U.S. patent application number 10/470487 was filed with the patent office on 2004-11-25 for nucleic hybridization assay method and device using a cleavage technique responsive to the complementary double strand or the single strand of nucleic acids or oligonucleotides.
Invention is credited to Yoo, Jae Chern.
Application Number | 20040234970 10/470487 |
Document ID | / |
Family ID | 19705035 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040234970 |
Kind Code |
A1 |
Yoo, Jae Chern |
November 25, 2004 |
Nucleic hybridization assay method and device using a cleavage
technique responsive to the complementary double strand or the
single strand of nucleic acids or oligonucleotides
Abstract
A cleavable signal element applicable to quantitative and
qualitative assay devices, using a cleavable technique specifically
responsive to a complementary double strand or single strand of
nucleic acids, and a nucleic acid hybridization assay method and
device using the cleavable signal element are provided. Using the
cleavable technique responsive to the complementary double strand
or single strand of nucleic acids, detection sensitivity to a
target nucleic acid can be increased, and diagnosis and detection
reliability can be improved twice through in-situ determinations.
Through simultaneous single nucleotide polymorphism (SNP) detection
and expression profile determination, more accurate diagnosis for
many diseases can be achieved. The assay device can be easily
modified to be suitable for detection with general laser-based
detection systems such as CD-ROM readers. Information read from the
assay device is digitized as software and transmitted to and
received by doctors and patients through a computer network or
wirelessly, which enables construction of remote diagnosis
systems.
Inventors: |
Yoo, Jae Chern;
(Kyungsangbuk-do, KR) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
19705035 |
Appl. No.: |
10/470487 |
Filed: |
February 17, 2004 |
PCT Filed: |
January 28, 2002 |
PCT NO: |
PCT/KR02/00126 |
Current U.S.
Class: |
435/6.12 ;
536/24.3 |
Current CPC
Class: |
C12Q 1/6823 20130101;
Y02A 90/10 20180101; B82Y 30/00 20130101; C12Q 1/6823 20130101;
C12Q 2533/101 20130101; C12Q 2525/131 20130101; C12Q 2521/301
20130101; C12Q 1/6823 20130101; C12Q 2565/625 20130101; C12Q
2525/131 20130101; C12Q 2521/301 20130101 |
Class at
Publication: |
435/006 ;
536/024.3 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 27, 2001 |
KR |
2001/3956 |
Claims
What is claimed is:
1. A cleavable signal element comprising: a restriction probe of a
single strand having a particular sequence cleavable by a
restriction enzyme specific to a double strand of a particular
sequence; and a capture probe of a single strand having a
complementary sequence to a target nucleic acid for diagnosis or
assay to form a double strand through hybridization to the target
nucleic acid, wherein one end of the restriction probe is attached
to a solid support substrate, and the other end of the restriction
probe is ligated to the capture probe, thus forming a
single-stranded, cleavable capture probe.
2. The cleavable signal element of claim 1, wherein when the
capture probe contacts a sample including the target nucleic acid
of the complementary sequence, the capture probe is double-stranded
through hybridization to the target nucleic acid, the restriction
probe is double-stranded through DNA extension using the target
nucleic acid hybridized to the capture probe as a primer with the
addition of a DNA polymerization solution, the double-stranded
restriction probe is cleaved by the restriction enzyme, and the
cleaved cleavable capture probe is removed from the solid support
substrate through washing, thus resulting in a cleaved signal
element; and when the capture probe contacts a sample not including
the target nucleic acid of the complementary sequence, the
single-stranded, cleavable capture probe remains attached to the
solid support substrate even after additions of the DNA
polymerization solution and the restriction enzyme and washing,
thus resulting in an uncleaved signal element.
3. The cleavable signal element of claim 2, wherein the DNA
polymerization solution comprises a solution of four dNTPs and a
DNA polymerase solution.
4. A cleavable signal element comprising a capture probe of a
single strand having a complementary sequence to a target nucleic
acid for diagnosis or assay to form a double strand through
hybridization to the target nucleic acid, wherein one end of the
capture probe is attached to a solid support substrate, and the
capture probe itself forms a single-stranded, cleavable capture
probe which is cleavable by a cleavage enzyme specifically
responsive to a double strand or single strand of nucleic
acids.
5. The cleavable signal element of claim 4, wherein when the
capture probe contacts a sample including the target nucleic acid
of the complementary sequence, the capture probe is double-stranded
through hybridization to the target nucleic acid, the
double-stranded capture probe is cleaved by the cleavage enzyme
specifically responsive to the double strand of nucleic acids, and
the cleaved cleavable capture probe is removed from the solid
support substrate through washing, thus resulting in a cleaved
signal element; and when the capture probe contacts a sample not
including the target nucleic acid of the complementary sequence,
the single-stranded, cleavable capture probe remains attached to
the solid support substrate even after the addition of the cleavage
enzyme and washing, thus resulting in an uncleaved signal
element.
6. The cleavable signal element of claim 5, wherein the cleavage
enzyme is a DNAse.
7. The cleavable signal element of claim 4, wherein when the
capture probe contacts a sample not containing the target nucleic
acid of the complementary sequence, the capture probe remains as a
single strand without hybridization, the single-stranded capture
probe is cleaved by the cleavage enzyme specifically responsive to
the single strand of nucleic acids, and the cleaved cleavable
capture probe is removed from the solid support substrate through
washing, thus resulting in a cleaved signal element; and when the
capture probe contacts a sample including the target nucleic acid
of the complementary sequence, the capture probe is double-stranded
through hybridization to the target nucleic acid, and the
double-stranded, cleavable capture probe remains attached to the
solid support substrate even after the addition of the cleavage
enzyme and washing, thus resulting in an uncleaved signal
element.
8. The cleavable signal element of claim 7, wherein the cleavage
enzyme is a nuclease.
9. The cleavable signal element of claim 8, wherein the nuclease is
derived from mung bean.
10. The cleavable signal element of claim 1 or 4, wherein the solid
support substrate is a plastic substrate, a glass substrate, a
silicon substrate, or a gold substrate.
11. The cleavable signal element of claim 1 or 4, wherein the solid
support substrate has a self-assembled monolayer (SAM) on the
surface.
12. The cleavable signal element of claim 1 or 4, wherein the
capture probe has a length ranging from about 5- to about
30-mers.
13. The cleavable signal element of claim 1 or 4, wherein a label
is attached to one end of the cleavable capture probe to form a
label-attached cleavable capture probe structure or to one end or
side of an uncleaved probe to form a label-attached uncleaved probe
structure, to increase detection sensitivity for an uncleaved
signal element.
14. The cleavable signal element of claim 13, wherein the label
comprises a metal microsphere, a conducting polymer, a fluorescent
dye, a magnetic microsphere, and a streptavidin-labeled
microsphere.
15. The cleavable signal element of claim 14, wherein the metal
microsphere is formed of a metal selected from the group consisting
of gold, silver, nickel, platinum, chromium, and copper.
16. The cleavable signal element of claim 15, wherein a gold
microsphere has a diameter ranging from about 1 nm to about 10
.mu.m.
17. The cleavable signal element of claim 13, wherein the
streptavidin-labeled microsphere is attached to the cleavable
capture probe via biotin.
18. A nucleic acid hybridization assay device comprising: a solid
support substrate; a plurality of cleavable signal elements
according to any of claims 1 through 17 attached to the solid
support substrate; and an internal or external detector which
detects a uncleaved signal element and a cleaved signal element
from the plurality of cleavable signal elements.
19. The nucleic acid hybridization assay device of claim 18,
wherein the detector comprises an optical device, an
electrochemical device, a mass measurement device, or a capacitance
and impedance measurement device.
20. The nucleic acid hybridization assay device of claim 19,
wherein the optical device detects fluorescence of the uncleaved
signal element and cleaved signal element.
21. The nucleic acid hybridization assay device of claim 19,
wherein the detector detects a differential reflective signal or a
is differential conductive signal of the uncleaved signal element
and the cleaved signal element.
22. The nucleic acid hybridization assay device of claim 21,
wherein the detector detects the differential reflective signal by
measuring the reflectance, absorbance, or scattering of light or a
laser beam incident on the uncleaved signal element and the cleaved
signal element.
23. The nucleic acid hybridization assay device of claim 21,
wherein the detector detects the differential conductive signal by
measuring the capacitance and impedance of the uncleaved signal
element and the cleaved signal element.
24. The nucleic acid hybridization assay device of claim 23,
wherein the capacitance and impedance measurement device measures
the frequency response characteristics of the uncleaved signal
element and the cleaved signal element.
25. The nucleic acid hybridization assay device of claim 23,
wherein the capacitance and impedance measurement device comprises
interdigitated array electrodes having at least one digit and
arranged on the solid support substrate.
26. The nucleic acid hybridization assay device of claim 25,
wherein the interdigitated array electrodes have an input port to
check for the frequency response characteristics, and the input
port is connected to an electronic control device which generates a
frequency signal of a constant bandwidth.
27. The nucleic acid hybridization assay device of claim 25,
wherein a plurality of cleavable signal elements are deposited on
the interdigitated array electrodes.
28. The nucleic acid hybridization assay device of claim 25,
wherein the plurality of cleavable signal elements are deposited
only in the space between the interdigitated array electrodes.
29. The nucleic acid hybridization device of claim 25, wherein the
interdigitated array electrodes are substantially formed of
gold.
30. The nucleic acid hybridization device of claim 18, wherein a
label-attached uncleaved probe structure is formed on the solid
support substrate by attaching a label to the uncleaved signal
element to increase the sensitivity of the detector.
31. The nucleic acid hybridization device of claim 18, wherein the
plurality of cleavable signal elements are deposited on the solid
support substrate in a spatially-addressable pattern.
32. The nucleic acid hybridization device of claim 31, wherein the
spatially-addressable pattern enables a single-sample assay for
multiple analytes, a multiple-sample assay for a single analyte, or
a multiple sample assay for multiple analytes.
33. The nucleic acid hybridization device of claim 31, wherein the
solid support substrate is a plastic substrate formed of a material
selected from the group consisting of polypropylenes,
polyacrylates, polyvinyl alcohols, polyethylenes,
polymethylmethacrylates, and polycarbonates.
34. The nucleic acid hybridization device of claim 33, wherein the
solid support substrate is formed of polycarbonates.
