U.S. patent application number 12/964814 was filed with the patent office on 2011-06-09 for electrically active combinatorial chemical (eacc) chip for biochemical analyte detection.
Invention is credited to Jacque H. Georger, JR., Xing SU, Lei B. Sun.
Application Number | 20110136693 12/964814 |
Document ID | / |
Family ID | 36603507 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110136693 |
Kind Code |
A1 |
SU; Xing ; et al. |
June 9, 2011 |
ELECTRICALLY ACTIVE COMBINATORIAL CHEMICAL (EACC) CHIP FOR
BIOCHEMICAL ANALYTE DETECTION
Abstract
Apparatus and methods are disclosed for electrically active
combinatorial-chemical (EACC) chips for biochemical analyte
detection. An apparatus includes a substrate that has an array of
regions defining multiple cells, wherein each of the cells includes
a reaction cavity that contains multiple functional binding groups.
A method of detecting an analyte providing the reaction cavity
between a source and a drain or a pair of electrodes, applying a
voltage and monitoring a parameter indicative of an analyte
characteristic. A process of fabricating an EACC include bonding an
analyte to the multiple functional binding groups of each reaction
cavity, and forming an analyte sensing structure including the
substrate.
Inventors: |
SU; Xing; (Cupertino,
CA) ; Sun; Lei B.; (Santa Clara, CA) ;
Georger, JR.; Jacque H.; (Holden, MA) |
Family ID: |
36603507 |
Appl. No.: |
12/964814 |
Filed: |
December 10, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11025502 |
Dec 28, 2004 |
7879764 |
|
|
12964814 |
|
|
|
|
Current U.S.
Class: |
506/16 ; 506/15;
506/18 |
Current CPC
Class: |
B01J 2219/00612
20130101; B01J 2219/00637 20130101; B01J 2219/00662 20130101; B01J
2219/00621 20130101; B01J 19/0046 20130101; B01J 2219/00596
20130101; B01J 2219/0061 20130101; B01L 2300/0819 20130101; B01J
2219/00704 20130101; C12Q 1/6837 20130101; B01J 2219/00628
20130101; B01J 2219/00659 20130101; B01J 2219/00608 20130101; B82Y
15/00 20130101; B01J 2219/00317 20130101; C40B 60/04 20130101; B01L
3/5085 20130101; B01J 2219/00527 20130101; B01J 2219/00653
20130101; B01L 2300/0636 20130101; B01J 2219/0063 20130101; G01N
33/5438 20130101; B01J 2219/00722 20130101; B82Y 30/00 20130101;
B01J 2219/00626 20130101; C12Q 1/6837 20130101; C12Q 2565/601
20130101 |
Class at
Publication: |
506/16 ; 506/15;
506/18 |
International
Class: |
C40B 40/06 20060101
C40B040/06; C40B 40/00 20060101 C40B040/00; C40B 40/10 20060101
C40B040/10 |
Claims
1. An apparatus, comprising a substrate including an array of
regions defining a plurality of cells, each of the plurality of
cells including a reaction cavity containing multiple functional
binding groups, wherein the array of regions comprises a first
gradient of a first functional binding group and a second gradient
of a second functional binding group, wherein a distance between
the first functional binding group and the second functional
binding group corresponds to an inter-molecular distance between
binding locations on a biochemical analyte.
2. The apparatus of claim 1, wherein the multiple functional
binding groups are coupled to the substrate via hybridized DNA.
3. The apparatus of claim 1, wherein the multiple functional
binding groups are coupled to the substrate via a cross-linked
polymer.
4. The apparatus of claim 1, wherein the multiple functional
binding groups are coupled to the substrate via a copolymer or
chain transfer polymer or a combination thereof.
5. The apparatus of claim 1, wherein the multiple functional
binding groups are coupled to the substrate via a thiol-based
reaction product.
6. The apparatus of claim 1, wherein the plurality of cells each
comprise an electrical sensing circuit.
7. The apparatus of claim 1, wherein the plurality of cells each
comprise an optical sensing structure.
8. The apparatus of claim 1, wherein the plurality of cells
comprise a protein chip having a feature size between 0.5 microns
and 500 microns.
9. The apparatus of claim 8, wherein the plurality of cells
comprise a protein chip having a feature size of less than
approximately 100 microns.
10. The apparatus of claim 8, wherein the plurality of cells
comprise a protein chip having a feature size of less than
approximately one micron.
11. The apparatus of claim 1, wherein the plurality of cells each
comprise an electrically-active, combinatorial-chemical (EACC) chip
for biochemical analyte detection.
12. The apparatus of claim 11, wherein said analyte detection
comprises probe-less detection.
13. The apparatus of claim 11, wherein said analyte detection
comprises creation of a binding site with one or more of the
multiple functional binding groups.
14. The apparatus of claim 13, wherein the groups comprise
non-polymeric components.
15. The apparatus of claim 1, wherein the first gradient is in a
first direction or in the opposite direction or a combination
thereof.
16. The apparatus of claim 15, wherein the second gradient is in a
second direction or in the opposite direction or a combination
thereof.
17. The apparatus of claim 16, wherein the second direction is
approximately orthogonal to the first direction.
18. The apparatus of claim 1, wherein the substrate comprises
silicon having a surface modified with silanes.
19. The apparatus of claim 18, wherein the silanes comprise
phenyl.
20. The apparatus of claim 1, wherein the multiple groups comprise
a positively-charged group and a negatively charged group.
21. The apparatus of claim 1, wherein the multiple groups comprise
a polar group and a nonpolar group.
22. The apparatus of claim 21, wherein the multiple groups comprise
a positively-charged group and a negatively charged group.
23. An apparatus, comprising a substrate including an array of
transistor sensors defining a plurality of cells including a
reaction cavities containing binding groups on the gates of
transistors, wherein the cells are configured for monitoring a
parameter indicative of an analyte characteristic when a voltage is
applied between the sources and drains of the transistors.
24. The apparatus of claim 23, further comprising a channel defined
by an analyte bonded to a self-assembled monolayer.
25. The apparatus of claim 23, comprising different chemical
structures disposed on the gates of the transistors and contacting
a sample.
26. The apparatus of claim 25, wherein a set of pre-determined
chemical structures are associated with a set of transistors.
