directly proportional to the mass excess of each cell in solution . a low flow rate enables higher - resolution frequency measurements by increasing the transit time of the cell through the device . as described in [ 1 ], we can weigh several hundred cells individually in a few minutes with a femtogram resolution and produce a histogram of cell masses . continuing to refer to fig1 , element 24 is a fluorescence detection module . microfluidic flow cytometers have been previously demonstrated by several laboratories [ 22 ]. we will adopt a similar methodology for our first generation systems . external excitation for conventional reporters ( e . g . gfp and rfp ) or immunostains will be aligned and focused in the microchannel 12 and external optics will be provided for collecting the resulting signal . however , since mass and density detection with the smr requires ˜ 1 second per cell , our fluorescent readout will be considerably slower than conventional flow cytometry . in later systems , the additional measurement time will be used to increase the intensity resolution and thereby enable detection and localization of reporters at low concentrations . as cells move through the system at very slow speeds relative to conventional facs machines , it will be possible to resolve spatial localization of fluorescence in addition to intensity . to this end , fluorescence peaks can be recorded as cells pass through a focused excitation beam , or an imaging system could be used to distinguish nuclear , cytoplasmic , and plasma membrane localization . many signaling mechanisms cause cytoplasmic proteins to accumulate in the nucleus or at the plasma membrane ( and vice versa ). the pump module 16 is an integrated microfluidic pump and may be a monolithic membrane pump . the analyte delivery module 18 may consist of one or more monolithic membrane “ bus valves .” among microfluidic valves , these three - way bus valves are particularly well suited for adding fluid to and removing fluid from a rotary channel [ paegel et al . microfluidic serial dilution circuit . anal chem ( 2006 ) vol . 78 ( 21 ) pp . 7522 - 7 ]. referring still to fig1 , a mass density module 23 detects the mass density of the cell . those of skill in the art will recognize that cell density can also be calculated from a measurement of cell mass if cell volume can be determined . it is preferred that the coulter principle be used to measure cell volume . in one embodiment , electrical current through the smr is monitored as the cell flows through it thereby enabling the cell &# 39 ; s volume and mass to be measured simultaneously . in another embodiment , current is measured through a channel that is separate from the smr . this channel is designed and optimized for the coulter principle . another approach for measuring volume is the volume exclusion method ( vem ) to measure the rate of change and absolute volume of growing cells at a single cell level . a volume exclusion method device must meet the metabolic needs of the cell . this requirement demands that at least one of the device surfaces be gas permeable or that fresh media is constantly introduced . if the system is in constant flow , then evaporation through this permeable surface will be negligible . the cell must be kept in media for the majority of the experiment and there must be means for temperature control of the fluid . the cell may be measured multiple times in order to improve precision . one approach to this requirement is to pass the cell back and forth through a fluorescent dye sensing zone or to cycle the cell through the sensing zone . another approach is to set up a method of continuous measurement that does not significantly affect cell volume . the rate at which the measurements are repeated depends on the rate at which the cell is growing and the signal - to - noise ratio of the device . by increasing the sampling rate , the statistical significance of the measurement will improve . both the vem technique and the coulter principle are independent of cell morthology and , with the appropriate design considerations , offer the sensitivity required to differentiate between linear and exponential cell growth in a single cell . the volume exclusion technique is disclosed in gray et . al ., “ a new method for cell volume measurement based on volume exclusion of a fluorescent dye ,” cytometry , vol . 3 , no . 6 , pages 428 - 434 ( 1983 ). there are two major requirements that must be met in order for the system disclosed herein to operate with optimum performance first , a large number of cells must be maintained throughout multiple cell cycles . microfluidic devices for circulating cells while maintaining order have not been demonstrated on a large scale . a goal of the present invention program will be to determine the maximum number of cells for which order can be maintained . while this is relatively straightforward to achieve for a single - layer microfluidic system , the system disclosed herein requires that cells travel through the smr , interconnect holes in the silicon smr substrate and several microfluidic valves . thus , these components will need to be designed in a way to avoid dispersion in cell velocity . cell order and velocity can be held constant by interspersing the cells with plugs of an immiscible material such as oil or air . if sized appropriately , these plugs would serve to compartmentalize the cells and maintain cell order as the cells and plugs travel through the various measurement modules , interconnect holes , and valves in the rotary channel . conversely , cell order and velocity can also be maintained by encapsulating each cell inside an aqueous droplet within a continuous oil phase . second , when a cell divides , it will be necessary to independently acquire measurements from each daughter cell . this requires that the cells be separated by a few hundred microns so they can be weighed individually by the smr . to achieve the separation , shear force will be introduced to undivided cells with controllable pneumatic valves . the contents of all of the references included herein and appended hereto are incorporated by reference herein in their entirety . 1 . burg , t . p ., m . godin , s . m . knudsen , w . shen , g . carlson , j . s . foster , k . babcock , and s . r . manalis , weighing of biomolecules , single cells and single nanoparticles in fluid . nature , 2007 . 446 ( 7139 ): p . 1066 - 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