35. The nucleic acid hybridization device of claim 31, wherein the
solid support substrate is formed of a circular disk.
36. The nucleic acid hybridization device of claim 31, wherein the
solid support substrate is formed of a rectangular disk.
37. The nucleic acid hybridization device of claim 35, wherein the
circular disk has a diameter of approximately 120 mm and a
thickness of approximately 1.2 mm.
38. The nucleic acid hybridization device of claim 35, wherein the
circular disk comprises: a central void to engage a rotational
drive means; a sample injection port through which a sample is
injected; and an annular and/or a spiral track in which the
plurality of cleavable signal elements are deposited in the
spatially-addressable pattern.
39. The nucleic acid hybridization device of claim 38, wherein an
address pattern that provides coded address information is formed
on the circular disk.
40. The nucleic acid hybridization device of claim 35, wherein the
circular disk comprises: a central void to engage a rotational
drive means; a sample injection port through which a sample is
injected; and a radial assay sector in which the plurality of
cleavable signal elements are deposited in the
spatially-addressable pattern.
41. The nucleic acid hybridization device of claim 40, wherein the
circular disk comprises a plurality of assay sectors.
42. The nucleic acid hybridization device of claim 41, wherein the
plurality of assay sectors are connected to respective separate
sample injection ports.
43. The nucleic acid hybridization device of claim 41, wherein the
plurality of assay sectors are connected to a common sample
injection port.
44. The nucleic acid hybridization device of claim 41, wherein the
plurality of cleavable signal elements are deposited in each of the
plurality of assay sectors in an appropriate pattern for a
single-analyte assay or a multiple-analyte assay.
45. The nucleic acid hybridization device of claim 35, comprising a
plurality of circular disks.
46. The nucleic acid hybridization device of claim 35, wherein the
circular disk includes in a central track a database associated
with bioinformatics required for diagnosis and assay
interpretation, and telephone numbers, web link information and
software required for remote diagnosis.
47. The nucleic acid hybridization device of claim 35, wherein a
detector is mounted on the circular disk.
48. The nucleic acid hybridization device of claim 47, wherein the
detector comprises a non-contact interface through which
information read from the cleaved signal element and the uncleaved
signal element is transmitted to an external central controller or
storage device.
49. The nucleic acid hybridization assay device of claim 48,
wherein the non-contact interface comprises an infrared interface
and an optical interface.
50. The nucleic acid hybridization assay device of claim 49,
wherein the infrared interface is an infrared sensor, and the
optical interface is a photosensor.
51. The nucleic acid hybridization assay device of claim 35,
wherein the circular disk simultaneously comprises at least one SNP
(single nucleotide polymorphism) assay sector for SNP detection and
at least one expression assay sector for expression profile
analysis.
52. The nucleic acid hybridization assay device of claim 51,
wherein the SNP assay sector and the expression assay sector are
arranged separate in an angular direction or in a radial
direction.
53. A bio-driver apparatus comprising: a rotary disk receiver onto
which the nucleic acid assay device according to any of claims 18
through 52 is to be loaded; a motor driver which rotates the disk;
a rotary connector which connects the motor driver to a central
void portion of the disk such that the disk is rotatable; and an
optical device to write data in or to read data from the disk.
54. The bio-driver apparatus of claim 53, further comprising a
central controller which transmits information read from the disk
by the optical device to an external storage unit, transmits
information to be written to the optical device, and generates and
outputs a variety of control signals for the motor driver and the
other elements.
55. The bio-driver apparatus of claim 53, wherein the rotary
connector comprises an upper rotor and/or a lower rotor, the upper
and lower rotors being pushed close to the top and bottom surfaces,
respectively, of the central void portion when the disk begins to
rotate.
56. The bio-driver apparatus of claim 53, wherein the optical
device detects a differential reflective signal by measuring the
reflectance, absorbance, or scattering of incident light or an
incident laser beam.
57. The bio-driver apparatus of claim 53, wherein the optical
device detects fluorescence.
58. A bio-driver apparatus comprising: a rotary disk receiver onto
which the nucleic acid assay device according to any of claims 18
through 52 is to be loaded; a motor driver which rotates the disk;
a rotary connector which rotatably connects the motor driver to a
central void portion of the disk; an external power connector which
powers and/or supplies a control signal to a detector mounted on
the disk; and a non-contact interface through which information
read by the detector is transmitted.
59. The bio-driver apparatus of claim 58, further comprising a
central controller which transmits information read from the disk
by the detector to an external storage unit and generates and
outputs a variety of control signals for the motor driver and the
other elements.
60. The bio-driver apparatus of claim 58, further comprising an
optical device to write data in or to read data from the disk.
61. The bio-driver apparatus of claim 58, wherein the detector
detects a differential conductive signal by measuring capacitance
and impedance.
62. The bio-driver apparatus of claim 58, wherein the rotary
connector comprises an upper rotor and/or a lower rotor, the upper
and lower rotors being pushed close to the top and bottom surfaces,
respectively, of the central void portion when the disk begins to
rotate.
63. The bio-driver apparatus of claim 58, wherein the power
connector comprises a brush that frictionally contacts the upper
and/or lower rotors in connection with an external power supply
unit, and each of the upper and lower rotors comprises an annular
electrode plate frictionally contacting the brush.
64. The bio-driver apparatus of claim 63, wherein the annular
electrode plate comprises at least one conductive arm connected
thereto, and the central void portion of the disk comprises a hole
to engage the at least one conductive arm and a circuit pattern
connected to the hole to power the detector mounted on the disk
and/or supply the control signal to the detector.
65. The bio-driver apparatus of claim 64, wherein the at least one
conductive arm has a spring at its one end that is connected to the
annular electrode plate.
66. The bio-driver apparatus of claim 58, wherein the power
connector comprises an electromagnet attached to the rotary disk
receiver in connection with the external power supply unit, and the
electromagnet induces an AC voltage to a wound coil on the disk so
that the detector is powered in a non-contact manner.
67. The bio-driver apparatus of claim 66, wherein the disk further
comprises a rectifier for rectifying the AC voltage induced to the
wound coil.
68. A remote diagnostic system comprising: the nucleic acid
hybridization assay device according to any of claims 18 through
52; an existing communication network; and a computer in which
software capable of controlling access to the existing
communication network and digitizing information read from the
nucleic acid hybridization assay device is installed, wherein the
digitized information from the nucleic acid assay hybridization
assay device is transmitted to a doctor or a hospital, and a
patient is provided with a prescription, through the existing
communication network.
69. The remote diagnostic system of claim 68, wherein the computer
comprises assay interpretive algorithms, bioinformatics
information, and self-diagnostics related software.
70. The remote diagnostic system of claim 68, wherein the computer
comprises software capable of uploading diagnostic information to
remote locations and device drivers.
71. The remote diagnostic system of claim 70, wherein the software
comprises educational information for patients on clinical assays,
a variety of wet sites and links enabling a patient to directly
communicate with a doctor or hospital based on his/her diagnosis
result.
72. The remote diagnostic system of claim 68, wherein the computer
comprises a camera and a microphone for viewing a patient's face
and listening to his/her voice.
73. The remote diagnostic system of claim 68, wherein the
diagnostic data based on the digitized information are displayed on
a computer monitor, the computer automatically or manually
transmits the diagnostic data to a specialist through the existing
communication network, and the patient waits for a prescription
from the specialist.
74. A nucleic acid hybridization assay method comprising:
hybridizing a capture probe to a target nucleic acid present in a
liquid sample by contacting the nucleic acid hybridization assay
device according to any of claims 18 through 52 with the liquid
sample; contacting the cleavable capture probe with a restriction
enzyme or cleavage enzyme which is specifically responsive to a
cleavable signal element depending on whether the capture probe and
the target nucleic acid are hybridized or not; washing the nucleic
acid hybridization assay device to remove the cleavable signal
element cleaved by the restriction enzyme or cleavage enzyme; and
detecting whether the uncleaved signal element or the cleaved
signal element exists on the solid support substrate.
75. The nucleic acid hybridization assay method of claim 74,
further comprising contacting the cleavage capture probe with a DNA
polymerization solution before contact with the restriction
enzyme.
76. The nucleic acid hybridization assay method of claim 75,
further comprising contacting the cleavage capture probe with a
3'-5' exonuclease solution before contact with the DNA
polymerization solution.
77. The nucleic acid hybridization assay method of claim 74,
further comprising attaching a label to the cleavable signal
element before contacting the capture probe with the liquid sample,
or to an uncleaved signal element after contacting the cleavable
capture probe with the restriction enzyme or cleavage enzyme.
78. The nucleic acid hybridization assay method of claim 74,
further comprising at least one wash step.
79. The nucleic acid hybridization assay method of claim 74,
wherein washing is performing by rotating the nucleic acid
hybridization assay device with or without addition of a detergent
solution, or by applying an external electric field.
80. A nucleic acid hybridization assay method comprising: (a)
injecting a sample into a sample injection port disposed near the
center of a disk in a nucleic acid hybridization assay device; (b)
rotating the disk and stopping the rotation of the disk when the
sample reaches an outer edge of the disk; (c) incubating the disk
in a stationary state at room temperature for hybridization; (d)
adding a buffer solution as a washing solution while rotating the
disk at a high speed, to wash the disk; (e) adding a DNA
polymerization solution containing a mixed solution of four dNTPs
and a DNA polymerase and incubating the disk in a stationary state
for DNA extension; (f) adding a solution of a restriction enzyme
specifically responsive to a double strand of a particular sequence
and incubating the disk in a stationary state, to cleave the double
strand; (g) washing the disk by rotating the disk at a high speed
with the addition of a buffer solution or by applying an external
electric or magnetic field; and (h) drying the disk and reading
information from the disk using a detector which is programmed to
detect a predetermined assay site on which a cleavable signal
element is deposited and comprises an optical device, an
electrochemical device, or a capacitance and impedance measurement
device.
81. The nucleic acid hybridization assay method of claim 80,
further comprising adding a 3'-5' exonuclease solution before step
(e) of DNA extension.