27. The apparatus of claim 26, wherein the cells are configured
such that binding patterns are translated into generated electrical
signals when source-drain voltages are applied that differ
depending on the different pre-determined chemical structures.
28. The apparatus of claim 27, further comprising a processor-based
analyzer for identifying analytes in the sample by analyzing
patterns of electrical signals generated by the transistors with
respect to the gate-associated chemical compositions.
29. The apparatus of claim 28, further comprising a pre-built
database of standard analytes, wherein the analyzer uses computer
pattern recognition in the identification.
30.-50. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This applications is a divisional of U.S. application Ser.
No. 11/025,502, filed Dec. 28, 2004. The foregoing application is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] Embodiments of the invention relate generally to the field
of biological and/or chemical sensing. More particularly,
embodiments of the invention relate to electrically active
combinatorial-chemical (EACC) chips for biochemical analyte
detection.
[0004] 2. Background Information
[0005] Currently, biological and chemical analyte detections are
based primarily on specific interaction between analytes and their
binding partners. To perform high throughput assays, a large number
of molecular probes need to be immobilized on a surface to form a
microarray. Such microarrays are sometimes referred to as bio-chips
(e.g., protein chips or gene chips). Preparing a large number of
specific polymeric probes (e.g., antibodies or nucleic acids) is,
however, both time-consuming and costly. Moreover, immobilizing the
polymeric probes in discrete small surface areas is technically
difficult and expensive. It is desired to have a more efficient
approach to preparing and immobilizing probes.
[0006] Traditional approaches to making biochips involve chemically
preparing polymeric probes and then subsequently spotting the
chemically prepared polymeric probes on the chips. However, the
minimum feature size attainable with these probes is typically
>100 um for a protein chip (array), or >1 um for a gene chip
(array). It is desired to have smaller feature sizes available in
the future. While higher density bio-chips are clearly desirable
from the perspective of both cost to manufacture and clinical
efficiency, fabricating higher density bio-chips based on smaller
polymeric probe feature sizes is both technically challenging and
time-consuming. It is desired to have an approach that will permit
the fabrication of chips based on smaller probe feature sizes.
[0007] Referring to FIGS. 1A and 1B, current biochips for direct
analyte detection (antibody chips, DNA chips, aptamer chips) are
based on interactions of analytes with their polymeric binding
partners (probes), each of the latter of which presents unique
intra molecular binding sites. Referring to FIG. 1A, a binding
partner (probe) 110 is immobilized on a substrate 120. The binding
partner 110 then binds with an analyte 130, thereby enabling the
detection of the analyte 130. This binding approach is based on the
principle of using a single, unique and large molecule for specific
binding of analytes. This approach is highly specific and accurate,
and generally involves small dimension(s). On the other hand, this
approach is very costly and time-consuming because of the need to
obtain analyte-specific probes or binding partners, and is
generally inflexible. Also, as only known probes are used to detect
known analytes; but not-yet-identified analytes are undetectable.
It is desired, therefore, to have an approach that can detect
unknown analytes.
[0008] Referring to FIG. 1B, two different types of analytes 140,
150 are dispersed across a substrate 160 by a buffer solvent flow.
The analytes 140, 150 are spatially segregated across a surface of
the substrate 160, thereby enabling separation of two different
analytes 140, 150. The resulting spatial segregation permits
detection of individual analytes. Separation in this instance is
based on the principle of buffer solvent flow. This approach is low
cost, fast, and flexible, but is less specific and less accurate
than is desired, and it involves large dimension(s). Another
technique might involve molecular migration in a gel
(electrophoresis) based on size and molecular weight.
[0009] Protein binding to a surface may be affected by the chemical
property of the surface. In this way, protein chips with different
binding surfaces have been produced. Chromagraphic and
spectrographic binding surface technologies have also been
evolving, wherein bio-chip detections are typically read by optical
methods. When the chip feature (spot) size becomes <1 um,
however, optical detection becomes impractical. It is desired to
have an approach that enables detection and reading with higher
density bio-chips.
[0010] Electronic sensors for biomolecule detection have also been
demonstrated. Although such electronic sensors have the potential
to overcome the spatial limitations of optical detection,
electronic sensors by themselves do not appear to obviate the
underlying feature size limitations of the polymeric probe-analyte
paradigm.
[0011] Self aligned monolayers have been demonstrated. The
formation of patterned coplanar monolayers (which can be termed
ultra thin films) and the use of those patterns to selectively bind
colloidal catalysts & plate electroless metals selectively at
high resolution are under investigation. Further research into the
formation of ultra thin films for the selective adhesion of various
types of biological cells is ongoing.
[0012] Heretofore, the requirements of a more efficient approach to
preparing and immobilizing probes, smaller probe feature sizes, the
ability to detect unknown analytes and the detection and reading of
higher density bio-chips have not been fully met. It is therefore
desired provide techniques that meet these goals.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0013] The drawings accompanying and forming part of this
specification are included to depict certain aspects of embodiments
of the invention. A clearer conception of the embodiments of the
invention, and of the components and operation of systems provided
with embodiments of the invention, will become more readily
apparent by referring to the exemplary, and therefore non-limiting,
embodiments illustrated in the drawings, wherein identical
reference numerals designate the same elements. The embodiments of
the invention may be better understood by reference to one or more
of these drawings in combination with the description presented
herein. It should be noted that the features illustrated in the
drawings are not necessarily drawn to scale. Brief descriptions are
provided below, followed a detailed description of the preferred
embodiments in view of the illustrative drawings.
[0014] FIGS. 1A and 1B illustrate conventional techniques for
binding and separating analytes.
[0015] FIG. 1C illustrates the use of a plurality of different
binding groups to detect an analyte, representing an embodiment of
the invention.
[0016] FIG. 2 illustrates top plan and partial cross section views,
respectively, of a combinatorial-chemical chip, representing an
embodiment of the invention.
[0017] FIGS. 3A-3C illustrate a combinatorial printing head, a side
view of filled reaction cavities and a side view of mixed self
assembled monolayers, respectively, representing embodiments of the
invention.
[0018] FIG. 4 illustrates four self assembled monolayer chemical
structures mapped across a two dimensional array, representing an
embodiment of the invention.
[0019] FIG. 5 illustrates a group of four multi-chemical gradient
areas, representing an embodiment of the invention.
