U.S. patent application number 09/363929 was filed with the patent office on 2001-12-06 for laboratory in a disk.
Invention is credited to VIRTANEN, JORMA.
Application Number | 20010048895 09/363929 |
Document ID | / |
Family ID | 21905358 |
Filed Date | 2001-12-06 |
United States Patent
Application |
20010048895 |
Kind Code |
A1 |
VIRTANEN, JORMA |
December 6, 2001 |
LABORATORY IN A DISK
Abstract
An apparatus is described that includes an optical disk, adapted
to be read by an optical reader, comprising a first sector having
substantially self-contained assay means for localizing an analyte
suspected of being in a sample to at least one, predetermined
location in the first sector and a second sector containing control
means for conducting the assay and analyte location information,
with respect to one or more analytes suspected of being in a
sample, accessible to the reader, wherein the presence or absence
of the analyte at said location is determinable by the reader using
the control means and the location information. Depending on the
nature of the assay, the disk will include fluid storage means,
fluid transfer means, such as one or more capillary ducts, valves,
batteries, dialyzers, columns, filters, sources of electric fields,
wires or other electrical conductive means such as metallic surface
deposits and the like.
Inventors: |
VIRTANEN, JORMA; (IRVINE,
CA) |
Correspondence
Address: |
David J Oldenkamp, Esq.
Oppenheimer Wolff & Donnelly LLP
2029 Century Park East, Suite 3800
Los Angeles
CA
90067
US
|
Family ID: |
21905358 |
Appl. No.: |
09/363929 |
Filed: |
July 29, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09363929 |
Jul 29, 1999 |
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09064636 |
Apr 21, 1998 |
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6030581 |
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09064636 |
Apr 21, 1998 |
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PCT/US98/04377 |
Feb 27, 1998 |
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60039419 |
Feb 28, 1997 |
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Current U.S.
Class: |
422/68.1 ;
422/50; 435/6.11; 435/7.1 |
Current CPC
Class: |
B01L 3/502715 20130101;
B01L 2400/0409 20130101; B01L 2300/023 20130101; B01L 3/502738
20130101; B01L 3/5027 20130101; B01L 3/545 20130101; B01L 2200/0673
20130101; B01L 2300/18 20130101; B01L 7/52 20130101; C12Q 1/6825
20130101; B01L 2300/06 20130101; B01L 2300/1855 20130101; B01L
2200/10 20130101; B01L 2400/0677 20130101; B01L 3/502746 20130101;
G01N 35/00069 20130101; B01L 2200/16 20130101; B01L 2300/1827
20130101; G01N 33/54373 20130101; B01L 2300/0803 20130101; B01L
2300/0867 20130101; B01L 2300/024 20130101; B01L 2400/0415
20130101; B01L 2400/0638 20130101; B01L 2300/0806 20130101; B01L
2300/1833 20130101; B01L 2200/0605 20130101; B01L 3/50273 20130101;
C12Q 1/6834 20130101; B01L 3/502753 20130101; B01L 2300/02
20130101; B01L 2300/0864 20130101; B01L 2300/1861 20130101; B01L
3/502784 20130101; B01L 2300/0645 20130101 |
Class at
Publication: |
422/68.1 ; 435/6;
435/7.1; 422/50 |
International
Class: |
G01N 015/06; G01N
001/00; C12Q 001/68; G01N 033/53 |
Claims
What is claimed is:
1. An optical disk, adapted to be read by an optical reader,
comprising a first sector having a substantially self-contained
assay means for binding or reacting an analyte suspected of being
in a sample to at least one, predetermined location in the first
sector and optionally a second sector containing a control means
for conducting the assay and analyte location information with
respect to one or more analytes suspected of being in a sample,
accessible to a reader, and wherein the presence or absence of the
analyte at said location is determinable by the reader using the
control means and the location information.
2. The optical disk of claim 1 which includes a sealable sample
entry port in fluid communication with the assay means.
3. An apparatus for conducting an assay comprising an optical disk,
a disk reader and an information processor, wherein the disk
comprises a first sector having a substantially self-contained
assay means for binding an analyte suspected of being in a sample
to at least one, predetermined location in the first sector and
optionally a second sector containing control information for
conducting the assay and analyte location information with respect
to one or more analytes suspected of being in the sample,
accessible to the reader and processable by the information
processor, wherein the disk is adapted to be read by the reader and
the information processor is adapted to determine the presence or
absence of the analyte at said location using the control
information and the location information.
4. The apparatus of claim 3 wherein the reader is a CD-ROM or a DVD
reader and wherein the reader is adapted to be coupled to an
information processor.
5. The apparatus of claim 4 wherein the information processor is a
personal computer.
6. The disk of claim 1 wherein the assay means comprises a fluid
storage means and a fluid transfer means formed in a disk
surface.
7. The disk of claim 6 wherein the fluid transfer means comprises a
capillary duct.
8. The disk of claim 7 wherein the fluid transfer means comprises a
valve.
9. The disk of claim 6 wherein the disk comprises an
electrochemical energy means.
10. The disk of claim 1 wherein the assay means comprises a sample
port, a sample preparation sector, an analyte separation sector and
an assay sector wherein the analyte is localized.
11. The disk of claim 6 wherein the fluid transfer means is
responsive to centrifugal force or an electric field.
12. The disk of claim 1 wherein the disk comprises a multiplicity
of first sectors adapted to analyze for a multiplicity of
analytes.
13. The disk of claim 1 further comprising a multiplicity of first
sectors adapted to analyze for the same analyte or different
analytes wherein each of said multiplicity of sectors is adapted
for fluid communication to a sample port.
14. An assay element comprising a substrate, a first
oligonucleotide bound to the substrate, a spacer molecule bound at
a first end to the first oligonucleotide by a second
oligonucleotide that is complementary to the first oligonucleotide,
wherein the spacer molecule further comprises a means for binding
to an analyte in a sample and having a second end that is
detectable by a detection means, the spacer molecule further
comprising a site intermediate the first and second ends that is
cleavable, the binding means having a first moiety between the
first end of the spacer molecule and the cleavage site for binding
to a first part of the analyte and a second moiety between the
second end of the spacer molecule and the cleavage site for binding
to a second part of the analyte.