82. A nucleic acid hybridization assay method comprising: (a)
injecting a sample into a sample injection port disposed near the
center of a disk in a nucleic acid hybridization assay device; (b)
rotating the disk and stopping the rotation of the disk when the
sample reaches an outer edge of the disk; (c) incubating the disk
in a stationary state at room temperature for hybridization; (d)
adding a buffer solution as a washing solution while rotating the
disk at a high speed, to wash the disk; (e) adding a solution of a
cleavage enzyme specifically responsive to a double strand or
single strand of nucleic acids and incubating the disk in a
stationary state, to cleave the double strand or single strand; (f)
washing the disk by rotating the disk at a high speed with the
addition of a buffer solution or by applying an external electric
or magnetic field; and (g) drying the disk and reading information
from the disk using a detector which is programmed to detect a
predetermined assay site on which a cleavable signal element is
deposited and comprises an optical device, an electrochemical
device, or a capacitance and impedance measurement device.
83. The nucleic acid hybridization assay method of claim 80 or 82,
further comprising attaching a label to the cleavable signal
element before sample injection, or to an uncleaved signal element
after strand cleavage, to increase detection sensitivity.
Description
TECHNICAL FIELD
[0001] The present invention relates to cleavable signal elements
using a cleavage technique specifically responsive to a
complementary double strand or single strand of nucleic acids,
which are applicable to quantitative and qualitative assay devices,
and a nucleic acid hybridization assay method and device using the
cleavable signal element.
BACKGROUND ART
[0002] To date, most clinical diagnostic assays for the detection
of small quantities of analytes in fluids have been conducted as
individual tests; that is, as single tests conducted upon single
samples to detect individual analytes. More recently,
multiple-sample preparation and automated reagent addition devices
and multiple-sample assay devices, either in parallel or in serial
procession, have been designed to improve efficiency and economy.
Such automated reagent preparation devices and automated multiplex
analyzers are often integrated into a single device.
[0003] Large-scale clinical laboratory analyzers of this type can
accurately perform hundreds of assays in one hour automatically or
semi-automatically. However, these analyzers are expensive and only
centralized laboratories and hospitals can afford them. Such
centralization necessitates sample transport to the laboratory or
hospital, and often precludes urgent or emergent analysis of
time-critical samples.
[0004] Thus, to address these problems, there is an increasing need
for clinical analyzers which are cheap and easy-to-handle for
everyone, such as clinical analyzers suitable for use at the
patient bedside or in the patient's home without dedicated
detectors. Blood glucose and pregnancy testers are well known
examples.
[0005] Although useful tests of this sort have been offered for
many years, a major breakthrough was the introduction of solid
phase immunoassays and other strip tests since 1980. Most notable
are Advance.RTM. test (Johnson & Johnson), TAM.TM. hCG assay
(Monoclonal Antibodies, Inc.), Clear Blue Easy.TM. (Unipath Ltd.),
and ICON (Hybritech). Commercially Available are Quantab.TM.
(Environmental Test Systems), AccuLevel.RTM. (Syva), AccuMeter.RTM.
(ChemTrak), Clinimeter.TM. (Crystal Diagnostics), and Q.E.D..TM.
(Enxymatics). One of the newest is a thermometer-type assay device
(Ertinghausen G., U.S. Pat. No. 5,087,556) that is not yet
commercially available. These systems can be used to assay blood
levels of therapeutic drugs and general chemical analytes such as
cholesterols.
[0006] One disadvantage, however, of each of these formats is that
only one, or a very limited number, of assays can conveniently be
performed simultaneously.
[0007] To fill the gap between massive analyzers and strip testers,
some small instruments have been developed. The most notable is
Eclipse ICA.TM. (Biotope, Inc.). This device is an automated
centrifugal immunoassay and chemistry system. Patient samples are
pipetted into cassettes that are placed into a rotary device.
Sixteen tests can be run in approximately 17 minutes. The results
are measured by UV/Nisible spectrometry or by fluorometry.
[0008] Despite these developments, there still exists a need for a
simple device that can easily be used for multiple quantitative
assays without a specialized detector.
[0009] <Spatially Addressable Probe Assays>
[0010] Recently, spatially addressable arrays of different
biomaterials have been fabricated on solid supports. These probe
arrays permit the simultaneous analysis of a large number of
analytes. Examples are arrays of oligonucleotides or peptides that
are fixed to a solid support and that capture complementary
analytes. One such system is described by Fodor et al., Nature,
Vol. 364, Aug. 5, 1993. Short oligonucleotide probes attached to a
solid support bind complementary sequences contained in longer
strands of DNA in liquid sample; the sequence of the sample nucleic
acids is then calculated by computer based on the hybridization
data so collected.
[0011] There remains a need for an economical system to fabricate
spatially addressable probe arrays in a simplified format that
provides both for ready detection and the ability to assay for
large numbers of test substances (i.e. analytes) in a fluid test
sample in a single step, or a minimum number of steps, or assay for
a single test substance or analyte in a large number of fluid test
samples.
[0012] <Spatially Addressable Laser-Based Detection
Systems>
[0013] Several devices permit spatially addressable detection of
digital information. In particular, several formats have been
developed based on differential reflectance and transmittance of
recording information.
[0014] In conventional audio or CD-ROM compact disks, digital
information or digitally encoded analog information is encoded on a
circular plastic disk by means of indentations in the disk.
Typically, such indentations are on the order of one-eighth to
one-quarter of the wavelength of the incident beam of a laser that
is used to read the information from the disk. The indentations on
the disk cause destructive interference within the reflected beam,
which corresponds to a bit having a "zero" value. The flat areas of
the disk reflect the laser beam back to a detector and the detector
gives a value of "one" to the corresponding bit.
[0015] In another convention, a change of intensity of a reflected
light beam gets a value of one while a constant intensity
corresponds to zero.
[0016] Since the indentations have been formed in the disk in a
regular pattern from a master copy containing a predetermined
distribution of bits of "zero" and bits of "one", the resultant
signal received by the detector is able to be processed to
reproduce the same information that was encoded in the master
disk.
[0017] The standard compact disk is formed from a 12-cm
polycarbonate substrate, a reflective metal layer, and a protective
lacquer coating. The format of current CDs and CD-ROMs is described
by the ISO 9660 industry standard.
[0018] The polycarbonate substrate is optical-quality clear
polycarbonate. In a standard pressed, or mass-replicated CD, the
data layer is part of the polycarbonate substrate, and the data are
impressed in the form of a series of pits by a stamper during the
injection molding process. The stamping master is typically
glass.
[0019] Pits are continuously spirally impressed in the CD
substrate. The reflective metal layer applied thereupon, typically
aluminum, assumes the shape of the solid polycarbonate substrate,
and differentially reflects the laser beam to the reading assembly
depending on the presence or absence of "pits." An acrylic lacquer
is spin-coated as a thin layer on top of the reflective metal layer
to protect it from abrasion and corrosion.
[0020] Although similar in concept and compatible with CD readers,
the information is recorded differently in a recordable compact
disk (CD-R). In CD-R, the data layer is separate from the
polycarbonate substrate. The polycarbonate substrate instead has
impressed upon it a continuous spiral groove as an address for
guiding the incident laser. An organic dye is used to form the data
layer. Cyanine or a metal-stabilized cyanine compound is generally
used to form the data layer. An alternative material is
phthalocyanine. One such metallophthalocyanine compound is
described in U.S. Pat. No. 5,580,696.
[0021] In CD-R, the organic dye layer is sandwiched between the
polycarbonate substrate and the metallized reflective layer,
usually 24 carat gold, but alternatively silver, of the media.
Information is recorded by a recording laser of appropriate
preselected wavelength that selectively melts "pits" into the dye
layer, causing the pits to become non-translucent. The reading
sensor reads the presence or absence of pits from refractivity
rather than differential reflectivity by physical pits in the
standard CD. As in a standard CD, a lacquer coating protects the
information layer.
[0022] Other physical formats for recording and storing information
have been developed based on the same concept as the compact disk:
creation of differential reflectance or transmittance on a
substrate to be read by laser. One such format is termed digital
versatile disk (DVD). A DVD looks like standard CD: it is a 120-mm
(4.75 inch) disk with a hole in the center for engaging a rotatable
drive mechanism. Like a CD, data is recorded on the disk in a
spiral trail of tiny pits, and the disks are read using a laser
beam. In contrast to a CD, which can store approximately 680
million bytes of digital data under the ISO 9660 standard, the DVD
can store from 4.7 billion to 17 billion bytes of digital data. The
DVD's larger capacity is achieved by making the pits smaller and
the spiral tighter, that is, by reducing the pitch of the spiral,
and by recording the data in as many as four layers, two on each
side of the disk. The smaller pit size and tighter pitch require
that the reading laser wavelength be smaller. While the smaller
wavelength is compatible with standard pressed CDs, it is
incompatible with current versions of the dye-based CD-R.
[0023] Thus, a single sided/single layer DVD can contain 4.7 GB of
digital information. A single sided/dual layer DVD can contain 8.5
GB of information. A Dual sided/single layer disk can contain 9.4
GB of information, while a dual sided/dual layer DVD contains up to
17 GB of information.
[0024] Depending on the capacity, the disk may have one to four
information layers. In the 8.5 GB and 17 GB options, a
semi-reflector is used in order to access two information layers
from one side of the disk. For the 8.5 GB DVD and 17 GB options,
the second information layer per side may be molded into the second
substrate or may be added as a photopolymer layer. In either case,
a semi-reflector layer is required to allow both information layers
to be read from one side of the disk. For the 17 GB DVD, it is
necessary to produce two dual-layer substrates, and bond them
together.
[0025] The DVD laser reader is designed to adjust its focus to
either layer depth so that both of them can be quickly and
automatically accessed.
[0026] All of the above-described formats require that the disk be
spun. The nominal constant linear velocity of a DVD system is 3.5
to 4.0 meters per second (slightly faster for the larger pits in
the dual layer versions), which is over 3 times the speed of a
standard CD, which is 1.2 mps.
[0027] <Detection Method of DNA Chips>
[0028] DNA chips refer to chips having highly immobilized DNA
probes of interest on solid substrates and are used for the
analysis of a gene expression profile, genetic defects, etc., in
samples. To investigate whether the sample contains a target
nucleic acid that binds to the probe immobilized on the substrate,
a detection system therefor is required.
[0029] Most currently-available genetic analysis DNA chips employ a
method of fluorescently labeling a sample DNA, reacting it with the
proves immobilized on the chip, and detecting the unreacted
fluorescent material remaining on the chip surface using a confocal
microscope or charge coupled device (CCD) imager (U.S. Pat. No.