[0020] FIGS. 6A and 6B illustrate structural diagrams of a field
effect sensor and a capacitance/impedance sensor, respectively,
representing embodiments of the invention.
[0021] FIG. 7 illustrates a structural diagram of a sensor for
static-electrical or capacitance/impedance measurements,
representing an embodiment of the invention.
[0022] FIGS. 8A-8C illustrate static-electrical detection of an
analyte, representing an embodiment of the invention.
[0023] FIG. 9A illustrates a schematic representation of a
substrate of silicon or glass modified with a self aligned
monolayer of trichlorophenylsilane, representing an embodiment of
the invention.
[0024] FIG. 9B illustrates a schematic representation of a first
self aligned monolayer (SAM) of trichlorophenylsilane exposed to
about 50 mJ of ultraviolet light at -250 nm in clean room air.
(.about.10% of dose to clear all phenyl groups), representing an
embodiment of the invention.
[0025] FIG. 9C illustrates a schematic representation of co-planar
self aligned monolayers after initial exposure and formation of a
second self aligned monolayer (SAM2), representing an embodiment of
the invention.
[0026] FIG. 9D illustrates a schematic representation of a final
composition of SAM, SAM2, & SAM3 example after using 2
exposures of about 50 mJ (initial exposure) and 100 mJ (subsequent
exposure) of ultraviolet light at .about.250 nm, representing an
embodiment of the invention.
[0027] FIGS. 10A and 10B illustrate DNA-based self-assembly
examples, representing embodiments of the invention.
[0028] FIG. 11 illustrates a cross-linked polymer example,
representing an embodiment of the invention.
[0029] FIGS. 12A and 12B illustrate co-polymerization and chain
transfer examples, respectively, representing embodiments of the
invention.
[0030] FIGS. 13A-13D illustrate thiol-PEG based examples,
representing embodiments of the invention.
[0031] The embodiments of the invention and the various features
and advantageous details thereof are explained more fully with
reference to these non-limiting embodiments that are illustrated in
the accompanying drawings and detailed in the following
description. Descriptions of well known starting materials,
processing techniques, components and equipment are omitted so as
not to unnecessarily obscure the embodiments of the invention in
detail. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration only
and not by way of limitation. Various substitutions, modifications,
additions and/or rearrangements within the spirit and/or scope of
the underlying inventive concept will become apparent to those
skilled in the art from this disclosure.
[0032] The descriptions herein of the invention and preferred and
alternative embodiments may be better understood in view of the
following definitions:
[0033] The term "non-polymer" refers to polyatomic organic
molecules that do not have repeated units that are either identical
or non-identical.
[0034] The term "protein chip" refers to a two or three dimensional
device that contains immobilized protein species (2 or more
proteins) that are arranged in regular patterns or irregular
patterns.
[0035] The term "optical sensing structure" refers to a device that
collects photons from other objects and converts them to electrical
signals.
[0036] The term "reaction cavity" refers to a 3D space that can
hold reactants and allow chemical or biochemical reactions to
proceed, typically measured in nanometer or micrometer scales.
[0037] The term "feature size" refers to the dimension(s) of an
individual feature of a given array. For example, a protein array
may have 100 protein spots. Thus the protein spots are the features
of the protein array. The dimension of a given spot is the feature
size of the spot. It can be measured by area, diameter or lengths
of sides.
[0038] The phrase "coupling via thiol-based reaction product"
refers to covalent bonding formation involving a hydrosulfide group
(--SH). It can happen between organic compounds or between a
thiol-containing organic compound and metals, such as gold and
silver.
[0039] A substrate of an apparatus in accordance with a preferred
embodiment includes an array of regions defining multiple cells.
Each of the cells includes a reaction cavity that contains multiple
functional binding groups. The substrate includes a solid material
that provides support as well as a functional surface. The
substrate can be made up of any of several materials, and
preferably inorganic materials such as silicon wafer, glass, metal
(aluminum, e.g.), or organic material such as plastic
(polycarbonate, e.g.). The surface of the substrate is preferably
coated with metal (gold) or a polymer (PEG) or both. Functional
groups on the surface may include mine groups or carboxyl
groups.
[0040] The multiple functional binding groups may be coupled to the
substrate via hybridized DNA, a cross-linked polymer, a copolymer,
a chain transfer polymer and/or a thiol-based reaction product. The
cells preferably each include an analyte sensing structure such as
an electrical sensing circuit or an optical sensing structure.
[0041] The cells may each comprise a protein chip or gene chip
having a feature size preferably between 0.5 microns and 500
microns, and preferably less than approximately 100 microns. The
cells may each comprise an electrically-active,
combinatorial-chemical (EACC) chip for biochemical analyte
detection. The analyte detection may be probe-less. The groups may
include non-polymeric components.
[0042] The array may include a first density gradient of a first
group in a first direction, and may further include a second
density gradient of a second group in a second direction. The
second direction may be approximately orthogonal to the first
direction. Moreover, four significant directions may include, e.g.,
from an overhead viewpoint, left to right, right to left, up to
down and down to up.
[0043] The substrate may comprise silicon having a surface modified
with silanes, wherein the silanes may comprise phenyl. The multiple
groups may include a positively-charged group and a negatively
charged group and/or a polar group and a non-polar group.
[0044] A method of detecting an analyte uses a substrate including
an array of regions defining multiple cells. Each of the cells
includes a reaction cavity containing multiple functional binding
groups. A channel may be defined between a source and a drain,
although not necessarily, or a region may be defined between a pair
of electrodes. A voltage is applied between the source and the
drain or the pair of electrodes. A parameter indicative of an
analyte characteristic is monitored when the voltage is applied.
Each of the cells may include an analyte bonded to a self-assembled
monolayer to define a channel or region between a source and drain
or pair of electrodes, respectively.
[0045] A method of making an analyte sensor uses a substrate
including an array of regions defining a plurality of cells each
including a reaction cavity. Multiple functional binding groups are
coupled to each reaction cavity. An analyte sensing structure is
formed including the substrate with the array of regions. An
analyte is preferably bonded to the multiple functional binding
groups of each reaction cavity.
[0046] The forming of the analyte sensing structure may include
foaming a source and a drain for each reaction cavity such that
each reaction cavity may define, although not necessarily, a
channel between the source and the drain, and coupling a voltage
source and monitoring system between the source and the drain, or
it may include forming a pair of electrodes for each reaction
cavity, and coupling a voltage source and monitoring system between
the pair of electrodes. It may also include forming an optical
sensing structure.