15. An assay component, adapted to be read by a CD-ROM or a DVD
reader comprising an optical disk and a substantially
self-contained assay means in the disk for binding an analyte
suspected of being in a sample to at least one, predetermined
location on the disk and means at said location for enabling
detection of the absence or presence of the analyte by the CD-ROM
or the DVD reader.
16. An optical disk, adapted to be read by a CD-ROM or a DVD reader
comprising a substantially self-contained assay means for
localizing an analyte suspected of being in a sample to at least
one, predetermined location on the disk and a means at said
location for detecting the absence or presence of the analyte by
the CD-ROM or the DVD reader.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to diagnostic assays and
methodology therefor. In particular, it relates to diagnostic assay
components configured on a compact optical disk and methodology for
the use thereof
BACKGROUND
[0002] There is an enormous need to make clinical assays faster,
cheaper and simpler to perform. Ideally patients should be able to
test themselves, if so desired. One-way towards this goal has been
through miniaturization and integration of various assay
operations. Currently, a number of bio-chip assays (so-called
because some are built using silicon chip photolithography
techniques) are commercially available or under development. All of
these approaches require a reading machine and a computer.
[0003] Disk-shaped cassettes used for clinical assays in
conjunction with UV/Vis spectrometry are also commercially
available. U.S. Pat. No. 15,122,284 describes a centrifugal rotor
that contains a number of interconnected fluid chambers connected
to a plurality of cuvettes. The rotor is adapted to be utilized
with a conventional laboratory centrifuge, and is formed of
materials that allow photometric detection of the results of assays
that have been carried out in the reaction cuvettes. A large number
of rotor configurations and related apparatus for the same or
similar types of analysis have been described. See for example U.S.
Pat. Nos. 5,472,603; 5,173,193; 5,061,381; 5,304,348; 5, 518,930;
5,457,053; 5,409,665; 5,160,702; 5,173,262; 5,409,665; 5,591,643;
5,186,844; 5,122,284; 5,242,606; and patents listed therein.
Lyophilized reagents for use in such systems are described in U.S.
Pat. No. 5,413,732.
[0004] The principles of a centrifugal analyzer have been adapted
into a disk that can be used. in a CD-drive like instrument (Mian,
et al., WO 97/21090 Application). Mian teaches a modified CD-drive
with a dual function: 1. It is used to read information stored in
the disk, and 2. It is used to rotate the disk. However, Mian does
not teach utilization of the reading capability of a CD-drive for
actual assay analysis.
[0005] Notwithstanding recent advances, there remains a need for a
simpler assay configuration that performs assays quickly,
efficiently, accurately and at low cost. The present invention
combines diagnostic assays with computers and compact disk
technology. In its most preferred embodiment, a computer with a
compact disk reader is the only instrument needed. All chemistry is
performed inside a compact disk that may be referred to as an
integrated biocompact disk (IBCD). The same compact disk is also
encoded with software, i.e., machine-readable instructional and
control information, that provides instructions to a computer prior
to, during and after the assay.
[0006] CDs or DVDs represent the most economical and in many ways
best information storage media. It must be noted that CD and DVD
are currently used acronyms that may change in the future even if
the underlying technology stays basically the same. A CD- or
DVD-drive is in several respects equivalent to a scanning confocal
microscope. At the same time these instruments are comparable to
good centrifuges, because in commercial drives the rotation
frequency is between 200-12,000 rpm and can be adjusted within
certain limits. Combining these three features into the same
analytical system results into great simplification as compared
with any other analytical technique. Yet, the performance is
comparable or better than in most competing methods. Although this
invention requires slightly modified CD-or DVD-drives, it is
possible to incorporate these changes into commercial drives. This
will enable Point-Of-Patient-Care (POPC) and home use of this
invention. Use of CD- or DVD-drives will allow accurate digital
analysis of any sample without any specific analytical
instrumentation.
SUMMARY OF THE INVENTION
[0007] In one aspect, the invention is directed to an optical disk,
adapted to be read by an optical reader, comprising a first sector
having a substantially self-contained assay means for binding an
analyte suspected of being in a sample to at least one
predetermined location in the first sector and optionally a second
sector containing control means for conducting the assay and
analyte location information, with respect to one or more analytes
suspected of being in a sample, accessible to a reader, wherein the
presence or absence of the analyte at said location is determinable
by the reader using the control means and the location information.
Depending on the nature of the assay, the disk may include fluid
storage means, fluid transfer means, such as one or more capillary
ducts, valves, batteries, dialyzers, columns, filters, sources of
electric fields, wires or other electrical conductive means such as
metallic surface deposits and the like.
[0008] The disk may have one or more sample entry ports to deliver
sample fluid to the assay sector. Such ports if present are
preferably sealable so that after application of the sample to the
disk, the sealed disk including the sample comprises a hermetically
sealed device that may be conveniently disposed of by conventional
means or other disposal mechanisms for dealing with biological
waste. Also, the assay sector of the disk is conveniently divided
into various subsections for sample preparation and analyte
separation. A waste receptacle subsection may be conveniently
provided as well. The assay sector may be divided into a
multiplicity of subsectors that each receives a sample. Each such
subsector may analyze for one or more analytes depending on the
particular application at hand.
[0009] In another aspect the invention is directed to an apparatus
for conducting an assay comprising an optical disk, a disk reader
and an information processor, the disk comprising a first sector
having substantially self-contained assay means for localizing an
analyte suspected of being in a sample to at least one,
predetermined location in the first sector and optionally a second
sector containing control information for conducting the assay and
analyte location information, with respect to one or more analytes
suspected of being in the sample, accessible to the reader and
processable by the information processor, wherein the disk is
adapted to be read by the reader and the information processor is
adapted to determine the presence or absence of the analyte at said
location using the control information and the location
information. The apparatus may include a reader having a CD-ROM or
DVD reader and an information processor, such as a personal
computer.
[0010] In still another aspect the invention is directed to an
optical disk, adapted to be read by a CD-ROM or DVD reader,
comprising a substantially self-contained assay means in the disk
for localizing an analyte suspected of being in a sample to at
least one, predetermined location on the disk and means at said
location for detection of the absence or presence of the analyte by
the CD-ROM or DVD reader.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic representation of a disk of this
invention.