6,141,096). However, such optical detection method is
disadvantageous in size reduction and cannot display digitized
outputs. For these reasons, research on the development of a new
detection method for electrical signal outputs is actively being
conducted.
[0030] Many research institutes, including Clinical Micro Sensors,
are researching the electrochemical detection of DNA hybridization
using a metal compound that is liable to oxidation/reduction (U.S.
Pat. Nos., 6,096,273, 6,090,933). Separate compounds containing
easily oxidizable/reducible metals form a complex upon DNA
hybridization, and the complex is electrochemically detected (Anal.
Chem., Vol., 70, pp. 4670-4677, 1998; J. Am. Chem. Soc., Vol. 119,
pp. 9861-9870, 1997; Analytica Chemica Acta, Vol, Vo. 2886, pp.
216-224, 1994; Bioconjugate Chem., Vol. 8, pp. 906-913, 1997).
However, this electrochemical method still needs separate
labeling.
[0031] Approaches to assay methods not using the fluorescent label
or any other labels have been actively made. As a result, a method
of measuring a difference in mass before and after binding using a
quartz crystal microbalance (Anal. Chem., Vol. 70, pp, 1288-1296,
1998), an assay method using matrix assisted laser description
ionization (MALDI) mass spectrometry (Anal. Chem., Vol. 69, pp.
4540-4546, 1997, U.S. Pat. No. 6,043,031) were developed.
[0032] Even a single-base difference can be analyzed using a
microfabricated cantilever, which is a mechanical sensor type for
measuring molecular binding force before and after binding of DNA
probe and target (Science, Vol., 288, pp. 316-318, 2000; Proc.
Natl., Acad. Sci. USA, 98, 2560, 2001). However, this method needs
additional equipment, such as a laser, for accurate measurement of
cantilever beam deflection.
[0033] The present invention relates to the field of diagnosis and
detection of small quantities of materials in fluids. It is an
object of the present invention to provide cleavable signal
elements using a cleavage technique specifically responsive to a
double strand or single strand of nucleic acids or oligonucleotides
having a complementary sequence, which are applicable to
quantitative and qualitative assay devices, and a nucleic acid
hybridization assay method and device using the cleavable signal
element.
[0034] It is another object of the present invention to provide an
accurate method and device of diagnosing a variety of diseases from
both single nucleotide polymorphism (SNP) detection and gene
expression profile obtained using the nucleic acid hybridization
assay device.
[0035] An analytical apparatus based on the nucleic acid
hybridization assay method and device using the cleavage technique
can be modified to use the standard laser-based detection system,
such as CD-ROM reader or DVD reader, and can be coupled to a
detector including an optical device, an electrochemical device, or
a capacitance and impedance measurement device. The analytical
apparatus and method according to the present invention are useful
in both detecting a number of individual analytes in a test sample
and detecting a single analyte in a large number of separate
samples.
[0036] It is still another object of the present invention is to
provide an remote diagnostic system providing convenience to both
patients and doctors by transmitting and receiving the information
read from the analytical apparatus and digitalized as computer
software, through an existing communication network, such as the
Internet.
DISCLOSURE OF THE INVENTION
[0037] It is an object of the present invention to provide a
nucleic acid hybridization assay method and device applicable to
quantitative and qualitative assay devices, which use a cleavage
technique specifically responsive to a complementary double strand
of nucleic acids of a particular sequence.
[0038] In the present invention, cleavage is performed using a
restriction enzyme specifically responsive to only a double strand
of a particular sequence. Hereinafter, the particular sequence is
referred to as a "restriction sequence", and each single strand of
the restriction sequence is referred to as a "restriction probe".
The restriction probe is additionally ligated to one end of a
capture probe that has a complementary sequence to a target DNA and
thus is hybridized to the target DNA, and then immobilized on a
substrate. The restriction probe and capture probe can be designed
collectively as a single probe. In forming a double strand from the
capture probe and restriction probe, the restriction probe is
cleaved by the restriction enzyme, and thus the restriction and
capture probes are collectively referred to as "cleavable capture
probes" or "cleavable signal elements".
[0039] Although use of the restriction enzyme that specifically
responds to a restriction sequence of the designed restriction
probe is well known in the field, it is preferable to design the
restriction probe such that the sequence of the restriction probe
ligated to one end of the capture probe does not overlap with the
sequence of the capture probe.
[0040] The sequence of the capture probe is determined to be
specific to an analyte of interest for diagnosis or analysis
purpose. As the capture probe contacts a sample containing a target
nucleic acid having a complementary sequence to the capture probe,
the capture probe forms a double strand with the target nucleic
acid through hybridization. At this time, the restriction probe
attached to one end of the capture probe still remains as a single
strand and does not form the double stand.
[0041] To form a double strand of the non-hybridized restriction
probe, a solution mixture containing four dNTPs and a DNA
polymerase required for DNA extension are added. Formation of a
double-strand of the restriction probe is achieved through DNA
extension using the target nucleic acid hybridized to the capture
probe as a primer.
[0042] Once the cleavable capture probe forms a complete double
strand through hybriziation of the capture probe to the target
nucleic acid and formation of the restriction probe double strand,
the double-stranded restriction probe is cleavable by the
restriction enzyme. After cleavage of the double strand by the
restriction enzyme, the cleaved signal element is removed from the
substrate through washing.
[0043] In contrast, when the capture probe contacts a sample that
does not have a complementary nucleic acid sequence to the capture
probe, the cleavable capture probe cannot form the double strand,
so it remains attached to the substrate after the addition of the
restriction enzyme and washing. The cleavable capture probe
remaining on the substrate is referred to as an "uncleaved probe".
To improve the sensitivity of a detector, a "label-attached
uncleaved probe" structure can be optionally formed on the
substrate by contacting the uncleaved probe with fluorescent labels
or other labeling elements such as metal microspheres.
[0044] After sample-to-probe contact, reaction using the DNA
polymerization solution and the restriction enzyme, and washing,
detection of the presence or absence of the uncleaved probe or
"label-attached uncleaved probe" structure on the substrate by a
detector including an optical device, an electrochemical device, a
mass measurement device, or a capacitance and impedance measurement
device, indicates the presence or absence of a particular
analyte.
[0045] As described above, "uncleaved probes (cleavable capture
probes not cleaved and adhering to the substrate)" or
"label-attached uncleaved probe" structures act as signal elements
for the presence of particular analytes. The presence of the
cleavable signal element (uncleaved signal element) on the
substrate means that sample does not contain a particular analyte,
and the absence of the cleavable signal element (cleaved signal
element) from the substrate means that sample contains a particular
analyte.
[0046] The present invention provides a nucleic acid hybridization
assay method using the cleavable capture probe as a cleavable
signal element.
[0047] In general, the assay method using the assay device and a
cleavage technique specifically responsive to a particular sequence
involves: contacting the assay device with a liquid sample;
reacting the cleavable signal probe with a DNA polymerization
solution to form a double strand; contacting the cleavable capture
probe with a restriction enzyme to cleave the double strand;
removing the cleaved double strand through washing; and detecting
the presence or absence of the cleavable signal element on a solid
support substrate using the detector including an optical device,
an electrochemical device, a mass measurement device, or a
capacitance and impedance measurement device, as described
above.
[0048] After washing, a label may be optionally attached to the
uncleaved probe remaining on the solid support substrate to form a
"label-attached uncleaved probe" structure. This label attachment
improves the sensitivity of the detector including an optical
device, an electrochemical device, a mass measurement device, or a
capacitance and impedance measurement device.
[0049] According to the present invention, the presence of a
particular analyte can be double checked through two steps, thereby
increasing assay reliability. As a first step, after contact with a
sample, whether the capture probe is double-stranded or not with
the target nucleic acid is determined. As a second step, after the
reaction with the DNA polymerization solution, the restriction
enzyme treatment, and the washing, whether the cleavable capture
probe remains on the substrate or not is determined in situ.
Therefore, the particular analyte can be detected with higher
reliability.
[0050] In another aspect, the present invention provides an assay
device comprising a solid support substrate on which a plurality of
cleavable capture probes (cleavable signal elements) are deposited
in a spatially-addressable pattern. Suitable materials for the
solid support substrate of the assay device include gold, glass,
and silicon, with polycarbonate being preferred. Alternative
examples of the solid support substrate include disks of any shape
compatible for detection using existing laser reflectance-based
detectors, including audio compact disk (CD) readers, CD-ROM
(compact disk read-only memory) readers, recordable CD readers, DVD
(digital versatile disk) readers, and the like.
[0051] In a preferred embodiment of the present invention, an
"uncleaved probe" or a "label-attached uncleaved probe" structure
and the cleaved double strand differentially reflect or scatter
incident light, in particular, incident laser light, which can be
adapted for detection using existing laser-reflectance based
detectors, including CD readers, CD-R readers, CD-ROM readers, or
DVD readers. Furthermore, according to the present invention, a
bioinformatics-related database for diagnosis and assay
interpretation, as well as hospital telephone numbers and web link
information for remote diagnosis, may be loaded to a spatial
address. In addition, the present invention enables personal
medical history management by writing the diagnosis result to a
CD-R or a hard disk.
[0052] The deposition of cleavable signal elements (cleavable
capture probes) on the assay device in a spatially-addressable
pattern permits a single-sample assay for multiple analytes, a
multi-sample assay for a single analyte, and a multi-sample assay
for multiple analytes.
[0053] Another aspect of the present invention provides a nucleic
acid assay device including cleavable signal elements responsive to
a variety of nucleic acid sequences. In view of this, the present
invention provides an assay method and device for assaying a
nucleic acid sequence present in a sample from the spatial address
of a signal generated upon contact with the nucleic acid containing
sample.
[0054] The present invention simultaneously provides single
nucleotide polymorphism (SNP) detection and expression profile
determination, thereby enabling more accurate diagnosis of many
kinds of diseases.
[0055] Another aspect of the present invention provides a remote
diagnostic system providing convenience to both patients and
doctors, which detects a plurality of cleavable signal elements
attached to a solid support substrate using the detector including
an optical device, an electrochemical device, a mass measurement
device, or a capacitance and impedance measurement device,
digitizes the detected information as computer software, and
transmits to both doctor and patient through an existing
communication network, such as the Internet.