[0047] The method may include modifying a surface of the substrate
with silanes, and the silanes may comprise phenyl. Modifications
methods may include any of a variety of techniques such as
adsorption or charge interaction.
[0048] A first gradient of a first group may be formed in a first
direction of the array, and a second gradient of a second group may
be formed in a second direction of the array. The first and second
directions may be orthogonal. Third and fourth directions would
include those opposite to the first and second directions.
[0049] An apparatus in accordance with an embodiment of the
invention includes a substrate that includes an array of regions
defining multiple cells, wherein each of the cells includes a
reaction cavity that contains multiple functional binding groups.
Another embodiment involves a method of detecting an analyte
comprising providing a substrate including an array of regions
defining multiple cells. Each of the cells includes a reaction
cavity containing multiple functional binding groups and defining a
channel between a source and a drain or defining a region between a
pair of electrodes. In a method in accordance with this embodiment,
a voltage is applied between the source and the drain or the pair
of electrodes, and a parameter indicative of an analyte
characteristic is monitored when the voltage is applied.
[0050] Another embodiment includes a process of fabricating an
electrically active combinatorial-chemical chip for biochemical
analyte detection comprising providing a substrate including an may
of regions defining multiple cells each including a reaction
cavity. Multiple functional binding groups are coupled to each
reaction cavity. In a process in accordance with this embodiment,
an analyte is bonded to the multiple functional binding groups of
each reaction cavity, and an analyte sensing structure is formed
including the substrate with the array of regions. Reaction
cavities may be coupled with different functional binding groups or
different molecules containing different groups.
[0051] To address the problems of creating a large number of
specific probes, immobilizing them in small surface areas and
applying the chips to samples containing unknown analytes, an
embodiment of the invention can adopt a "probe-less" approach. An
embodiment of the invention can vary surface properties to
selectively attract proteins and/or other molecules. An embodiment
of the invention can include creating a binding site with several
small molecules (binding components). Small molecules and/or
binding components are intended to mean nonpolymeric molecules
(e.g., can be hetero-oligomers). To achieve this, a limited number
of binding components (e.g., groups or molecules, covalently
attached or adsorbed) can be used in different ratios and densities
to obtain a large number of different chemical matrices that have
different binding potentials to different analytes. Biochips made
by this method can be termed combinatorial chemical (CC) chips.
[0052] An embodiment of the invention can use multiple small
compounds (binding components) to assemble arrays of combinatorial
chemical matrices for specific analyte binding and detections.
Detections can be achieved optically, electronically or
electrically. Thus, an embodiment of the invention can eliminate
costly and time-consuming specific probe generation and also allow
detection of not-yet-identified analytes. An embodiment of the
invention is useful for sample profiling, and it is particularly
useful for the analyses of proteins as well as other
bioanalytes.
[0053] Referring to FIG. 1C, a basic element of an AECC chip
embodiment of the invention is depicted. A substrate 170 can
provide structural support. A first binding group 181 is coupled to
the substrate 170. A second binding group 182 is also coupled to
the substrate 170 at an intermolecular distance from the first
binding group 171. An analyte 190 binds to both the first binding
group 181 and the second binding group 182. The inter-molecular
distance between the first binding group 181 and the second binding
group 182 corresponds to the inter-molecular distance between the
binding locations on the analyte 190. This embodiment of the
invention is based on the principle of using different molecules
(binding groups) for specific binding of analytes. This embodiment
of the invention is very flexible, very compact, sensitive, fast,
reasonably specific and accurate. The identification of the analyte
depends on the binding pattern of the analyte in the reaction
cavities of the apparatus and prior information derived from known
analytes
[0054] An embodiment of the invention can include: a chip surface
divided into multiple subareas (regions), each said sub-area can be
coated with a combination of different binding components, said
binding components can be organic compounds; said different binding
components can vary in size, composition, and arrangement of
functional groups; the ratios and densities of said binding
components can be different among different sub-areas and these
subareas can be identifiable (indexed) by X-Y coordinators.
[0055] In an embodiment of the invention, binding of an analyte on
a sub-area can require the presence of 2 or more binding
components. An electrical potential can be applied individually to
or sensed individually from each sub-area; analyte binding can be
detected electrically or electronically; and these detection
methods can be used for analyzing (profiling) of biological or
chemical samples.
[0056] An embodiment of the invention can include a chip having a
planar surface with an array of sub-areas; each of the sub-areas
can have 1 or more micro or nano-wells (i.e., reaction cavities).
Under each such sub-area or well, there can be an electronic sensor
and/or electrical structures(s) (e.g., transistors or electrodes
for electrical detections). Different chemicals (for instance, 2,
3, 4 or more) can be applied on the surface. When used in different
ratios and different densities, a large number of combinations of
chemicals (permutations) can be created. A simple way to generate
different ratios is to create different gradients from the binding
compounds, each of the gradients corresponding to one of the
binding components.
[0057] Referring to FIG. 2, a multi-chemical-gradients (MCG) chip
200 embodiment of the invention is depicted. The top portion of the
figure depicts a top plan view and the bottom portion of the figure
depicts a partial cross section view. In this embodiment, A, B C
and D are 4 different chemical compounds. As depicted, A-B
gradient(s) vary from between right and left and are represented by
the horizontal double ended arrow. As depicted, C-D gradient(s)
vary from between top and bottom and are represented by the
vertical double ended arrow. In this embodiment, a surface 210 of a
substrate 220 of the chip 200 includes an array of regions, each of
which defines a sub-area 230. Each of the sub-areas 230 includes a
sensor unit 240 which in turn includes a nano-well 250 (reaction
cavity) and a semiconductor or electrical sensor 260.
[0058] Referring to FIGS. 3A-3C, the chemicals (e.g., compounds of
the binding components and solvents/vehicles) can be delivered to
the surface of the wells by printing methods. Referring to FIG. 3A,
a printing head 310 is coupled to a pair of mixers 320 each of
which is in-turn coupled to a pair of reservoirs 330. The printing
head 310 can deliver a predetermined ratio of A/B/C/D to a
substrate surface 340. Referring to FIG. 3B, a plurality of filled
binding cavities 350 is arranged above a plurality of sensors 360
on the substrate. Referring to FIG. 3C, preferably, self-assembled
mono-layers (SAM) 370 are formed in each well (reaction cavity
350). For example, the bottom of the well can be coated with gold,
a thiol-polyethylene glycol (PEG) derived compound can be used as a
base component and compounds with similar (or the same) base
structure(s) together with additional functional groups on the
other end of the similar base structure molecules are the binding
components and used together with base component to form a mixed
SAM (self assembled monolayer). The functional groups associated
with the binding components play the binding roles in analyte
binding.