[0012] FIG. 2 A is a more detailed schematic representation of a
sample preparation and assay sector of the disk, illustrating the
overall layout of a typical assay sector.
[0013] FIG. 2 B is a schematic representation of an ubiquitous
assay sector that is capable of performing immunoassays, DNA
testing, cell counting, spectrophotometric assays and electrolyte
analysis.
[0014] FIG. 3 is a schematic representation of a disk of this
invention illustrating a multiplicity of assay sectors, each having
an individual sample inlet port.
[0015] FIG. 4 is a more detailed schematic representation of one of
the assay sectors illustrated in FIG. 3.
[0016] FIG. 5 is a schematic representation of a chemically
actuated battery useful in the present invention.
[0017] FIG. 6 is a schematic representation of a structure to
provide a dialysis function in the disk of this invention.
[0018] FIG. 7 is a schematic representation of a column that may be
included in the disk of this invention.
[0019] FIG. 8 is a schematic representation of an electrically
controlled valve useful in the present invention.
[0020] FIG. 9 is a schematic representation of a reagent train
configured in joined capillary ducts that is useful in the present
invention.
[0021] FIG. 10 is a schematic representation of an array of linear
assay sites that are conveniently located in a flow channel in the
assay sector of the disk of this invention.
[0022] FIG. 11 A-C is a schematic representation of a variation of
an assay element that is particularly useful for the detection of
viral and bacterial particles and cells using the general
methodology of site specific localization of the substance to be
detected.
[0023] FIG. 12 A-C is a schematic representation of a variation of
the detection methodology in which opaque particles are utilized in
the place of the reflective particles and bound to a reflective
surface. Zig-zag lines represent oligonucleotides, but can be any
recognition molecules, such antibodies. Particles are in this
example plastic spheres, but can be liposomes, cells, etc.
[0024] FIG. 13 is a schematic representation of an assay element of
the invention illustrating the spacer molecule, with component
sidearms and cleavage site, bound to a disk surface at one end and
to a reporter element (gold or latex sphere) at its other end.
[0025] FIG. 14 A is schematic representation of a first assay
element of this invention, at an early stage during the assay
procedure.
[0026] FIG. 14 B is schematic representation of a second assay
element of this invention, at an early stage during the assay
procedure.
[0027] FIG. 14 C is a schematic representation of the assay element
in FIG. 14 A wherein analyte molecules have bound the sidearms
forming a connective loop between the sides of the cleavage
site.
[0028] FIG. 14 D is a schematic representation of the assay element
in FIG. 14 B wherein analyte molecules have not bound to the
sidearms and no connective loop has formed between the sides of the
cleavage site.
[0029] FIG. 14 E is a schematic representation of the assay element
in FIG. 14 C after the spacer molecules have been cleaved. The
reporter element remains attached to the disk surface at a discrete
site.
[0030] FIG. 14 F is a schematic representation of the assay element
in FIG. 14 D after the spacer molecules have been cleaved. The
reporter element is detached from the disk surface and free to be
washed away from its discrete site.
[0031] FIG. 15 is a schematic representation of a cuvette assembly.
Four cuvettes and their associated reagent and sample preparation
chambers as well as light sources are shown in this example.
[0032] FIG. 16 is a schematic representation of a capillary array
that can be used to perform isoelectric focusing.
[0033] FIG. 17 is a schematic representation of an apparatus for
measuring exact volumes.
DETAILED DESCRIPTION OF THE INVENTION
[0034] A schematic overall representation of an integrated
bio-compact disk (IBCD) is set forth in FIG. 1. The disk
(Bio-Compact Disk, BCD) may be virtually of any shape and size. For
most practical applications it is circular having a diameter of
10-1000 mm, most advantageously 20-200 mm and a thickness of 0.1-20
mm, most advantageously 0.5-3 mm. The disk 10 contains two sectors:
an assay sector 11 and a software sector 12. A central hole 13 is
provided for location in a compact disk reader. Software for
controlling the assay may be on a separate disk. However, it is
preferred to have the software on the disk associated with an assay
for a particular analyte or analytes to minimize the opportunity
for human error when performing the assay. The possible components
and unit operations of the IBCD are presented in the following
description.
[0035] The disk rotates typically up to 16,000 rpm in conventional
CD-ROM or DVD readers. In all CD-ROM and DVD readers the speed is
adjustable within certain limits (200-16,000 rpm). However, for
some operations it may be advantageous to utilize rotations at
differing speeds, for example 1000-10,000 rpm, and most preferably
2000-5000 rpm. For any particular assay, the controlling software
dictates the rotation regimen during the analysis. This regimen,
the speeds and timing, including times in which perhaps no rotation
occurs to allow for incubation, electrophoresis, isoelectric
focusing, etc., is controlled to deliver reagents and sample to
appropriate sites on the assay sector as dictated by the assay
protocols. Available rotational speeds do allow for a significant
centrifugal force that may be used to move liquids. Another energy
source that may be easily used in the IBCD is chemical energy. A
most suitable form of chemical energy is released by a battery in
the form of electrical energy. Mechanical and chemical energy allow
the operation of many kinds of components. Important components of
a IBCD may include one or more of the following: capillaries,
containers, filters, dialysis membranes, chromatographic columns,
electrophoretic gels, valves, any micromechanical or electronic
components including microprocessors, electrodes, especially enzyme
electrodes, cuvettes, and assay elements. The possible unit
operations carried out by the components include the following:
centrifugation, filtering, transfer of liquids, mixing of liquids,
dialysis, column separations, heating, cooling, electroconvection,
electrophoresis, and analyte detection and signaling thereof.
[0036] The IBCD is conveniently made from two pieces comprising
upper and lower halves. The lower half may contain almost all the
components, while the upper half may be a flat cover containing
only a few components, such as electrodes and wires. The number of
layers in this invention may be more than two and many components
may also be pre-made as modules. Especially reagent containers,
cuvette assemblies, columns, micromechanical components, light
sources, and microprocessors are advantageously assembled as
modules. Various features may be printed onto the soft plastic.
Various components may be glued, either by thermal or UV-curing,
melted together, connected by complementary mechanical features,
mechanically clamped or simply enclosed inside a larger component.