[0056] The assay device and method according to the present
invention uses cleavable capture probes as cleavable signal
elements for detection of analytes in fluid test samples. Binding
of the analyte preselected for detection enables cleavage of the
cleavable capture probe at its restriction probe portion and
removal of the cleavable capture probe from the substrate
surface.
[0057] When the sample does not contain the target nucleic acid,
the cleavable capture probe remains attached to the substrate
surface. Therefore, the presence or absence of the cleavable signal
element (cleavable capture probe) can be used as digital (binary)
information indicating whether a particular analyte exists or not
in the sample. A differential signal between the uncleaved signal
element and the cleaved signal element indicates whether the
particular analyte exists or not in the sample.
[0058] In a preferred embodiment, the signal element according to
the present invention reflects or scatters incident light and is
light addressable. Binding of the analyte preselected for detection
enables cleavage and removal of the signal element. Reflection or
scattering of incident light, in particular, incident laser light,
from the uncleaved signal element indicates the absence of a
particular analyte in the sample, and reflection or scattering from
the cleaved signal element indicates the presence of the analyte in
the sample.
[0059] The cleavable signal elements of the present invention are
particularly adapted for detection using existing laser
reflectance-based detectors, including CD readers, CD-ROM readers,
laser disk readers, DVD readers, and the like. The cleavable signal
elements of the present invention thus permits the ready adaptation
of existing assay chemistries and existing assay schemes to
detection using-existing laser reflectance-base detectors. This
leads to substantial cost savings per assay over standard assays
using dedicated detectors.
[0060] Applicable assay examples are immunoassays, cell counting,
genetic detection assays based upon hybridization, genetic
detection assays based upon nucleic acid sequencing, nucleic acid
sequencing itself, and the like. The present invention thus allows
distribution of assay devices to research laboratories, physician's
offices, and individual homes that must currently be performed at
centralized locations.
[0061] The spatially addressable capabilities of the laser
reflectance-based detectors currently used to detect and interpret
information encoded on CDs and the like confer particular
advantages on assays adapted to use the cleavable reflective signal
elements of the present invention.
[0062] Thus, patterned deposition of multiple signal elements on a
single support or substrate, coupled with use of a detector capable
of addressing the spatial location of these individual signal
elements, permits the concurrent assay of a single sample for
multiple different analytes, multiple samples for a single analyte,
or multiple samples for multiple analytes. The present invention is
thus further directed to assay devices, commonly referred to herein
as disk, bio-compact disks, bio-DCs, or bio-DVDs, comprising
spatially addressable combinations (diverse geometries) of
cleavable signal elements of different analyte specificity. Among
such useful combinations are those that increase the predictive
value or specificity of each of the individual assays, combinations
that inculpate or exculpate particular diagnoses in a differential
diagnosis, combinations that provide broad general screening tools,
and the like.
[0063] Patterned deposition of multiple signal elements with
identical specificity further permits the detection, using a single
assay device, of large concentration ranges of a single analyte. It
is thus another aspect of the present invention to provide assay
devices comprising spatially addressable cleavable signal elements
of identical specificity, the physical location of which is capable
of conveying concentration information.
[0064] The spatially addressable capabilities of the laser
reflectance-based digital detectors further permits the combination
of interpretive software and the assay elements themselves on a
single assay device. Another aspect of the present invention,
therefore, is an assay device upon which software is encoded in an
area spatially distinct from the patterned deposition of cleavable
signal elements. The software may include information important for
correct tracking by the incident laser, assay interpretive
algorithms, standard control values, bioinformatics information,
self-diagnostics, and the like. The software may include device
drivers and software capable of uploading the diagnostic
information to remote locations. The software may include
educational information for patients on clinical assays, and may be
adapted for chosen audiences. The software may include a variety of
web sites and links, for example, a web site enabling a patient to
communicate with a doctor or hospital based on his/her diagnosis
result.
[0065] To increase detection sensitivity to reflection variations
in the nucleic acid hybridization assay according to the present
invention, one end of the cleavable capture probe (cleavable signal
element) is covalently bound to the substrate, and the other end of
the cleavable capture probe is labeled with, for example, a
conducting polymer (e.g., polyaniline), a fluorescent label, or a
metal microsphere, to form a label-attached signal element
("label-attached cleavable capture probe"), thereby increasing a
reflectivity variation relative to the cleaved signal element.
[0066] In another preferred embodiment, the cleavable signal
element according to the present invention provides information
(signal) on the presence or absence of analytes in the sample to a
capacitance and impedance measurement device for measuring
conductance variations. Binding of the analyte preselected for
detection enables removal of the cleavable signal element through
cleavage. A conductance difference between the uncleaved signal
element and the cleaved signal element signals whether the analyte
exists or not in the sample. After washing, a "label-attached
uncleaved probe" structure may be optionally formed on the
substrate by contacting the uncleaved signal element with a label,
such as a metal microsphere, to increase detector sensitivity.
[0067] To increase detection sensitivity to conductance variations
in the nucleic acid hybridization assay according to the present
invention, one end of the cleavable capture probe is bound to the
substrate, and the other end of the cleavable capture probe is
labeled with, for example, a conducting polymer (e.g.,
polyaniline), a fluorescent label, or a metal microsphere, to form
a label-attached signal element ("label-attached cleavable capture
probe"), thereby increasing a conductance (capacitance and
impedance) variation relative to the cleaved signal element.
[0068] Another object of the present invention is to provide a
nucleic acid hybridization assay method and device using a cleavage
enzyme specifically responsive to a complementary double strand of
nucleic acids, which are applicable to quantitative and qualitative
assay devices.
[0069] In the present invention, cleavage is achieved by a cleavage
enzyme, such as a DNAse, specific to double stands. In this case,
only a capture probe acts as a cleavable signal element without a
restriction probe. After the capture probe forms a double stand,
the DNAse cleaves the double strand at a capture probe portion.
Therefore, the capture probe functions as a "cleavable signal
element"
[0070] Once a double strand that is cleavable by the DNAse is
formed through hybridization of the capture probe to a target
nucleic acid, the double strand is cleaved by the DNAse and
separated from the substrate through washing.
[0071] In contrast, when the capture probe contacts a sample not
including a complementary sequence to the capture probe, the
capture probe does not form the double strand and thus remains as
an uncleaved capture probe, attached to the substrate even after
the addition of the DNAse and washing. After washing, a
"label-attached uncleaved probe" structure may be optionally formed
on the substrate by contacting the uncleaved signal element with a
label, such as a fluorescent label or a metal microsphere, to
increase detector sensitivity.
[0072] After contacting the sample, reaction with the DNAse, and
washing, detection of the presence of the uncleaved probe or
"label-attached uncleaved probe" on the substrate by the detector
including an optical device, an electrochemical device, a mass
measurement device, or a capacitance and impedance measurement
device, indicates whether the analyte is present or not in the
sample. Therefore, the "uncleaved probe" or "label-attached
uncleaved probe" acts as an analyte presence/absence signal
element. The presence of the cleavable signal element (uncleaved
signal element) on the substrate indicates that sample does not
contain a particular analyte, and the absence of the cleavable
signal element (the cleaved signal element remain) indicates that
sample contains a particular analyte.
[0073] Still another object of the present invention to provide a
nucleic acid hybridization assay method and device using a cleavage
enzyme specifically responsive to a single strand of nucleic acids,
which are applicable to quantitative and qualitative assay
devices.
[0074] In the present invention, cleavage is achieved by a cleavage
enzyme, such as a nuclease, for example, derived from mung bean,
specific to single strands. In this case, only a capture probe acts
as a cleavable signal element without a restriction probe.
[0075] When the capture probe contacts a sample not including a
complementary sequence to the capture probe, the capture probe does
not form a double strand and remains as a single strand which is
cleavable by the nuclease. The single strand is cleaved by the
nuclease and separated from the substrate through washing.
[0076] In contrast, when the capture probe is double-stranded
through hybridization to a target nucleic acid, the capture probe
remains as an uncleaved probe, attached to the substrate even after
the addition of the nuclease and washing.
[0077] After washing, a "label-attached uncleaved probe" structure
may be optionally formed on the substrate by contacting the
uncleaved signal element with a label, such as a fluorescent label
or a metal microsphere, to increase detector sensitivity.
[0078] After contacting the sample, reaction with the nuclease, and
washing, detection of the presence of the uncleaved probe or
"label-attached uncleaved probe" on the substrate by the detector
including an optical device, an electrochemical device, a mass
measurement device, or a capacitance and impedance measurement
device, indicates whether the analyte is present or not in the
sample. Therefore, the "uncleaved probe" or "label-attached
uncleaved probe" acts as an analyte presence/absence signal
element. The presence of the cleavable signal element (uncleaved
signal element) on the substrate indicates that sample contains a
particular analyte, and the absence of the cleavable signal element
(the cleaved signal element remain) indicates that sample does not
contain a particular analyte.
[0079] Hereinafter, the structure of the present invention provided
to achieve the above objects will be described.
[0080] To achieve an object of the present invention, there is
provided a cleavable signal element comprising: a restriction probe
of a single strand having a particular sequence cleavable by a
restriction enzyme specific to a double strand of a particular
sequence; and a capture probe of a single strand having a
complementary sequence to a target nucleic acid for diagnosis or
assay to form a double strand through hybridization to the target
nucleic acid, wherein one end of the restriction probe is attached
to a solid support substrate, and the other end of the restriction
probe is ligated to the capture probe, thus forming a
single-stranded, cleavable capture probe.
[0081] In the present invention, when the capture probe contacts a
sample including the target nucleic acid of the complementary
sequence, the capture probe is double-stranded through
hybridization to the target nucleic acid, the restriction probe is
double-stranded through DNA extension using the target nucleic acid
hybridized to the capture probe as a primer with the addition of a
DNA polymerization solution, the double-stranded restriction probe
is cleaved by the restriction enzyme, and the cleaved cleavable
capture probe is removed from the solid support substrate through
washing, thus resulting in a cleaved signal element. In contrast,
when the capture probe contacts a sample not including the target
nucleic acid of the complementary sequence, the single-stranded,
cleavable capture probe remains attached to the solid support
substrate even after additions of the DNA polymerization solution
and the restriction enzyme and washing, thus resulting in an
uncleaved signal element. Preferably, the DNA polymerization
solution comprises a solution of four dNTPs and a DNA polymerase
solution.
