Electricity: measuring and testing – Impedance – admittance or other quantities representative of... – Lumped type parameters
Reexamination Certificate
2000-08-22
2002-09-10
Le, N. (Department: 2858)
Electricity: measuring and testing
Impedance, admittance or other quantities representative of...
Lumped type parameters
C324S692000, C324S521000, C324S515000, C349S033000, C436S806000
Reexamination Certificate
active
06448794
ABSTRACT:
This application claims priority under 35 U.S.C. §119(a) to Chinese Application No. 00104350.1, entitled APPARATUS AND METHOD FOR HIGH THROUGHPUT ELECTROROTATION ANALYSIS, filed on Mar. 15, 2000 and Chinese Application No. 00124086.2, entitled APPARATUS AND METHOD FOR HIGH THROUGHPUT ELECTROROTATION ANALYSIS, filed on Aug. 18, 2000.
FIELD OF THE INVENTION
The present invention concerns the field of electrorotation and biophysics. More specifically, embodiments include micromachined or microfabricated electrorotation chips, which produce a rotating electric field and can evaluate the electrorotation behavior of biological and non-biological particles; an apparatus for analyzing the electrorotation properties of such particles; and methods of use thereof.
BACKGROUND OF THE INVENTION
Modern biotechnological and pharmaceutical approaches use a variety of analytic techniques to ascertain the behavior or identity of a molecule, cell, or biological particle. For example, investigators frequently use microbead-based multiplexed assays for analyzing the types and concentrations of target molecules in an “unknown” solution (see, e.g., Fulton R. J. et al,
Clinical Chemistry
, 43:1749-1756, (1997)). The target molecules may include antigens, antibodies, oligonucleotides, receptors, peptides, or enzyme substrates and may be labeled with a reporting molecule (e.g., a fluorescent molecule). Typically, in such an assay, different types of microbeads are used. Each type of microbead can be distinguished from others based on physical and chemical properties such as color, size, fluorescence molecule types and fluorescence intensity. The surfaces of different types of microbeads containing different types of molecules, i.e., each type of microbead is surface-activated or coated with one type of molecule. The molecules coating the surface of the microbeads may also be antibodies, antigens, oligonucleotides, receptors, peptides, enzyme substrates. Ideally, each molecule coating the surface of the microbeads interacts with one class of target molecule in the “unknown” solution.
Next, the surface-coated microbeads of many types are mixed together and are incubated with the “unknown” solution to allow the target molecules from the “unknown” solution to interact with the immobilized molecules on the microbeads. Prior to the incubation, all the target molecules are pre-labeled with certain types of reporter molecules (e.g., fluorescent molecules). Following the incubation, the detection of the reporter bound to the different types of microbeads is performed. By one approach, flow cytometry is used to analyze the levels of fluorescence on the individual microbeads and also determine the type of microbead detected. Because a “one-to-one” correspondence between the labeled target molecule and the immobilized molecule can be made, the identity and concentration of the target molecule in the “unknown” solution can be determined. A high-throughput format is desirable for these assays to expedite the analysis.
Modern approaches to pharmaceutical development also involve the analysis of interactions between a target molecule and molecules, cells, or particles of interest. For example, cell-based techniques are frequently used to screen chemical compound libraries for new drugs. The screening process typically employs cells having a target molecule to which an interaction with a drug-candidate is sought. The target molecule-containing cells are loaded into different reaction wells and are exposed to chemical compounds from a compound library. The cells are incubated with the chemical compounds for a specified time and then are evaluated for an interaction between the chemical compound and the target molecule. The detection of the interaction can be accomplished in many ways and fluorescence-based systems are frequently used. A similar assay determines the response of specific cell types to various amounts of chemical compounds and times of exposure. This type of assay can also be performed with a fluorescence-based detection system but the experimental set up allows for the determination of quantitative information. Desirably, a high-throughput system is also used for these assays to expedite drug development. The present invention relates to biological analyses which utilize electrorotation to characterize the behavior or identity of a molecule, cell or particle. Electrorotation analyses involve observing the behavior of molecules, cells, and particles, as well as complexes of these materials in a electric field that is applied so as to cause a rotation of the studied material. “Electrorotation” is a term of art that refers to the rotation of a material in an electric field. When a material is subjected to a rotating field, it becomes electrically polarized and the induced polarizations interact with the applied rotating fields, which produces a rotating torque that drives the rotation of the material. The rotation behavior (e.g., rotation rate and direction) depends on the frequency of the rotating field and electrical properties that are specific for the material being rotated. The measurement of rotational behavior can be expressed as a function of the frequency of the applied rotating field. From these measurements, the electrical properties (e.g., electrical conductivity and permittivity) unique to the material can be derived.
A number of electrorotation-based techniques have been developed for distinguishing particle types and analyzing particle properties. For example, U.S. Pat. No. 4,626,506 discloses an approach to distinguish cells that are secreting a cellular substance (e.g., proteins, hormones, etc) from non-secreting cells in the suspension. Further, U.S. Pat. No. 4,634,669 discloses an approach to differentiate two groups of particles based on observing different rotating directions of the particles when the frequencies of the applied rotating field are varied. Still further, U.S. Pat. No. 4,801,543 discloses an approach to analyze different groups of particles based on exposing the particles to two superimposed, simultaneous rotating electrical field forces with opposite rotation directions. Additionally, International Publication No. WO 93/16383 discloses an approach for detecting target molecules, which involves forming a complex between micro-particles and a target species and observing the difference in electrorotation properties between the original micro-particles and the complexes.
More discussion of electrorotation and its uses can be found in, for example, Arnold & Zimmermann,
Z. Naturforsch
., 37c:908-915, (1982); Fuhr et al.,
Stud. Biophys
. 108: 149-164, (1985); Gimsa et al., in
Physical characterization of biological cells
, Schutt W, Klinkmann H and Laprecht I and Wilson T editors, Gesundheit, Berlin, pp 295-323, (1991a); Huang et al.,
Phys. Med. Biol
., 37: 1499-1517, (1992); Huang et al.,
Biochim. Biophys. Acta
1282:76-84, (1996); Wang et al.,
Biochim. Biophys. Acta
. 1193: 330-344, (1994); Gascoyne, Becker FF and Wang,
Bioelectrochem. Bioenerg
. 36: 115-125, (1998); and Huang Y, et al.,
Biochim. Biophys. Acta
1417: 51-62 (1999).
BRIEF SUMMARY OF THE INVENTION
The present invention concerns the manufacture and use of electrorotation chips and an apparatus for high-throughput analysis of many different types of particles. Advantageously, embodiments can be used for high-throughput analysis of the rotation behaviors of different particle types on a single electrorotation chip, which allows for a rapid determination of the electrical properties of many different particles. Embodiments can also be used for high-throughput analysis of molecule-molecule, molecule-particle, and particle-particle interactions. For example, the invention can be used for cell-based, high-throughput screening for potential drug molecules from a chemical compound library.
Embodiments include an electrorotation chip comprising a substrate and a plurality of electrorotation units disposed on said substrate, wherein each of said electrorotation units comprises a plurality of electrode elements that
Cheng Jing
Liu Litian
Wang Xiao-Bo
Wu Lei
Xu Junquan
Aviva Biosciences Corporation
Deb Anjan K.
Knobbe Martens Olson & Bear LLP
Le N.
LandOfFree
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