[0059] Referring to FIG. 4, four schematic examples of
combinatorial chemical structures are depicted. The organic
compounds used as binding components have different functional
groups. Positively charged (PC) compounds are typically compounds
with amino groups. Negatively charged (NC) compounds can be those
containing carboxyl groups, sulfate group and phosphate groups.
Compounds which are hydrophobic (nonpolar (NP)) can be those with
benzyl ring structures and alkyl chains. Other compounds that are
hydrophilic (polar (p)) can also be used, such as compounds with
hydroxyl group, amine group, or organic compounds with hetero-atoms
(e.g., nitrogen, oxygen). Organic compounds with halogen atoms can
also be used. Compounds with reactive compounds groups may also be
used, such as compounds with a thiol group, or an aldehyde group.
Short peptides, including non-natural amino acids and oligo
nucleotides (including those with modified structures) can be used
together with other organic compounds.
[0060] Other factors can also be considered in fabricating CC
chips: for example, molecular chain length, position of functional
groups, distance between functional groups, number of functional
groups per molecule, ratio of mixed functional groups per molecule,
arrangement of mixed functional groups on a molecule. These factors
are important in generating 3-dimensional binding sites.
[0061] Referring to FIG. 5, CC chip can also be made with more than
4 chemical conditions (independent variables). For instance, the
binding components (functional groups can be structurally arranged
in a molecule to provide a contextual condition. In this way, an
additional condition can be molecular chain length. The position of
functional groups in a molecule can be another condition. The
distance between function groups can be a condition. The number of
functional groups per molecule can be a condition. the ratio of
mixed functional groups per molecule can be a condition. The
arrangement of mixed functional groups in a molecule can also be a
condition. Also, the total density of functional groups on surface
(region) can be a condition.
[0062] Referring to FIGS. 6A-6B, 7 and 8A-8C, different
electrical/electronic sensors can be used together with a CC chip.
Optical sensors can also be used together with a CC chip. In the
case of an active electrical/electronic sensor, the chip can be
termed an electrically active CC chip or EACC chip. For example,
field-effect-transistor sensors, capacitance and impedance sensors
and/or static-electric sensors can be integrated in the chip.
Ideally, there is a sensor associated with each reaction cavity and
each of the sensors is controlled independently.
[0063] Referring to FIG. 6A, a field effect measurement embodiment
is depicted. An analyte 610 in a reaction cavity 620 with aqueous
buffer is bonded to an SAM layer 630 to define a channel 640 on a
substrate 650. The channel 640 is located between a source 645 and
a drain 655 which are both coupled to a voltage source and
monitoring system 660. Referring to FIG. 6B, a capacitance or
impedance measurement embodiment is depicted. The analyte 610 is
again bonded to the SAM layer 630 to define the channel 640 on the
substrate 650. In this embodiment, the channel 640 is located
between a first electrode 670 and a second electrode 675 which are
both coupled to a voltage source and monitoring system 660.
[0064] Referring to FIG. 7, a co-planar electrode static-electrical
or capacitance/impedance measurement embodiment is depicted. A self
aligned monolayer 710 is connected to a bottom surface electrode
720. The bottom surface electrode 720 is coupled to a source
connector 725. A top surface electrode 730 is located opposite the
self aligned monolayer 710 across a reaction cavity with aqueous
buffer.
[0065] Sample binding: any biochemical or chemical samples can be
used, provided chips with affinity surfaces are used. Conditions
for sample binding and washing can be similar to those used in
standard chromatography procedures: ion exchange, size exclusion,
affinity binding, reverse phase binding (e.g., varying pH, ionic
strength, solvent concentration) Sample concentrations, binding
time and washing conditions can also be modified from the standard
procedures. A microfluidic system (or micro electromechanical
system (MEMS)) can be combined with the chip.
[0066] Detection: Field effect, capacitance and impedance can be
monitored for each reaction cavity, provided suitable
electrical/electronic structures are made in the chip. An external
chip reader is preferably used to collect and analyze the data.
FIGS. 8A-8C illustrate an example of detection based on
static-electric attraction. After selective binding and washing, an
electrical potential is applied between a top surface of the chip
810 and a bottom surface of the chip 820. After drying by vacuum,
electrical charges are built up around the molecules. The charges
make the molecules move (fly) toward the top surface. Because the
top plate can have a transistor (charge detector) corresponding to
those in the bottom plate, molecular charging and flying can be
regulated and detected independent of those in other reaction
cavities.
[0067] Several transistors can be in a cell (reaction cavity) with
the gates of the transistors coupled to the binding molecules. An
SAM layer may not be necessary due to the importance of the
distance between the analytes and the gate surface (i.e., the
closer the better). The binding of the analytes close to the gate
can affect electron distribution and thus the conductance of the
transistor (between source and drain). Another type of structure in
a cell (cavity) is a combination of electronic sensor (transistor)
and electrical sensor (electrode for impedance measurement).
[0068] Data interpretation can be based on the premise that no
specific probes or binding partners are required. Therefore, data
obtained should be compared to reference or control samples or to
normalized data. Algorithms can be trained and used to address
particular problems.
[0069] Embodiments of the invention are applicable to clinical,
research, pharmaceutical, agriculture, and environmental
protection. Samples may need fractionation or enrichment before
contacting a chip. Different chips can be used for the same sample
to get complete information of interest.
[0070] The invention can include modifying the surface of glass or
silicon with silanes that contain phenyl or other aromatic moieties
that have absorption at about 260 nm and below. These materials can
form a self assembling monolayer (SAM) using standard
microelectronics processing techniques such as those used to
promote adhesion of photoresists in standard high volume
manufacturing (HVM) processing.