[0082] Another cleavable signal element according to the present
invention comprises a capture probe of a single strand having a
complementary sequence to a target nucleic acid for diagnosis or
assay to form a double strand through hybridization to the target
nucleic acid, wherein one end of the capture probe is attached to a
solid support substrate, and the capture probe itself forms a
single-stranded, cleavable capture probe which is cleavable by a
cleavage enzyme specifically responsive to a double strand or
single strand of nucleic acids.
[0083] In this case, when the capture probe contacts a sample
including the target nucleic acid of the complementary sequence,
the capture probe is double-stranded through hybridization to the
target nucleic acid, the double-stranded capture probe is cleaved
by the cleavage enzyme specifically responsive to the double strand
of nucleic acids, and the cleaved cleavable capture probe is
removed from the solid support substrate through washing, thus
resulting in a cleaved signal element. In contrast, when the
capture probe contacts a sample not including the target nucleic
acid of the complementary sequence, the single-stranded, cleavable
capture probe remains attached to the solid support substrate even
after the addition of the cleavage enzyme and washing, thus
resulting in an uncleaved signal element. Preferably, the cleavage
enzyme is a DNAse.
[0084] When the capture probe contacts a sample not containing the
target nucleic acid of the complementary sequence, the capture
probe remains as a single strand without hybridization, the
single-stranded capture probe is cleaved by the cleavage enzyme
specifically responsive to the single strand of nucleic acids, and
the cleaved cleavable capture probe is removed from the solid
support substrate through washing, thus resulting in a cleaved
signal element. In contrast, when the capture probe contacts a
sample including the target nucleic acid of the complementary
sequence, the capture probe is double-stranded through
hybridization to the target nucleic acid, and the double-stranded,
cleavable capture probe remains attached to the solid support
substrate even after the addition of the cleavage enzyme and
washing, thus resulting in an uncleaved signal element. Preferably,
the cleavage enzyme is a nuclease, more preferably, derived from
mung bean.
[0085] In the cleavable signal elements according to the present
invention, the solid support substrate may be a plastic substrate,
a glass substrate, a silicon substrate, or a gold substrate.
Preferably, the solid support substrate has a self-assembled
monolayer (SAM) on the surface. Preferably, the capture probe has a
length ranging from about 5- to about 30-mers.
[0086] To increase detection sensitivity for an uncleaved signal
element, it is preferable that a label is attached to one end of
the cleavable capture probe to form a label-attached cleavable
capture probe structure or to one end or side of an uncleaved probe
to form a label-attached uncleaved probe structure, to increase
detection sensitivity for an uncleaved signal element. In this
case, the label may comprise a metal microsphere, a conducting
polymer, a fluorescent dye, a magnetic microsphere, and a
streptavidin-labeled microsphere. Preferably, the metal microsphere
is formed of a metal selected from the group consisting of gold,
silver, nickel, platinum, chromium, and copper. Preferably, a gold
microsphere has a diameter ranging from about 1 nm to about 10
.mu.m. Preferably, the streptavidin-labeled microsphere is attached
to the cleavable capture probe via biotin.
[0087] To achieve another object of the present invention, there is
provided a nucleic acid hybridization assay device comprising: a
solid support substrate; a plurality of cleavable signal elements
according to any of the cleavable signal elements described above
attached to the solid support substrate; and an internal or
external detector which detects a uncleaved signal element and a
cleaved signal element from the plurality of cleavable signal
elements.
[0088] It is preferable that the detector comprises an optical
device, an electrochemical device, a mass measurement device, or a
capacitance and impedance measurement device. Preferably, the
optical device detects fluorescence of the uncleaved signal element
and cleaved signal element.
[0089] It is preferable that the detector detects a differential
reflective signal or a differential conductive signal of the
uncleaved signal element and the cleaved signal element.
Preferably, the detector detects the differential reflective signal
by measuring the reflectance, absorbance, or scattering of light or
a laser beam incident on the uncleaved signal element and the
cleaved signal element.
[0090] Alternatively, the detector may detect the differential
conductive signal by measuring the capacitance and impedance of the
uncleaved signal element and the cleaved signal element. In this
case, preferably, the capacitance and impedance measurement device
measures the frequency response characteristics of the uncleaved
signal element and the cleaved signal element.
[0091] In the nucleic acid hybridization assay device according to
the present invention, the capacitance and impedance measurement
device may comprise interdigitated array electrodes having at least
one digit and arranged on the solid support substrate. Preferably,
the interdigitated array electrodes are substantially formed of
gold. Preferably, the interdigitated array electrodes have an input
port to check for the frequency response characteristics, and the
input port is connected to an electronic control device which
generates a frequency signal of a constant bandwidth.
[0092] In the nucleic acid hybridization assay device according to
the present invention, a plurality of cleavable signal elements may
be deposited on the interdigitated array electrodes, preferably
only in the space between the interdigitated array electrodes.
[0093] In the nucleic acid hybridization device according to the
present invention, to increase the sensitivity of the detector, it
is preferable that a label-attached uncleaved probe structure is
formed on the solid support substrate by attaching a label to the
uncleaved signal element.
[0094] Preferably, the plurality of cleavable signal elements are
deposited on the solid support substrate in a spatially-addressable
pattern, more preferably, to enable a single-sample assay for
multiple analytes, a multiple-sample assay for a single analyte, or
a multiple sample assay for multiple analytes.
[0095] In the nucleic acid hybridization device according to the
present invention, it is preferable that the solid support
substrate is a plastic substrate formed of a material selected from
the group consisting of polypropylenes, polyacrylates, polyvinyl
alcohols, polyethylenes, polymethylmethacrylates, and
polycarbonates. Among those materials for the solid support
substrate, polycarbonates are more preferred. Preferably, the solid
support substrate is formed of a circular disk or a rectangular
disk. The circular disk may have a diameter of approximately 120 mm
and a thickness of approximately 1.2 mm. The nucleic acid
hybridization assay device according to the present invention may
include a plurality of circular disks.
[0096] It is preferable that, in the nucleic acid hybridization
device according to the present invention, the circular disk
comprises: a central void to engage a rotational drive means; a
sample injection port through which a sample is injected; and an
annular and/or a spiral track in which the plurality of cleavable
signal elements are deposited in the spatially-addressable pattern.
Preferably, an address pattern that provides coded address
information is formed on the circular disk.
[0097] Alternatively, the circular disk in the nucleic acid
hybridization device according to the present invention may
comprise: a central void to engage a rotational drive means; a
sample injection port through which a sample is injected; and a
radial assay sector in which the plurality of cleavable signal
elements are deposited in the spatially-addressable pattern.
Preferably, the circular disk comprises a plurality of assay
sectors. The plurality of assay sectors may be connected to
respective separate sample injection ports or to a common sample
injection port. The plurality of cleavable signal elements are
deposited in each of the plurality of assay sectors in an
appropriate pattern for a single-analyte assay or a
multiple-analyte assay. Therefore, the nucleic acid hybridization
assay device according to the present invention is applicable for a
single-sample assay for multiple analytes, a multi-sample assay for
a single analyte, and a multi-sample assay for multiple
analytes.
[0098] It is preferable that the circular disk includes in a
central track a database associated with bioinformatics required
for diagnosis and assay interpretation, and telephone numbers, web
link information and software required for remote diagnosis.
[0099] In the nucleic acid hybridization device according to the
present invention, it is preferable that a detector is mounted on
the circular disk. The detector may comprise a non-contact
interface through which information read from the cleaved signal
element and the uncleaved signal element is transmitted to an
external central controller or storage device. Preferably, the
non-contact interface comprises an infrared interface and an
optical interface. As an example, the infrared interface may an
infrared sensor, and the optical interface may be a
photosensor.
[0100] Preferably, the circular disk in the nucleic acid
hybridization assay device according to the present invention
simultaneously comprises at least one SNP (single nucleotide
polymorphism) assay sector for SNP detection and at least one
expression assay sector for expression profile analysis. In this
case, the SNP assay sector and the expression assay sector may be
arranged separate in an angular direction or in a radial
direction.
[0101] To achieve still another object of the present invention,
there is provided bio-driver apparatus comprising: a rotary disk
receiver onto which any nucleic acid assay device described above
is to be loaded; a motor driver which rotates the disk; a rotary
connector which connects the motor driver to a central void portion
of the disk such that the disk is rotatable; and an optical device
to write data in or to read data from the disk.
[0102] Preferably, the bio-driver apparatus further comprises a
central controller which transmits information read from the disk
by the optical device to an external storage unit, transmits
information to be written to the optical device, and generates and
outputs a variety of control signals for the motor driver and the
other elements.
[0103] In the bio-driver apparatus according to the present
invention, it is preferable that the rotary connector comprises an
upper rotor and/or a lower rotor, the upper and lower rotors being
pushed close to the top and bottom surfaces, respectively, of the
central void portion when the disk begins to rotate.
[0104] In the bio-driver apparatus according to the present
invention, the optical device may detect fluorescence, preferably,
a differential reflective signal by measuring the reflectance,
absorbance, or scattering of incident light or an incident laser
beam.
[0105] Alternatively, the present invention provides a bio-driver
apparatus comprising: a rotary disk receiver onto which any nucleic
acid assay device described above is to be loaded; a motor driver
which rotates the disk; a rotary connector which rotatably connects
the motor driver to a central void portion of the disk; an external
power connector which powers and/or supplies a control signal to a
detector mounted on the disk; and a non-contact interface through
which information read by the detector is transmitted.
[0106] Preferably, the bio-driver apparatus further comprises a
central controller which transmits information read from the disk
by the detector to an external storage unit and generates and
outputs a variety of control signals for the motor driver and the
other elements.
[0107] Preferably, the bio-driver apparatus further comprises an
optical device to write data in or to read data from the disk.
Software including, for example, bioinformatics information, can be
written in or read from the disk.
[0108] Preferably, the detector detects a differential conductive
signal by measuring capacitance and impedance.
[0109] Preferably, the rotary connector comprises an upper rotor
and/or a lower rotor, the upper and lower rotors being pushed close
to the top and bottom surfaces, respectively, of the central void
portion when the disk begins to rotate.
[0110] Preferably, the power connector comprises a brush that
frictionally contacts the upper and/or lower rotors in connection
with an external power supply unit, and each of the upper and lower
rotors comprises an annular electrode plate frictionally contacting
the brush. One of the upper and lower rotors may be used. In this
case, two opposite nodes of the power supply unit are connected to
one brush. When both of the upper and lower rotors are used,
brushes contacting the upper and lower rotors may respectively
connected to the opposite nodes of the power supply unit.