Some areas may be treated, for instance, with ammonia plasma to
render these areas hydrophilic. The surface may be further treated
by various molecules that render the surface inert or alternatively
give it specific adsorption properties. Silylation is a general
method for the treatment of surfaces (Virtanen, J. A., Kinnunen, P.
K. J. and Kulo, A., "Organosilanes and their hydrolytic polymers as
surface treatment agents for use in chromatography and
electronics," U.S. Pat. No. 4,756,971). Covalent attachment of
detergents will reduce the adsorption of proteins, such as albumin,
and will also reduce the adsorption of soluble proteins. Metal
electrodes and wires may be evaporated onto desired areas. Masks or
resists may be used to localize the plasma treatment or metal
deposition. Capillary ducts and fluid storage and retention
compartments may be machined into the optical disks or formed by
chemical means or in injection molding operations. As shown with
reference to FIG. 2, the assay sector may contain a sample inlet
port 14. The sample port is preferably sealable so that at the disk
is effectively sealed, except for necessary venting to allow for
fluid flow, to protect from any biological hazards. By various
means, e.g. centrifugal force and like means that are well known in
the art, a portion of the sample is metered to a sample preparation
site 15, that may contain reagents and the like in order to conduct
the assay. Alternatively, or in conjunction with reagents already
in the sample preparation segment, a reagent train 16 may be
provided to deliver, as needed, the necessary reagents in the
proper order to the sample preparation segment. Additional details
of the reagent train are shown in FIG. 9. It may be necessary to
separate the analyte from the sample, at least partially, and this
may be done in a sample separation segment designated generally as
17. A battery 18 is provided if electrical energy is required for
the separation process. Additional details of the battery are shown
in FIG. 5 and described below. The resultant sample is then
transferred to the assay site 19. In a preferred embodiment of the
invention, the assay site contains an assay element as described in
greater detail below. The analyte binds to a predetermined location
on the disk if it is present in the sample, and the presence of the
analyte is detected by the reader from information that identifies
the particular analyte with the location at which it is bound. A
waste compartment is provided to collect overflow of reagents or
sample that exceeds metered amounts for use in the assay and the
various compartments and fluid transfer channels are vented
appropriately to allow for fluid flow throughout the surface of the
assay sector.
[0037] In one embodiment of the invention, a multiplicity of assay
sectors 21, 22, 23, etc. as shown in FIG. 3 may be provided, each
sector connected to an individual sample inlet port 24, 25, 26
respectively. The operation of each sector is substantially as
described above although different assays may be conducted at the
same time in individual sectors either for a multitude of analytes
or a multitude of patients. The details of a particular sector are
shown in greater detail in FIG. 4, where the various possible
components are identified by the same numbers as used in the
foregoing description.
Components
[0038] As shown in FIG. 5, a battery may be provided that consists
simply of two metal layers, such as copper and zinc, which are in
the lower and upper half, respectively. During storage they are
separated by air. When the disk is rotated, the space between these
two metals is filled by dilute mineral acid, depending on the
nature of the metal electrodes. In the case of copper and zinc,
this may be dilute sulfuric acid, containing copper ions and the
battery is activated. This battery generates a voltage of 1.5 V for
only about 1 hour. However, this is more than enough to complete
the analysis. Longer lasting batteries may be made, if necessary,
from other materials or thicker metal layers. Importantly, allowing
water to flow into the space between the metal layers deactivates
the battery. The activation and deactivation cycle may be repeated
several times. Several batteries may be coupled in series to
increase the potential, if necessary. Optionally, photodiodes may
be included into the circuitry. In this case, the computer
controlling the assay is provided with information about the active
circuits. Also, a miniaturized, pre-fabricated battery may be
utilized and activated by closing the electrical circuit with a
salt, e.g. sodium chloride, solution.
[0039] Capillaries preferably are used to transfer liquid and air.
Also, very small volumes of liquid may be stored in capillaries.
Preferably, air capillaries are hydrophobic, while capillaries that
come into contact with water are hydrophilic. As necessary,
capillaries may have circular or rectangular cross-sections.
Typical depths are between 10 .mu.m and 500 .mu.m, while widths are
between 50 .mu.m and 2 mm. Air capillaries utilize the larger
dimensions to prevent any formation of a pressure gradient, unless
otherwise desired. The velocity of the flow depends on the
frequency of the rotation of the IBCD, the dimensions of the
capillary and the viscosity and density of the liquid. Physical
properties of the liquid are dictated by the assay and the
frequency of rotation is limited to a certain extent by the CD-ROM
or DVD reader. Thus, the dimensions of the capillary are used to
adjust the speed of the liquid transfer. The capillary ducting may
be provided with "bottlenecks," i.e., restrictions in the
cross-sectional areas of the capillary, to control the velocity of
the liquid as necessary. Hydrophilicity and hydrophobicity may be
used for the same purpose.
[0040] The exact dimensions of the capillary network and chambers
may be designed by using the Navier-Stokes equation:
.rho.v=.rho.-.gradient.p+.mu..gradient..sup.2v
[0041] where .rho. is the density, p is the pressure, v is the
velocity, b is the body force field, .mu. is the viscosity and
.gradient.is the differential operator del (Mase, Continuum
Mechanics, McGraw-Hill, 1970). Pressure is a scalar field, while v
and b are vector fields. Commercial computer software for solving
of the Navier-Stokes equation in complicated geometries is
available.
[0042] Containers or compartments formed in the disk are used for
sample input, to store reagents, to perform reactions and to
collect waste. Their depth is about 1-2000 .mu.m, preferably about
10-800 .mu.m and they may have any shape possible, although
circular or rectangular cross-sections are preferred. Compartments
are hydrophilic, except for one end of the waste container which
has an air capillary that is hydrophobic. Reaction compartments may
be formed with electrodes for heating, electroconvection of
electrochemical purposes. Electrodes are preferably evaporated gold
films. Compartments may also have valves that are operated by
electricity or chemically as described below. Storage containers
may be metal coated, preferably gold coated, to prevent the
penetration of the water into the plastic. Reagents may also be
prepacked into cassettes, which are virtually impermeable. These
cassettes may be closed during storage and opened manually, by
puncturing or by opening a valve or plug when the sample cassette
is place in the disk. Opening of the cassette may also be
facilitated by centrifugal force when the IBCD starts to rotate. In
any case, proper liquid flow is maintained during the assay by
computer control via CD or DVD-reader.