[0071] Referring to FIG. 9A, an embodiment of the invention is
depicted as including a self aligned monolayer (SAM) on a substrate
of silicon, silica, or metal oxides. In the case of simple aromatic
groups R can be, e.g., hydrogen, amine, ethylenediamine, cyano,
methyl, or fluorine groups. Therefore, the starting SAM can have
many different chemical characteristics that determine the surface
energy, polarity, and capability to attach additional moieties to
or just be a relatively inert reaction well characterized starting
surface for further modification using deep ultra violet (DW)
light. An embodiment of the invention can use a phenyl group
(R.dbd.H) for the example depicted in FIGS. 9A-9D.
[0072] Once the substrate has been treated it can be exposed (flood
or using high resolution mask) on a standard and readily
commercially available DUV scanner or stepper. Because the Si--C
bond is the weakest bond in the SAM what occurs is the breakage of
that bond and the phenyl group is volatilized. In ambient
atmosphere, the Si--: radical reacts with O2 & H2O to form
SiOH. It is important to note that this is the same surface as the
initial substrate surface, but before the formation of the SAM, and
it is now one Si atom taller. The dose in mJ/cm2 to completely
remove all of the phenyl groups is well documented in several
publications and is on the order of 200 to 1000 mJ/cm2 and is also
dependent on the type of aromatic and organic group chosen for the
original SAM. For instance, it can be assumed that 500 mJ/cm2 is
the dose to remove all the phenyl groups.
[0073] Referring to FIG. 9B, the resulting surface after the
substrate and SAM has been exposed to 50 mJ/cm2 is depicted. After
exposure .about.10% of the surface is now available for a 2.sup.nd
SAM to be formed. It is most important to note in this example that
2nd SAM material may have no aromatic group and, therefore, will
not be affected by subsequent DUV exposures because it has
substantially no absorption in the DUV spectrum, i.e., above -200
nm. In this example, perfluorooctyldimethylchlorosilane is used
(SAM2) as the next SAM formation material. Treatment of the exposed
surface with SAM2 will yield a new surface containing .about.10% of
SAM2 and 90% of the original phenyl silane SAM as depicted in FIG.
9C.
[0074] In this example, one additional exposure/SAM formation using
a trimethoxysilane N-(2-aminoethyl-3-aminopropyl)trimethoxysilane
SAM3 is performed, but this process could be continued to build a
very large variety of well defined surfaces. In this example a 2nd
exposure is 100 mJ and will remove .about.20% of the remaining
phenyl groups and following treatment with SAM3 will create a
surface with .about.20% SAM3 .about.10% SAM2 and .about.70% of the
original SAM as depicted in FIG. 9D.
[0075] This procedure could be continued to put more SAMs of known
concentration on the surface and subsequent surface chemistry can
be done to attach bio-relevant chemistry such as antibodies, DNA,
or RNA, to the appropriate R group on the SAM. The surface can thus
be patterned in arrays very easily and even have high resolution
(<100 nm line/space) within an array. This embodiment of the
invention makes it feasible to make arrays of well defined surface
chemistry with minimal reticles or masks.
[0076] For instance, if it were desired to make small areas (100 um
square) of well defined, but different surface concentrations of
the three SAMs described above on bare Si metal oxides or glass,
the following technique could be used. The surface can be treated
with photosensitive trichlorophenylsilane and then exposed via a 10
um.times.10 um array with dose increments of 5 mJ/cm2 (i.e.,
.about.1% the does assumed above to be required to remove all the
phenyl groups) over a range from 0-500 mJ/cm2. Then SAM2 formation
can be performed resulting in 10.times.10 array containing a ratio
of the 1.sup.st two SAMs of from approximately 0% to approximately
100% across the array. The 2.sup.nd exposure can then be performed
but in reverse spatial arrangement of the increments, or with any
desired dose range, to yield many different surfaces of known
composition. Specifically, with a reverse exposure starting at 500
mJ and going to 0 in the same increments, the result would be
10.times.10 array that contains .about.100% SAM2 & SAM3, but
with .about.0% of the original SAM.
[0077] However, in another instance, an embodiment of the invention
could utilize the same range and not reverse dose, and this would
result in an array with .about.100% original SAM and with the SAM2
& SAM3 increasing in concentration by 1% each until they reach
.about.50% each of the surface concentration half way through the
array. From then on SAM2 would continue to increase by 1% and SAM3
would decrease by 1% with .about.0% of original same making up the
concentration of the surface until 100% SAM2 is reached at last
exposure field. The variations on this sub-generic scenario are
enormous. The same examples described above can work with 193 nm
exposure which will make the aromatic photosensitive SAMs more
efficient but will also make many of the non-aromatic SAMs slightly
sensitive to each of the subsequent exposures, but since they are
so much less absorbing they will be much less involved in the
photochemical cleavage, and therefore they are just accounted for
in determining the final composition of the surface.
[0078] While not being limited to any particular performance
indicator or diagnostic identifier, preferred embodiments of the
sensor array can be identified one at a time by testing for the
presence of sensing with respect to a known concentration of target
analyte. The test for the presence of sensing can be carried out
without undue experimentation by the use of a simple and
conventional impedance spectroscopy experiment. Among the other
ways in which to seek embodiments having the attribute of sensing
guidance toward the next preferred embodiment can be based on the
presence of a characteristic IR spectroscopy signal.
[0079] Embodiments of the electrically active
combinatorial-chemical chip for biochemical analyte detection can
be identified by scanning electron microscope (SEM) cross-sections.
Embodiments of the electrically active combinatorial-chemical chip
for biochemical analyte detection can also be identified by
material analysis of devices containing sensors using techniques
such as Auger spectroscopy and/or dynamic secondary ion mass
spectroscopy.
[0080] Embodiments of the invention can include impedance
spectroscopy, amperommetry, voltammetry and other electrochemical
techniques used to generate a response from adsorbed analyte
through the electrodes/probes. Embodiments of the invention can
include the use of optical techniques such as FTIR spectroscopy can
be used to identify the functional groups of analyzed chemicals
species.
[0081] Specific embodiments of the invention will now be further
described by the following, nonlimiting examples which will serve
to illustrate in some detail various features. The following
examples are included to facilitate an understanding of ways in
which embodiments of the invention may be practiced. It should be
appreciated that the examples which follow represent embodiments
discovered to function well in the practice of embodiments of the
invention, and thus can be considered to constitute preferred modes
for the practice of embodiments of the invention. However, it
should be appreciated that many changes can be made in the
exemplary embodiments which are disclosed while still obtaining
like or similar result without departing from the spirit and scope
of embodiments of the invention. Accordingly, the examples should
not be construed as limiting the scope of embodiments of the
invention.