[0111] It is preferable that the annular electrode plate comprises
at least one conductive arm connected thereto, and the central void
portion of the disk comprises a hole to engage the at least one
conductive arm and a circuit pattern connected to the hole to power
the detector mounted on the disk and/or supply the control signal
to the detector. In this case, the at least one conductive arm may
a spring at its one end that is connected to the annular electrode
plate.
[0112] In the bio-driver apparatus according to the present
invention, it is preferable that the power connector comprises an
electromagnet attached to the rotary disk receiver in connection
with the external power supply unit, and the electromagnet induces
an AC voltage to a wound coil on the disk so that the detector is
powered in a non-contact manner. In this case, the disk further
preferably comprises a rectifier for rectifying the AC voltage
induced to the wound coil.
[0113] To achieve yet still another object of the present
invention, there is provided a remote diagnostic system comprising:
any nucleic acid hybridization assay device according to the
present invention described above, an existing communication
network such as the Internet; and a computer in which software
capable of controlling access to the existing communication network
and digitizing information read from the nucleic acid hybridization
assay device is installed, wherein the digitized information from
the nucleic acid assay hybridization assay device is transmitted to
a doctor or a hospital, and a patient is provided with a
prescription, through the existing communication network.
[0114] In the remote diagnostic system according to the present
invention, the computer may comprise assay interpretive algorithms,
bioinformatics information, and self-diagnostics related software.
Preferably, the computer comprises software capable of uploading
diagnostic information to remote locations and device drivers. In
this case, the software may include educational information for
patients on clinical assays, a variety of wet sites and links
enabling a patient to directly communicate with a doctor or
hospital based on his/her diagnosis result.
[0115] It is preferable that the computer comprises a camera and a
microphone for viewing a patient's face and listening to his/her
voice. It is preferable that the diagnostic data based on the
digitized information are displayed on a computer monitor, the
computer automatically or manually transmits the diagnostic data to
a specialist through the existing communication network, and the
patient waits for a prescription from the specialist.
[0116] To achieve another object of the present invention, there is
provided a nucleic acid hybridization assay method comprising:
hybridizing a capture probe to a target nucleic acid present in a
liquid sample by contacting any nucleic acid hybridization assay
device according to the present invention described above, with the
liquid sample; contacting the cleavable capture probe with a
restriction enzyme or cleavage enzyme which is specifically
responsive to a cleavable signal element depending on whether the
capture probe and the target nucleic acid are hybridized or not;
washing the nucleic acid hybridization assay device to remove the
cleavable signal element cleaved by the restriction enzyme or
cleavage enzyme; and detecting whether the uncleaved signal element
or the cleaved signal element exists on the solid support
substrate.
[0117] Preferably, the nucleic acid hybridization assay method
further comprises contacting the cleavage capture probe with a DNA
polymerization solution before contact with the restriction enzyme.
As a result, the restriction probe forms a double strand through
DNA extension using the target nucleic acid hybridized to the
capture probe as a primer. Preferably, the nucleic acid
hybridization assay method further comprises contacting the
cleavage capture probe with a 3'-5' exonuclease solution before
contact with the DNA polymerization solution. As a result, a
portion of the target nucleic acid that remains as a single strand
without hybridization to the capture probe is cleaved, so that the
target nucleic acid can act as the primer.
[0118] It is preferable that the nucleic acid hybridization assay
method further comprises attaching a label to the cleavable signal
element before contacting the capture probe with the liquid sample,
or to an uncleaved signal element after contacting the cleavable
capture probe with the restriction enzyme or cleavage enzyme. When
the label attachment is applied before contacting the sample, it is
preferable that the label is attached during the synthesis of the
capture probe or after the immobilization of the capture probe to a
solid support substrate. Preferably, simple washing is performed
between contact with the restriction enzyme and the label
attachment.
[0119] It is preferable that the nucleic acid hybridization assay
method further comprises at least one wash step. In the nucleic
acid hybridization assay method, washing may be performing by
rotating the nucleic acid hybridization assay device with or
without addition of a detergent solution, or by applying an
external electric field.
[0120] One embodiment of the nucleic acid hybridization assay
method according to the present invention comprising: (a) injecting
a sample into a sample injection port disposed near the center of a
disk in a nucleic acid hybridization assay device; (b) rotating the
disk and stopping the rotation of the disk when the sample reaches
an outer edge of the disk; (c) incubating the disk in a stationary
state at room temperature for hybridization; (d) adding a buffer
solution as a washing solution while rotating the disk at a high
speed, to wash the disk; (e) adding a DNA polymerization solution
containing a mixed solution of four dNTPs and a DNA polymerase and
incubating the disk in a stationary state for DNA extension; (f)
adding a solution of a restriction enzyme specifically responsive
to a double strand of a particular sequence and incubating the disk
in a stationary state, to cleave the double strand; (g) washing the
disk by rotating the disk at a high speed with the addition of a
buffer solution or by applying an external electric or magnetic
field; and (h) drying the disk and reading information from the
disk using a detector which is programmed to detect a predetermined
assay site on which a cleavable signal element is deposited and
comprises an optical device, an electrochemical device, or a
capacitance and impedance measurement device.
[0121] It is preferable that the nucleic acid hybridization assay
method further comprises adding a 3'-5' exonuclease solution before
step (e) of DNA extension. As a result, a portion of the target
nucleic acid that remains as a single strand without hybridization
to the capture probe is cleaved, so that the target nucleic acid
can act as the primer.
[0122] Another embodiment of the nucleic acid hybridization assay
according to the present invention comprises: (a) injecting a
sample into a sample injection port disposed near the center of a
disk in a nucleic acid hybridization assay device; (b) rotating the
disk and stopping the rotation of the disk when the sample reaches
an outer edge of the disk; (c) incubating the disk in a stationary
state at room temperature for hybridization; (d) adding a buffer
solution as a washing solution while rotating the disk at a high
speed, to wash the disk; (e) adding a solution of a cleavage enzyme
specifically responsive to a double strand or single strand of
nucleic acids and incubating the disk in a stationary state, to
cleave the double strand or single strand; (f) washing the disk by
rotating the disk at a high speed with the addition of a buffer
solution or by applying an external electric or magnetic field; and
(g) drying the disk and reading information from the disk using a
detector which is programmed to detect a predetermined assay site
on which a cleavable signal element is deposited and comprises an
optical device, an electrochemical device, or a capacitance and
impedance measurement device.
[0123] To increase detection sensitivity, the nucleic acid
hybridization assay methods described above may further comprise
attaching a label to the cleavable signal element before sample
injection, or to an uncleaved signal element after strand cleavage.
When the label attachment is applied before contacting the sample,
it is preferable that the label is attached during the synthesis of
the capture probe or after the immobilization of the capture probe
to a solid support substrate. Preferably, simple washing is
performed between contact with the restriction enzyme and the label
attachment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0124] FIGS. 1A through 2C show alternative embodiments of the
attachment of a plurality of cleavable signal elements to a
derivatized site of a variety of substrates, in particular:
[0125] FIG. 1A is a schematic representation of the attachment of a
plurality of cleavable capture probes (cleavable signal elements)
at a derivatized site on the plastic (carbonate) substrate of an
assay device by covalent bonds, where n of (CH).sub.n is an integer
greater than zero;
[0126] FIG. 1B is a schematic representation of a nucleic acid
hybridization assay shortly after introduction of a sample
containing nucleic acids;
[0127] FIG. 1C is a schematic representation of a step after the
procedure of FIG. 1B, in which oligonucleotides present in the
sample have bound to complementary oligonucleiotide of a first
capture probe to form a double strand, but have not bound to
oligonulcleotide of a second capture probe, where the rectangular
box denotes the complementary double strand formation;
[0128] FIG. 1D is a schematic representation of a step after the
procedure of FIG. 1C, in which a single-stranded restriction probe
ligated to the end of the first capture probe is double-stranded
using a DNA polymerization solution, in particular, the restriction
probe forms a double strand through DNA extension using the
complementary target nucleic acid attached to the first capture
probe in the step of FIG. 1C as a primer, whereas the second
capture probe still remains as a single strand;
[0129] FIG. 1E is a schematic representation of a step after the
assay procedure of FIGS. 1C and 1D, in which the restriction probe
ligated to the end of the first capture probe that has formed the
double strand is cleaved by contact with a restriction enzyme, and
the cleavable first capture probe double-stranded through the
complementary hybridization is removed from the substrate surface,
whereas the second capture probe still remains as a single strand
on the substrate surface;
[0130] FIG. 1F is a schematic representation of the removal of the
first capture probe cleaved in the procedure of FIG. 1E by
washing;
[0131] FIG. 1G is a schematic representation of the formation of a
"label-attached uncleaved probe" structure by contacting the second
capture probe with a label, for example, an SSB protein, in which
the second capture probe is tethered to the substrate surface while
the first capture probe is removed from the substrate surface
through washing, which provides differential signals as well as
spatially-addressable differential reflective signals to a detector
including an optical device, an electrochemical device, a mass
measurement device, or a capacitance and impedance measurement
device;
[0132] FIG. 2A is a schematic representation of an embodiment of
the nucleic acid hybridization assay according to the present
invention to increase the sensitivity of a detector including an
optical device, an electrochemical device, a mass measurement
device, or a capacitance and impedance measurement device, in which
a plurality of cleavable capture probes (cleavable signal elements)
are covalently bound to a derivarized site of the plastic substrate
(polycarbonate) surface of an assay device, and metal microspheres,
conducting polymers, or fluorescent labels are attached to the
other free end of the cleavable capture probes;
[0133] FIG. 2B is a schematic representation of the cleavage and
removal of the first capture probe by washing; and
[0134] FIG. 2C is a schematic representation of the labeling of the
other free end of the cleavable capture probes with a
streptavidin-labeled magnetic microbead;
[0135] FIGS. 3A through 3D show alternative embodiments of the
spatially addressable arrangement of the cleavable signal
elements;
[0136] FIGS. 4A through 4E show alternative embodiments of the
supply of power to a rotating assay device (disk);
[0137] FIG. 4F shows an implementation of detection of
analyte-specific signals generated by the assay device using an
optical device;
[0138] FIGS. 5A through 51 show alternative embodiments of the
detection of analyte-specific signals generated by the assay device
of FIG. 3C using a capacitance and impedance measurement device
having interdigitated array electrodes;
[0139] FIGS. 6A and 6B show alternative embodiments of
implementation of the differential reflection between an uncleaved
signal element and a cleaved signal element;
[0140] FIGS. 6C through 6E show alternative embodiments of
implementation of the differential conductance (impedance or
capacitance) between the uncleaved signal element and the cleaved
signal element using the interdigitated array electrodes;
[0141] FIG. 7A illustrates the arrangement of four separate assay
sectors in an assay device, each containing a different cleavable
signal element, to assay in parallel a single sample for four
different analytes;
[0142] FIG. 7B shows an embodiment of the arrangement of the assay
device of FIG. 7A on a disk;
[0143] FIG. 8 shows an embodiment of a remote diagnostic system
according to the present invention, in which the information read
by a detector of the assay device is digitalized as computer
software and mutually transmitted to and received by a patient and
a doctor through an existing communication network, for example,
the Internet;
[0144] FIG. 9 illustrates a washing method by external electric
field application;
[0145] FIGS. 10A through 10K illustrate embodiments of attachment
of cleavable signal elements to different types of substrate
surfaces for an assay device;
[0146] FIGS. 11A and 11B show alternative embodiments of an assay
device using the cleavable signal element according to the present
invention for diagnosing a variety of diseases through both single
nucleotide polymorphism (SNP) detection and gene expression profile
determination;
[0147] FIG. 12 shows the reaction mechanism of the 3'-5'
exonuclease;
[0148] FIGS. 13A and 13B are photographs showing the results of an
analysis in Example 2 optically measured by atomic force microscopy
(AFM); and
[0149] FIG. 14 is a graph of the impedance measured in Example
2.