[0043] The liquid flow during the assay may be monitored by using a
reflective element. The reflective element utilizes the laser that
is in the CD or DVD-reader and the fact that even when the liquid
is transparent its reflective index is significantly different from
that of air. Thus the laser light is reflected back to the CD or
DVD-reader in the presence of air and in some other direction in
the presence of liquid, or vice versa. Another method of monitoring
the liquid flow is to use an active light source, such as an LED or
semiconductor laser. Such a light may be powered by the presence of
an electrically conductive liquid, such as plasma or buffer, acting
to close an electronic circuit.
[0044] A LC-display may be used to transmit information from the
IBCD to the CD or DVD-drive and to the computer. LC-display may
have a large number of pixels that reflect light when there is a
potential over the LC-film. These pixels may be, for instance,
linearly organized, so that in one end low potential is needed for
the reflection of the light, while on the other end the potential
must be much higher for the same result. A CD or DVD-drive is able
to localize the reflective pixels and accordingly, the potential in
the circuit can be measured. Potential change can be due to an
electrochemical process in one of the electrochemical cells. For
example, an electrode coated with cholesterol oxidase will generate
hydrogen peroxide in the presence of cholesterol. Hydrogen peroxide
will change the potential of the circuitry and cholesterol may be
quantitated.
[0045] Filters may be used to remove large particles, such as
cells, dust, etc. from the soluble sample. Accordingly, filters are
most preferably included as part of the sample inlet compartment.
Filters may be formed from porous plastic, glass, cross-linked
cotton or cellulose, etc. These materials may be in the shape of
rods or similar shapes depending on the particular use to which
they are being put. Plastics, such as Teflon, may be used as
films.
[0046] Since chaotropic agents are often used to denature
oligonucleotides during sample preparation, it is advantageous to
provide a means of dialysis in the disk to remove the salt prior to
the assay being performed. As shown in FIG. 6, a dialysis unit is
prepared by putting a dialysis membrane 27 on either one or both
halves (top and bottom) of a compartment formed in the disk 10.
Taking into account the small volumes, the buffer that is already
inside the dialysis membrane is usually sufficient and typically no
buffer is needed on the side of the membrane opposite the fluid
layer.
[0047] A column may be prepared, such as shown in FIG. 7, by
filling a compartment 28 with a desired gel, adsorbent or ion
exchanger, e.g. silica gel, Sephadex, etc. (the particular material
is chosen for the particular application for which it is used) and
putting a filter 29 in the other end. Examples of potential uses,
include separating smaller molecules from larger ones and
fractionating hydrophilic and hydrophobic compounds. An ion
exchange column is especially useful for the separation of nucleic
acids from other biomolecules. The columns lend themselves to other
uses that may be convenient or necessary for conducting any
particular assay.
[0048] FIG. 8 illustrates a valve, designated generally as 30, that
may be located in one end of a column or a reaction container,
which has two outlet capillaries 31 and 32. In addition, there are
two electrodes, 33 and 34, which are not charged initially at the
position illustrated and a conductive, metallic foil 35 that is
adapted to close one or the other of the capillaries depending on
its position relative to each capillary. The metal foil is biased
to close one of the capillaries when no current is flowing and
operates to open the previously closed capillary and close the
other capillary when current flows. As an example, the valve is
made from a thin gold foil, which is mechanically pressed against
the other outlet capillary and is electrically connected to the
closest electrode. When the battery is activated the gold foil is
repelled by the closest electrode and attracted by the other
electrode. As a result the gold foil is pressed against the other
outlet. Other conductive metallic foils may be used, but a metal
that is conductive and non-corroding is preferred for most
operations. The battery may be deactivated as explained earlier and
the valve is then switched back to its original position.
[0049] The laser of CD-R or CD-RW-drives has power up to 10 mW that
can heat objects to high temperatures, even to 600.degree. C. The
power is strong enough to puncture holes in several materials,
including plastics. Plastic should contain a dye that absorbs the
laser light. Thermal expansion may be used for reversible valving.
For instance, the bending of bimetallic foils is extremely
sensitive to the temperature.
[0050] Piezoelectric material may be used as a valve.
Piezoelectricity may also be used for measuring extremely small
volumes of liquids, for example nanoliters of the sample can be
divided between different assays.
[0051] Valve-like operations may also be performed chemically by
deposition from solution of a solid chemical compound and/or
dissolution of a deposited, solid compound. The first outlet of
such a valve is closed by deposition of a chemical compound inside
the capillary. The compound may be, for example, silver chloride.
The chloride ions may be in the main fluid stream while in separate
side capillaries are pure water and silver nitrate in water. The
side capillaries are configured such that first the water and then
the silver nitrate are added to the main fluid stream containing
the chloride. The moment the silver ions arrive at the intersection
it is clogged, effectively acting as a closed valve. Alternatively,
a capillary may be initially clogged by the solid form of a soluble
compound, such as sodium chloride. Addition of any aqueous solution
dissolves the sodium chloride clog and the capillary is opened.
[0052] The assay element is preferably utilized in the assay site
of the present invention. Briefly, the assay element (FIG. 13)
includes a cleavable spacer 61 covalently attached at one end 60 to
the disk surface 59 and at the other end 62 to a reporter element
65. The preferred embodiments of the reporter element described
herein include reflective gold spheres or opaque latex spheres.
Also included are two recognition elements 63a, 63b, hereafter
referred to as sidearms which are covalently attached to each
spacer such that the one sidearm is connected to each side of the
spacer's cleavage site 64. The preferred embodiments of the
sidearms described herein include oligonucleotides, antibodies and
oligonucleotide-antibody conjugates. The assay elements may be used
to detect the presence of an analyte and create a signal thereof
through either a positive or negative recognition event (FIG. 14).