EXAMPLES
Example 1
[0082] Referring to FIGS. 10A and 10B, a DNA-based self-assembly
structure is provided for affinity binding array. A single-stranded
oligonucleotide with one or more "coding regions" and a binding
ligand A1 is immobilized on one of the spots on an array surface
(FIG. 10A). The binding ligands preferably include small molecules
such as biotin, pyridine, furan, imidazole, pyran, benzene, purine,
pyrimidine, benzoic acid, aniline, styrene, phenol, typtophan, or
another compound of interest or as may be understood by those
skilled in the art. The oligo nucleotide is from approximately 20
to approximately 100 bases long, and each coding region contains
from approximately 10 to approximately 20 DNA bases with specific
sequences; while the binding ligands are small molecules such as
biotin, pyridine, furan, imidazole, pyran, benzene, purine,
pyrimidine, benzoic acid, aniline, styrene, phenol, typtophan, or
any other compounds of interest. The ligand can be attached to the
oligonucleotide through known chemistry, e.g., N-hydroxysuccinimide
ester (NHS) mediated conjugation (FIG. 10B), 1-Ethyl-3-(3-dimethyll
aminopropyl)carbodiimide (EDC) catalyzed amide formation or
reductive amination.
[0083] In general, there are several ways to immobilize DNA. A
first would be charge attraction on a positively charged surface
such as a surface coated with polysine. A second would be covalent
attachment through a molecular end such as a thiol attachment
reaction with a metal. An armine group on DNA will react with a
carboxyl group on a surface. A third way involves specific binding.
For example, biotin on DNA may be captured by streptavidin on the
surface.
[0084] After the immobilization, a second single-stranded
oligonucleotide with one or more "coding regions" and ligand B1 was
contacted with the substrate. The oligo nucleotide can be 20-100
bases long, and each coding regions can contains 10-20 DNA bases
with specific sequences One of the coding regions of the second
oligonucleotide should be complimentary to one of the coding
region. Under hybridization condition (for example, incubation at
37.degree. C. for 1 hour in 2.times.SSC buffer: 0.03 M sodium
citrate, 0.3 M NaCl, pH approx. 7.0,) the two oligo nucleotide's
will hybridize and thus the two ligands A1 and B1 will be
localized.
[0085] Additional steps can be performed to add more
ligand-oligonucleotides to the surface, resulted in a collection of
ligands A1, B1, C1 . . . localized in the said array spot at
certain orientation. On other array spots, different ligands or
different combination of ligands can be applied to create another
unique collection of ligands. Thus, an affinity array based on DNA
self-assembly can be generated.
[0086] Solvents that can be used in the ligand incorporation step
and the spotting step include water, salt buffer solution such as
SSC, and organic solvent in which oligonucleotide is soluble, such
as methanol and DMF (dimethyl Formamide). For the hybridization
step, buffer solutions such as SSC, citrate, borate and phosphate
with up to 50% of Formamide or urea can be used. The same format
can be used to make PNA and RNA self-assembly arrays as well.
Example 2
[0087] Referring to FIG. 11, an embodiment of the invention
includes a cross-linked polymer based structure for affinity
binding array. Small molecule ligand A1 was incorporated into a
cross-linkable polymers such as poly(acrylamide)-co-poly(acrylic
acid), and other ligand B1, C1, was incorporated into other
cross-linkable polymers individually. The cross-linkable polymer
was selected from synthetic polymers such as polyacrylamide,
polyacrylic acid, polyallyamine, polyvinylalcohol, and natural
polymers such as polysaccharide and DNA.
[0088] Referring to FIGS. 12A and 12B, two possible incorporation
methods are copolymerization (FIG. 12A) and chain-transfer (FIG.
12B). In the co-polymerization method, a reactive monomer was added
to the polymerization mix and the resulted co-polymer was reacted
with ligand A1, B1, through the reactive function group NHS-ester.
In the chain transfer method, the ligands were linked to a
chain-transfer reagent such as thiol, and hence incorporated at the
end of the polymer chain. Other polymer functionalization methods
such as post-polymerization reaction can also be used.
[0089] A mixture of the ligand-loaded cross-linkable polymer was
deposited on an array spot, and a cross-linker such as
1,4-diaminobutane was introduced after the polymers were activated
by EDC (1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide). The
resulting cross-linked polymer had the attached ligands (A1, B1, C1
. . . ) localized in the array spot. Using this method, each array
spot was deposited with a different set of ligands, and an affinity
array was built.
[0090] Preferred and alternative solvents that were and/or could be
used in the ligand incorporation step, the spotting step, and
cross-linking steps include water-based solvents such as water and
buffer solutions such as citrate, borate and phosphate; and organic
solvent such as DMF (dimethyl Formamide), ethyl alcohol, methanol,
iso-propanol, and THF (tetrahydrofuran). Other cross-linking
methods such as glutaraldehyde crosslinking of mines or
photo-initiated radical crosslinking of poly-acrylamide can also be
used.
Example 3
[0091] Referring to FIGS. 13A-13D, an embodiment of the invention
includes making combinatory chemical structures. PEG-based thiol
compounds with combinatory chemical structures were made from
thiol-PEG-mine or thiol-PEG-carboxylate.
DCC=N,N'-dicyclohexylcarbodiimide. The molecular weight of these
thiol-PEG chemical structures was from 200 to 5000, with 3-100
repetitive PEG units. The above reactions were performed in water,
water-based buffer solution such as sodium phosphate solution, and
organic solvents such as DMF and THF.
[0092] Generally, PEG polymers may be advantageously used, as in
example 3, primarily to fill the surface spaces or gaps on a chip.
It can reduce non-specific binding. If one end of the PEG polymers
have thiol groups that can be attached to a gold surface, the PEG
molecules can self-assemble into a monolayer on the gold surface.
If a portion of the PEG polymers have functional groups (for
example, examine groups), then the chip surface will be
functionalized by the amine groups whose density is determined as a
% of the amine-containing PEG polymers.
[0093] A practical application of embodiments of the invention that
has value within the technological arts is integrating chemical
and/or biological sensing with computing and communication. There
are virtually innumerable uses for embodiments of this
invention.
