Optical waveguides – Optical waveguide sensor
Reexamination Certificate
1999-03-02
2002-04-23
Healy, Brian (Department: 2874)
Optical waveguides
Optical waveguide sensor
C385S014000, C385S115000, C385S116000, C385S117000, C385S120000, C385S147000, C385S901000, C433S149000, C436S164000, C436S172000, C436S800000, C436S805000
Reexamination Certificate
active
06377721
ABSTRACT:
FIELD OF THE INVENTION
The present invention is generally concerned with biosensors, biosensor arrays, and sensing apparatus, and sensing methods for the analysis of chemical and biological materials. More particularly, the invention is directed to biosensors, biosensor arrays, sensing apparatus and sensing methods which employ cells and mixed populations of cells for analysis of chemical and biological materials.
BACKGROUND OF THE INVENTION
It is generally recognized that important technical advances in chemistry, biology and medicine benefit from the ability to perform microanalysis of samples in minute quantities. However, making analytical measurements on minute quantities has long been a challenge due to difficulties encountered with small volume sample handling, isolation of analytes, and micro-analysis of single-cell physiology.
Nanoliter, picoliter, and femtoliter volume studies have been explored in a range of applications involving in vitro and in vivo cellular investigations [R. M. Wightman, et al.,
Proc. Natl. Acad. Sci. U.S.A.
88:10754(1991); R. H. Chow, et al.
Nature
356:60(1992); T. K. Chen, et al.
Anal. Chem.
66:3031(1994); S. E. Zerby, et al.,
Neurochem.
66:651(1996); P. A. Garis, et al.
J. Neurosci.
14:6084(1994); G. Chen, et al.,
J. Neurosci.
15:7747(1995)], electrochemistry [R. A. Clark, et al.,
Anal. Chem.
69(2):259(1997)], matrix-assisted laser desorption—ionization mass spectrometry [S. Jespersen, et al.,
Rapid Commun. Mass Spectrom.
8:581(1994)], micro-column liquid chromatography [I. A. Holland, et al.,
Anal. Chem.
67:3275(1995); M. D. Oates, et al.,
Anal. Chem.
62:1573(1990)], micro-titration [M. Gratzl. et al
Anal. Chem.
65:2085(1993); C. Yi, et al.,
Anal. Chem.
66:1976(1994)], and capillary electrophoresis [M.Jansson, et al.,
J. Chromatogr.
626:310(1992); P. Beyer Hietpas, et al.
J.Liq.Chromatogr.
18:3557(1995)].
Clark, et al. [
Anal. Chem.
69(2):259(1997)] has disclosed a method for fabricating picoliter microvials for electrochemical microanalysis using conventional photolithographic masking and photoresist techniques to transfer mold polystyrene microvials on silicon wafer templates. These microvials typically exhibit non-uniformity in size and shape due to the difficulty in controlling the resist etching of the molding surface and the transfer molding process.
Park, et al. [
Science
276:1401(1997)] has disclosed a modified lithographic method for producing arrays of nanometer-sized holes using polystyrene-polybutadiene, ordered, diblock copolymers as masks in reactive ion etching of silicon nitride. This multi-step method is capable of producing arrays of picoliter-sized holes which are typically 20 nanometers in diameter and 20 nanometers deep with a spacing of 40 nanometers. Hole densities of up to 10
11
holes/cm
2
are disclosed. The range of sizes and spacings of the holes produced by this method is limited by the size of the copolymer microdomains. Uniformity of hole size and spacing is difficult to maintain with this method due to difficulties in controlling the etching method employed to form the holes.
Deutsch, et al. [
Cytometry
16:214(1994)] have disclosed a porous electroplated nickel microarray comprised of micron-sized conical holes in blackened nickel plate. Hole sizes range from a 7 um upper diameter to a 3 um lower diameter with an 8 um depth. The array is used as a cell carrier for trapping individual cells while studying the responses of individual cells to changes in their microenvironment. In U.S. Pat. No. 4,772540, Deutsch, et al., have also disclosed a method for making such an array using a combined photoresist and electroplating technique.
Corning Costar Corp. (Acton, Mass.) produces a commercial microwell array for miniaturized assays under the trademark PixWell™. These arrays are made from microformed glass plates and comprise 40 um diameter by 20 um deep tapered wells with a well density of 4356 wells/cm
2
.
Microwell arrays have particular utility in the study of living cells. In cell research, the measurement of responses of individual cells to changes or manipulations in their local environment is desirable. Any method or device designed for such studies must provide for the capability of maintaining cell viability, identifying the location of individual cells, and correlating response measurements with individual cells.
Due to the availability of viable fluorescent probes for intracellular studies, fluorescence measurements of living cells have significant utility in the study of cell functions. Thus fluorescence optical measurements are often utilized in cell studies where three generic methods of cell measurement are available, comprising bulk measurements of cell populations, dynamic measurements of cell populations or individual cells, and static measurements of individual cells.
The characteristics of an entire cell population as a whole can be studied with bulk measurements of sample volumes having a plurality of cells. This method is preferred where cell populations are very homogeneous. A generally recognized limitation of this method is the presence of background fluorescence which reduces the sensitivity of measurements and the inability of distinguishing differences or heterogeneity within a cell population.
Flow cytometry methods are often employed to reduce problems with background fluorescence which are encountered in bulk cell population measurements [M. R. Gauci, et al.,
Cytometry
25:388(1996); R. C. Boltz, et al.,
Cytometry
17:128(1994)]. In these methods, cell fluorescence emission is measured as cells are transported through an excitation light beam by a laminar flowing fluid. Flow cytometry methods may be combined with static methods for preliminary sorting and depositing of a small number of cells on a substrate for subsequent static cell measurements [U.S. Pat. No. 4,009,435 to Hogg, et al.; Kanz, et al.,
Cytometry
7:491(1986); Schildkraut, et al.,
J. Histochem Cytochem
27;289(1979)].
Gauci, et al., disclose a method where cell size, shape and volume is measured by light scattering and fluorescent dyes are utilized to determine protein content and total nucleic acid content of cells. This method further provides for counting and sizing various cells at a rate of approximately 100 cells per second.
Flow cytometry techniques are generally limited to short duration, single measurements of individual cells. Repetitive measurements on the same cell over time are not possible with this method since typical dwell times of a cell in the excitation light beam are typically a few microseconds. In addition, the low cumulative intensity from individual cell fluorescence emissions during such short measurement times reduces the precision and limits the reliability of such measurements.
Regnier, et al., [
Trends in Anal. Chem.
14(4):177(1995)] discloses an invasive, electrophoretically mediated, microanalysis method for single cell analysis. The method utilizes a tapered microinjector at the injection end of a capillary electrophoresis column to pierce an individual cell membrane and withdraw a sample of cytoplasm. The method measures cell contents, one cell at a time. The method is generally limited to the detection of easily oxidized species.
Hogan, et al., [
Trends in Anal. Chem.
12(1):4(1993)] discloses a microcolumn separation technique which may be utilized in combination with either a conventional gas chromatograph-mass spectrometer, micro thin layer chromatography or high pressure liquid manipulation of small cellular volumes. The sensitivity of the method is limited and may require pre-selection of target compounds for detection.
Static methods are generally the preferred method for measurements on individual cells. Measurement methods range from observing individual cells with a conventional optical microscope to employing laser scanning microscopes with computerized image analysis systems [see L. Hart, et al.,
Anal. Quan
Taylor Laura C.
Walt David R.
Flehr Hohbach Test Albritton & Herbert LLP
Foster David C.
Healy Brian
Silva Robin M.
Trustees of Tufts College
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