A positive recognition event (FIG. 14A, C and E) occurs when an
analyte 66 binds to both sidearms 63a, 63b resulting in the
completion of a connective loop 67 between the two sides the spacer
bisected by the cleavage site 64. A negative recognition event
(FIG. 14B, D and F) occurs when analyte 66 binds to only one or
neither of the sidearms 68a, 68b and consequently no loop is made
connecting the two sides of the spacer. When a positive recognition
event is followed by cleavage of the spacers, an unbroken
connection from disk to reporter element remains intact (FIG. 14E).
On the other hand, cleavage of the spacers in an assay element
following a negative recognition event results in the reporter
elements being disconnected from the disk (FIG. 14F). Thus,
negative recognition results in loose reporter elements that are
easily washed away whereas positive recognition results in the
reporter elements being retained in their discrete assay sectors.
In either case, the results may be observed immediately by CD-ROM
or DVD reader.
[0053] Further embodiments of the invention are described herein
that utilize both reflective or opaque reporter molecules, and
positive and/or negative recognition events to carry out a broad
range of possible assays. For example, in some assays the sidearms
may be connected before a sample is added and binding of the
analyte acts to disconnect the sidearms. In this case, a positive
recognition event results in the disappearance of the reporter
element, while a negative recognition event results in the reporter
element being retained.
[0054] Other possible embodiments of the assay element described
herein do not include cleavable spacers with sidearms. In one such
alternative scheme the surface of the IBCD is coated by metal,
preferably by gold, and the analyte connects the opaque particles,
such as latex beads, or dye loaded liposomes on the metal
surface.
Opaque Spheres as Assay Elements
[0055] Previous assay elements are based on the binding of
reflective particles to the transparent surface of the IBCD. The
situation may be reversed so that opaque particles are bound to a
reflective surface. This approach is especially advantageous when
large cells are assayed and is illustrated generally in FIG.
12.
[0056] A metal film is deposited onto the plastic surface.
Information may be coded into this metal layer as it is done in
conventional CDs. This information may include spatial addresses or
other information related to the assay. The metal layer is further
covered by a plastic layer. This is then aminated, as described
previously, and instead of gold spheres, large latex spheres 58
(10-50 .mu.m diameter), which contain a dye, are attached to the
substrate via spacer molecules as previously described. These latex
spheres are partially coated with recognition molecules as
described above for gold spheres. Cell recognition binds the latex
spheres to the substrate even after the spacers are cleaved, and
the dye in the spheres prevents the reflection of the laser light
from the metal layer. Alternatively, if a proper fluorescent dye
and wavelength of laser light are used, the fluorescent emission of
the spheres may be used to monitor the assay. This requires a
specialized instrument and will be facilitated by blue lasers when
they become available for use in CD-ROM or DVD-readers.
[0057] In the simplest version of the cell detection assay, the
latex spheres are not connected with the IBCD before the assay, but
are added after the cells are bound to the IBCD. The latex sphere
suspension is added, the recognition molecules on the spheres bind
to the proper cells and these cells are immobilized. These latex
spheres may then be observed by reduced reflectance using the
CD-ROM or DVD-reader.
Complementary Binding of Spacers
[0058] One drawback of the covalent binding of spacers is that the
disk is not easily regenerated after the spacers are cleaved. If
the spacers are instead connected to the substrate with
complementary oligonucleotides, the disk can be regenerated after
an assay is completed. The spacers or their residues are removed by
heating or by using chaotropic agents. The duplexes that bind
spacers are denatured and the disk can be cleaned. The disk retains
the oligonucleotides that were binding old spacers. All
oligonucleotides on one assay site are identical. They may be
different in different assay sites, or they may be identical on the
whole IBCD. New spacers having oligonucleotides complementary to
those on the IBCD are added. After incubation the complementary
oligonucleotides of the spacer and the IBCD hybridize. The excess
spacers are washed away. In this case the oligonucleotide sidearms
may be attached to the spacers before the spacers are attached to
the surface. Gold spheres are then added, they are bound by the
thiol groups or disulfide bridges of the spacers, and the disk is
ready to be used again.
[0059] A cuvette is used for UV/Vis spectrophotometric,
fluorescence or chemiluminscence assays. A cuvette in the BCD is
basically a capillary that is located between a light source and a
photodetector. Light can be guided by mirrors and waveguides. The
number of cuvettes in the BCD varies between 0-10,000 and most
advantageously between 0-50 per assay sector. The sample arrives
into the most cuvettes via a sample preparation chamber. These
chambers may contain preloaded reagents or reagents are stored in
separate chambers and are mixed with the sample while it arrives
into the sample preparation chamber. Sample and reagents may be
heated electrically by infrared radiation that is generated by a
photodiode. After the incubation period the sample is transferred
into the cuvette. The transmitted or emitted light is measured by a
photodetector. In this invention the photodetector is most
advantageously inside the CD or DVD drive.
[0060] Light sources for spectrophotometric assays are most
advantageously photodiodes or semiconductor lasers. It is possible
to use the light source of the CD or DVD drive. However, currently
these instruments use only one wavelength that corresponds to
infrared or red light. If an internal light source of the CD or DVD
drive is used, the photodiode or laser in FIG. 15 is replaced by a
mirror. Although several assays can be performed by using infrared
or red light, it is advantageous for most applications to use
additional light sources. For example, an array of photodiodes can
be fabricated so that red, yellow, green and blue light can be
generated. It is possible to design a photodiode for any given
wavelength and accordingly, the number of photodiodes can be up to
300 to cover the whole UV/visible spectral range. Laser generate
more power and are better focused than photodiodes and they are
preferred. Especially microcavity and nanodot lasers are very
small, and they can be fabricated to emit almost any wavelength.
The light sources can be fabricated as a module that can be
attached onto the disk before and removed after the use of the
BCD.
Unit Operations
[0061] Next are described the unit operations: centrifugation,
filtering, transfer of liquids, mixing of liquids, dialysis, column
separations, heating, cooling, electroconvection and
electrophoresis.
[0062] Centrifugal force is the main force used to transfer liquids
in the IBCD. It may also be used for centrifugation, which is
important when calls are separated from plasma. In this case, it is
advantageous to include a filter with the sample intake
container.
[0063] In the transfer of liquids, order and timing are important.