[0094] Embodiments of the invention are cost effective and
advantageous for at least the following reasons. An embodiment of
the invention obviates the need for specific probe synthesis as
well as site-specific immobilization and only a limited number of
small chemicals can be used in different combinations. An
embodiment of the invention can have the advantage of simplifying
chip fabrication. An embodiment of the invention can have a compact
size and high capacity. Electrical detection allows fabrication of
chips with very high sub-area density (protein chip with feature
size<1 um), no optical system and thus chip reader can be very
compact. An embodiment of the invention can be flexible and
applicable to samples of different compositions and from different
sources. An embodiment of the invention can have the advantage of
detecting not-yet-identified analytes in that unknown compounds can
bind to the EACC chips and be detected. In general, embodiments of
the invention improve quality and/or reduce costs compared to
previous approaches.
[0095] The terms "a" or "an", as used herein, are defined as
including one and more than one. The term plurality, as used
herein, is defined as including two and more than two. The term
another, as used herein, is defined as at least a second or more.
The terms "comprising" (comprises, comprised), "including"
(includes, included) and/or "having" (has, had), as used herein,
are defined as open language (e.g., requiring what is thereafter
recited, but open for the inclusion of unspecified procedure(s),
structure(s) and/or ingredient(s) even in major amounts. The terms
"consisting" (consists, consisted) and/or "composing" (composes,
composed), as used herein, close the recited method, apparatus or
composition to the inclusion of procedures, structure(s) and/or
ingredient(s) other than those recited except for ancillaries,
adjuncts and/or impurities ordinarily associated therewith. The
recital of the term "essentially" along with the terms "consisting"
or "composing" renders the recited method, apparatus and/or
composition open only for the inclusion of unspecified
procedure(s), structure(s) and/or ingredient(s) which do not
materially affect the basic novel characteristics of the
composition. The term coupled, as used herein, is defined as
connected, although not necessarily directly, and not necessarily
mechanically. The term any, as used herein, is defined as all
applicable members of a set or at least a subset of all applicable
members of the set. The term approximately, as used herein, is
defined as at least close to a given value (e.g., preferably within
10% of, more preferably within 1% of, and most preferably within
0.1% of). The term substantially, as used herein, is defined as
largely but not necessarily wholly that which is specified. The
term generally, as used herein, is defined as at least approaching
a given state. The term deploying, as used herein, is defined as
designing, building, shipping, installing and/or operating. The
term means, as used herein, is defined as hardware, firmware and/or
software for achieving a result. The term program or phrase
computer program, as used herein, is defined as a sequence of
instructions designed for execution on a computer system. A
program, or computer program, may include a subroutine, a function,
a procedure, an object method, an object implementation, an
executable application, an applet, a servlet, a source code, an
object code, a shared library/dynamic load library and/or other
sequence of instructions designed for execution on a computer or
computer system.
[0096] All the disclosed embodiments of the invention disclosed
herein can be made and used without undue experimentation in light
of the disclosure. Embodiments of the invention are not limited by
theoretical statements recited herein. Although the best mode of
carrying out embodiments of the invention contemplated by the
inventor(s) is disclosed, practice of the embodiments of the
invention is not limited thereto. Accordingly, it will be
appreciated by those skilled in the art that the embodiments of the
invention may be practiced otherwise than as specifically described
herein.
[0097] It will be manifest that various substitutions,
modifications, additions and/or rearrangements of the features of
the embodiments of the invention may be made without deviating from
the spirit and/or scope of the underlying inventive concept. It is
deemed that the spirit and/or scope of the underlying inventive
concept as defined by the appended claims and their equivalents
cover all such substitutions, modifications, additions and/or
rearrangements.
[0098] All the disclosed element's and features of each disclosed
embodiment can be combined with, or substituted for, the disclosed
elements and features of every other disclosed embodiment except
where such elements or features are mutually exclusive. Variation
may be made in the steps or in the sequence of steps defining
methods described herein.
[0099] Although the sensor array described herein can be a separate
module, it will be manifest that the sensor array(s) may be
integrated into the system with which it is (they are) associated.
Similarly, although the hand held device described herein can be a
separate module, it will be manifest that the hand held device(s)
may be integrated into the system with which it is (they are)
associated.
[0100] The sensor array may comprise an array of transistor
sensors. Different chemical structures (groups or molecules) may be
disposed on the gates of the transistors. A sample preferably
contacts each of the gate-associated chemical structures. A set of
pre-determined chemical structures are associated with a set of
transistors. Different analytes interact with the pre-determined
chemical structures differently, such that the patterns are unique.
Binding patterns are translated into electrical signals by the
transistors. The analytes in the sample may be identified by the
pattern of electrical signals of the transistors with respect to
the gate-associated chemical compositions. A database is preferably
pre-built using standard analytes, and computer pattern recognition
is used in the identification.
[0101] The individual components need not be formed in the
disclosed shapes, or combined in the disclosed configurations, but
could be provided in all shapes, and/or combined in all
configurations. The individual components need not be fabricated
from the disclosed materials, but could be fabricated from all
suitable materials. Homologous replacements may be substituted for
the substances described herein. Agents that are both chemically
and physiologically related may be substituted for the agents
described herein where the same or similar results would be
achieved.
[0102] While an exemplary drawings and specific embodiments of the
present invention have been described and illustrated, it is to be
understood that that the scope of the present invention is not to
be limited to the particular embodiments discussed. Thus, the
embodiments shall be regarded as illustrative rather than
restrictive, and it should be understood that variations may be
made in those embodiments by workers skilled in the arts without
departing from the scope of the present invention as set forth in
the appended claims and structural and functional equivalents
thereof.
[0103] In methods that may be performed according to the invention
and/or preferred embodiments herein and that may have been
described above and/or claimed below, the operations have been
described in selected typographical sequences. However, the
sequences have been selected and so ordered for typographical
convenience and are not intended to imply any particular order for
performing the operations.
[0104] The appended claims are not to be interpreted as including
means-plus-function limitations, unless such a limitation is
explicitly recited in a given claim using the phrase(s) "means for"
and/or "step for." Subgeneric embodiments of the invention are
delineated by the appended independent claims and their
equivalents. Specific embodiments of the invention are
differentiated by the appended dependent claims and their
equivalents.
* * * * *