In order to insure the proper sequence of arrival to a certain
reaction site, liquid trains, such as illustrated in FIG. 9, may be
created. In one embodiment, two main capillaries, 36 and 37, are
provided that are in fluid communication with each other via
connecting capillaries 38, 39 and 40. One of the main capillaries
is an air channel to allow for fluid flow and typically is rendered
hydrophobic. The other main channel carries the reagents in liquid
form and typically is hydrophilic. The connecting capillaries and
associated cavities may serve to store the reagents, generally
designated 41, 42 and 43, and maintain their relative locations
with respect to each other. The fluid compartment to which they are
directed and their timing of delivery is controlled by their
respective locations, the size of the capillaries, the density and
viscosity of the fluids and the rotational speed of the disk. The
liquids are separated by small air bubbles to prevent mixing,
unless mixing is desired. To prevent pressure gradients air
capillaries are connected upstream with all liquid capillaries. To
further prevent the liquids from entering the air capillaries,
these are hydrophobic.
[0064] Mixing of two solutions is performed by merging two
capillaries in a Y-shaped formation. This alone provides good
mixing. To guarantee more efficient mixing a capillary may have
small periodic enlargements after the merge. It must be noted that
rotation of the IBCD results in efficient mixing in the
containers.
[0065] In dialysis the liquid is in contact with the membrane
containing the buffer. The molecular weight cutoff of the membrane
may be chosen to be between 300-500,000 Dalton. Because only a very
thin layer of the liquid is in contact with the dialysis'membrane,
the dialysis is very fast. However, the ratio of the liquid to
buffer is only between 1:10 and 1:100 so that the dialysis is not
quantitative. For most purposes it is sufficient.
[0066] Gel, adsorption and ion exchange chromatographies are all
possible. The various molecular species are fractionated by the
chromatographic media and exit the capillary separately as in
conventional chromatography. Using a valve, certain fractions may
be selected and guided into an assay element.
[0067] Heating is best done electrically. Upper and lower
electrodes are separated by about 500 .mu.m. If the solution
contains ions, the system is virtually short circuited and heats
up. Heating may be terminated by removing ions either from the
battery or from the container. Constant temperature can be achieved
by including a thermostat into the circuitry. A bimetallic element
is a very simple thermostat that can close a circuit below a preset
temperature and open it at higher temperature. Another heating
mechanism is provided by the laser of the CD or DVD-drive.
Especially, CD-R-drives have powerful lasers. Either the top or the
bottom of the cavity can have a liquid crystalline film that is
isolated by a transparent layer, if necessary. On the other side of
the cavity is a reflective layer. When the temperature of the
cavity is below the main transition temperature the liquid crystal
will scatter the light and no reflection is observed. Above the
main transition temperature the light is reflected back and the
heating can be discontinued and it is less effective anyway.
Cooling is preferably provided by endothermic dissolution, i.e.,
the absorption of heat by the presence of a dissolving substance.
The cooling solution and the solution to be cooled should be
separated by a thin aluminum, copper, silver or gold film. Cooling
may also be produced by passive air cooling. This method cools only
to ambient temperature, but for most purposes this is enough.
Cooling and heating may also be alternated in a cyclical fashion,
either in one cavity or in a serially alternating sequence of
heating and cooling cavities. This allows PCR amplifications to be
performed inside the IBCD.
[0068] Electroconvection, electrophoresis and isoelectric focusing
may each be utilized in particular applications. In
electroconvection the material is transferred without trying to
separate it into components. In electrophoresis the separation is
the main purpose. The separation is facilitated by the use of a gel
that prevents convection. Because distances are very short, the
available field strength is sufficient for proper electrophoresis.
For the same reason the necessary time for separation is fairly
short and may be on the order of 1-5 minutes, or even less than 1
minute. Useful electroconvection may be performed in few seconds.
Isoelectric focusing is basically electrophoresis in a pH gradient.
A pH gradient may be created by an array of parallel capillaries,
each of which contains a different buffer so that the pH changes
gradually. This is demonstrated in FIG. 16. A large part of the
buffer will remain in the capillaries and this will guarantee the
existence of the pH gradient during the isoelectric focusing. After
the focusing is completed the components can be moved along the
capillaries by centrifugal force or an orthogonal electrophoresis
can be performed. This method allows almost complete fractionation
of human plasma proteins (Anderson, Tracy and Anderson, "The Plasma
Proteins," 2.sup.nd Ed., Vol. 4, Academic Press, Inc., 1984).
[0069] A particularly advantageous configuration of an assay site
is illustrated in FIG. 10. The assay element contains the spacer
molecules and the reflective spheres as described previously but
does so in a linear array that may be conveniently located in one
or more of the capillary channels at the assay site of the disk. As
has been described, analyte binds to the spacer molecules that have
side arms receptive to or complementary to the analyte (as
illustrated in A) and after washing the analyte that has bound is
located at specific locations of the array (as illustrated in B).
The presence of the bound analytes is determined by conventional
address determination as with conventional compact disk readers and
associated software as has been described.
EXAMPLE 1
Assay Sector for Oligonucleotide Analysis (FIG. 2, Assay
Sector)
[0070] A sample that contains DNA is mixed with sodium dodecyl
sulfate to lyse the cells. This solution is transferred into the
container denoted "Sample in" and the disk is rotated. The sample
is filtered and mixed with a mixture of complementary
oligonucleotides. These oligonucleotides are complementary to those
to be analyzed and also have a thiol group at one end.
Hybridization is allowed to proceed in the container denoted
"Sample prep." Optionally, this container may be heated (not shown
in Figure). After appropriate incubation, the disk is rotated.
While the sample is transferred into the container denoted "Sample
sep." it is mixed with a nuclease S solution delivered from a side
capillary. The mixture is allowed to incubate in the "Sample sep."
container which has two gold electrodes and a valve as has been
illustrated in FIG. 8. The lower electrode is coated with spacers
having isothiocyanate end groups. These bind to the thiol
containing oligonucleotides several of which are hybridized with
the sample. All unhybridized parts of DNA are digested and washed
away. The battery then becomes operational. This is adjusted by the
speed by which the acid and copper ions flow into the empty
battery. The container heats up, the bound oligonucleotides are
released and the valve is switched.
[0071] The oligonucleotides are flushed into the assay area. After
suitable incubation the ligase arrives into the assay area and the
two sidearms on the spacer molecule are connected, if the sample
contains the proper oligonucleotide. The labile spacers are cut. If
spacers contain siloxane groups the cutting is done by addition of
fluoride ions.
[0072] The loose gold spheres are washed away by rotating the IBCD
at high speed. Reading may be performed immediately.
EXAMPLE 2
Assay Element for the Detection of Cells and Viruses
[0073] Alternative embodiments of the assay element described
elsewhere herein are useful for the detection of viral and
bacterial particles, cells and other particles that are larger than
the oligonucleotides, antibodies, antigens and the like that have
been described previously. Viruses are typically nearly spherical
particles having a diameter less than 0.5 .mu.m. Bacteria are
commonly either spherical or rod shaped. Their largest dimension is
less than 2 .mu.m excluding flagella and other similar external
fibers. These pathogens are smaller or about the same size as the
gold spheres used to detect them, and their interaction with two
sidearms of the spacer may be limited. For this reason these
sidearms are connected with the surface of the IBCD and the gold
sphere instead of with the spacer as illustrated in FIG. 11. The
gold sphere is attached to a spacer molecule 45 at one end of the
spacer molecule and the other end of the spacer is attached to the
surface of the substrate 46. The spacer molecule is provided with a
typical cleavage site 47, for example a siloxane moiety, as has
been previously described. In contrast to prior described
embodiments where the side-arms are attached to the spacer molecule
between the substrate and the cleavage site and the gold sphere and
the cleavage site, side arms are attached to the gold sphere and to
the surface of the substrate. For illustration purposes, in FIG. 11
oligonucleotides 48 and 49 are attached to the surface of the
substrate and oligonucleotides 50 and 51 are attached to the
surface of the gold sphere. Then complementary oligonucleotides are
conjugated with members of a specific binding pair, designated as
52, 53, 54, and 55 are attached to the oligonucleotides on the
substrate and the gold sphere as illustrated. This gives much more
space for the cells to bind with the antibodies or other
recognition molecules.
[0074] The spacers each still have at least one cleavage site. They
are, in all respects identical to those described previously except
that they have no attached sidearm molecules. When the cell for
example arrives at the assay site, if it contains moieties that
form specific binding pairs with their respective complementary
members, a connective loop is formed between the gold sphere and
the substrate. When the spacer molecule is cleaved, the gold sphere
is retained on the substrate and the presence of the cell may be
detected as previously described. However, if no specific binding
pairs are formed, upon cleavage of the spacer, the gold sphere does
not remain attached to the substrate and is removed.
[0075] Antibodies or other recognition molecules may be attached to
the substrate in a manner similar to that with which the spacers
are attached. All spacers on the IBCD are identical and are
attached at the same time to the amino groups or analogous active
groups on the surface. About half of the amino groups are used for
the attachment of the spacers. The other half is used to couple
recognition molecules to the substrate. If all recognition
molecules on the surface of the IBCD are similar, they may be
attached at the same time as spacers. Alternatively, if the
recognition molecules are specific for each assay site, they may be
dispensed locally by contact printing, ink-jet printing or
microcapillary deposition.
[0076] After the gold spheres are attached to the thiol groups in
the spacers, the other recognition molecules are attached, also via
thiol groups, to the gold spheres. For this purpose these
recognition molecules are first conjugated with a spacer containing
a protected thiol or amino group. The amino group may be
derivatized so that a thiol group is introduced. The various
recognition molecules to be attached to the gold spheres are
dispensed in a manner similar to that with which the other
recognition molecules were attached with the surface of the
IBCD.
[0077] The recognition molecules may be oligonucleotides. These
oligonucleotides may be further hybridized with complementary
oligonucleotide-biomolecule conjugates. This approach allows
attachment of sensitive and reactive biomolecules, for example,
proteins containing several amino or thiol groups.
[0078] The recognition molecules bound to the gold spheres are free
to diffuse around the sphere although they are tightly bound. The
cell that is recognized by both recognition molecules completes a
connective loop that binds the gold sphere to the surface of the
IBCD. After cleaving the spacer, the gold sphere is retained and
detected by the CD-ROM or DVD reader.
[0079] A multiplicity of different recognition molecules in the
same assay site may be used. The advantage of this approach is that
all known mutants of a certain pathogen species may be detected on
one assay site. The various mutants also may be characterized on
different assay sites containing specific recognition
molecules.
[0080] The IBCD is a universal analyzer. It is easy to use and in
its most advanced form it contains all reagents and only the sample
is added. It can be used in clinical laboratories, hospitals,
doctors' offices, and in the home. In home use the information can
be loaded into a doctor's office via the internet. The IBCD can be
designed so that the genetic signature of each patient is measured
every time. About 35 polymorphism points are enough to give every
person a unique "bar-code" . This eliminates possible mistakes due
to mixing of tubes or labels. Assays that can be performed include,
but are not limited to immunoassays, DNA testing, cell counting and
cell shape measurement, detection of cancerous cells in tissue
samples, blood chemistry and electrolyte analysis. Other
applications include mass screening of drug candidates, food and
environmental safety analysis, and monitoring pathogens and toxins
in a battlefield.
EXAMPLE 3
[0081] Turbidimetric Assay of Lipase Activity
[0082] The reagent cavity contains 15 .mu.L of stabilized triolein
(250 .mu.M) emulsion that contains sodium deoxycholate (30 mM) and
CaCl.sub.2 (100 .mu.M) at pH 9.0 in TRIS buffer (25 mM). The sample
preparation chamber contains lyophilized porcine colipase (0.5
.mu.g). Two microliters of serum is taken into the sample
preparation chamber (using apparatus as shown in FIG. 17) together
with stabilized triolein and other reagents. Part of the mixture (5
.mu.L) is further transferred into a cuvette. Because the exit
capillary goes toward the center of the disk, the counterpressure
will prevent further flow. Absorbance at 340 nm is read at one
minute intervals. The .DELTA.A/min is a measure of lipase
activity.
[0083] While this invention has been described with respect to some
specific embodiments, it is understood that modifications thereto
and equivalents and variations thereof will be apparent to one
skilled in the art and are intended to be and are included within
the scope of the claims appended hereto.
* * * * *