DETAILED DESCRIPTION OF THE INVENTION
[0150] The present invention, including a nucleic acid
hybridization assay method and device, using a cleavage technique
specifically responsive to a complementary double strand or single
strand of nucleic acids or oligonucleotide will be described in
greater detail with reference to the appended drawings.
[0151] <Spatially Addressable Cleavable Signal Elements>
[0152] The reaction mechanism of a cleavable signal element
according to the present invention, termed a "bio-bit", can be
easily understood by reference to FIGS. 1A through 1C. Referring to
FIG. 1A, a plastic substrate 20 has a substrate 21 to which are
attached cleavable capture probes 34 and 35. The substrate 20 can
be formed of a porous or solid substrate using a variety of
materials including plastic, glass, mica, silica, and the like, but
plastic is most preferred for reasons of economy, ease of
derivatizaton for attaching the cleavable signal elements to the
surface, and compatibility with existing laser reflection-based
detectors, such as CD-ROM and DVD readers. Suitable plastics
include polypropylenes, polyacrylates, polyvinyl alcohols,
polyethylenes, polymethylmethacrylates, and polycarbonates, with
polypropylenes and polycarbonates being preferred and
polycarbonates being the most preferred.
[0153] The cleavable signal elements 34 and 35 include respective
capture probes 34b and 35b and respective restriction probes 34a
and 35a. The surface 21 of the substrate 21 can be derivatized to
provide covalent bonding to each of the cleavable signal elements
34 and 35. To protect the cleavable capture probes 34 and 35 from
direct contact with the surface 21 of the substrate 21, a monomer
layer of non-reactive molecules, for example, an alkane chain
(CH.sub.2).sub.n, can be formed on any substrate according to the
present invention. As an example, an alkane chain
((CH.sub.2).sub.n) 31 attached to one end of the restriction probe
34a is shown.
[0154] The alkane chain 21 at one end of each restriction probes
34a and 35a is attached to the surface 21 via an amide linkage. The
restriction probes 34a and 35a of the cleavable signal elements 34
and 35 have a cleavage site that is susceptible to cleavage by a
restriction enzyme in double-strand formation.
[0155] Analyte specificity is conferred upon the cleavable signal
element by the sequence of the capture probes 34b and 35b. The
capture probes 34b and 35b includes an oligonucleotide of 5- to
20-mers, preferably 8- to 17-mers, most preferably 8- to 12-mers,
but longer oligonucleotides (cDNA) can be used. A large number of
cleavable signal elements 34 and 35 are present at particular
derivatized sites on the surface 21 of the substrate 20 of an assay
device called a "bio-disk".
[0156] In the present invention, the oligonucleotides of the
capture probes 34b and 35b bind with the complementary single
strands of nucleic acids present in a test sample. In other words,
the complementary oligonucleiotides form double strands, each
including a specific binding pair.
[0157] As shown in FIGS. 1A through 2C, the cleavable signal
elements (cleavable capture probes) 34 and 35 at different sites on
the assay device surface have discrete oilgonucleotide sequences.
In FIG. 1A, the first and second cleavable signal elements 65 and
66 have oligonucleotides 35b and 34b, respectively.
[0158] As shown in FIGS. 1B and 1C, when contacted with a test
sample containing an oligonucleotide 36, the complementary
oligonucleotide 35b binds with the oligonucleotide 36 present in
the test sample to form a double strand (referred to as also a
"capture double strand") 37, as shown in FIG. 1C. If there no
complementarity between the sample oligonucleotide 36 and the
oligonucleotide 34b, there is no binding between those groups as
illustrated in FIG. 1C. The capture probe 35b forms the double
strand 37 with the sample oligonucleotide 36, whereas the
restriction probe 35a at one end of the capture probe 35b currently
remains as a single strand.
[0159] To form a double strand of the restriction probe 35a at one
end of the capture probe 35b that have formed the double strand 37,
as shown in FIG. 1D, a DNA polymerization solution is added. The
restriction probe 35a is double-stranded via DNA extension using a
target nucleic acid hybridized to the capture probe 35b as a
primer. The restriction double strand 38 formed through the DNA
extension is shown in FIG. 1B.
[0160] In FIG. 1E, cleavage of the restriction double strand 38 by
addition of a restriction enzyme after formation of the capture and
restriction double strands 37 and 38 in the steps of FIGS. 1C and
1D is shown.
[0161] After the restriction double strand 38 is cleaved at the
cleavage site 38c, the first cleavable signal element 65
specifically bound with the test sample is detached and removed
from the surface 21 by washing. This is illustrated in FIG. 1F. If
it is desired to assay multiple samples for a single
oligonucleotide, the captures probes at different sites will
generally have the same oligonucleotide sequence. Presence or
absence of a cleavable signal element (after cleavage) on the
surface 21 may be detected from differential reflectance of
incident light, in particular, incident laser light, or by a
capacitance and impedance measurement device capable of measuring
conductivity variations.
[0162] FIG. 1G illustrates an alternative embodiment of labeling
after the washing to increase the sensitivity of a detector. The
uncleaved signal element 66 remaining on the substrate 20 after the
step of FIG. 1F is brought into contact with a label 39, for
example, an SSB protein, to form a "label-attached uncleaved probe"
structure and thus increase a difference in reflectivity or
conductivity between the cleaved signal element 65 and the
uncleaved signal element 66, thereby resulting a higher sensitivity
of the detector.
[0163] FIG. 2A is a schematic representation of an embodiment of
the nucleic acid hybridization assay according to the present
invention to increase the sensitivity of a detector including an
optical device, an electrochemical device, a mass measurement
device, or a capacitance and impedance measurement device. As shown
in FIG. 2A, a plurality of cleavable capture probes (cleavable
signal elements) are covalently bound to a derivarized site of the
plastic substrate (polycarbonate) surface of an assay device, and a
label 40, such as a metal microsphere, conductive polymer, or
fluorescent label is attached to the other free end of the
cleavable capture probes.
[0164] The label 40, such as the metal microsphere, act as a
reflective signal generation element to permit detection of the
presence of the first and second cleavable signal elements 65 and
66 coupled to the substrate 20 of the assay device. Suitable
materials for the reflective signal generation element include gold
(Au), silver (Ag), nickel (Ni), chromium (Cr), platinum (Pt), and
copper (Cu), but Au is preferred due to its nature to easily and
strongly bind to a thiol (SH)-group attached to one end of the
cleavable signal elements 34 and 35. The metal microsphere may be
formed of solid metal, metal-coated plastic, or glass bead. Any
reflective materials, instead of metal, can be used.
Au-microspheres directly bind to a thiol group attached to one end
of the cleavable signal elements 34 and 35.
[0165] In FIG. 2A, the first and second cleavable signal elements
65 and 66 have the oligonucleiotides 35b and 34b, respectively.
When contacted with a test sample containing an oligonucleotide,
the complementary oligonucleotide 35b binds with the
oligonucleotide present in the test sample to form a double strand
(not shown). If there no complementarity between the sample
oligonucleotide and the oligonucleotide 34b, there is no binding
between those groups. The capture probe 35b forms the double strand
with the sample oligonucleotide, whereas the restriction probe 35a
at one end of the capture probe 35b currently remains as a single
strand.
[0166] To form a double strand with the restriction probe 35a at
one end of the capture probe 35b that have formed the double
strand, a DNA polymerization solution is added. The restriction
probe 35a is double-stranded via DNA extension using a target
nucleic acid of the hybridized capture probe as a primer. After
formation of the complete double strand, the double-stranded
restriction probe is cleaved by addition of a restriction
enzyme.
[0167] After the double-stranded restriction probe is cleaved at
the cleavage site 38c, the first cleavable signal element 65
specifically bound with the test sample and the label 40 are
detached and removed from the surface 21 by washing. This is
illustrated in FIG. 2B. Presence or absence of a cleavable signal
element (after cleavage) on the surface 21 may be detected from
differential reflectance of incident light, in particular, incident
laser light, and by a capacitance and impedance measurement device
capable of measuring conductivity variations.
[0168] FIG. 2C shows an embodiment of the labeling of cleavable
capture probes with the label 39, which may be a metal microsphere,
conducting polymer, or fluorescent label, in FIG. 1G. In FIG. 2C, a
plurality of cleavable signal elements are covalently bound to a
derivatized site of the surface 21 of the assay device substrate
20, and the other end of the cleavable signal elements is labeled
with the label 39, such as a metal microsphere, conducting polymer,
or fluorescent label, via avidin 51 and biotin 50.
[0169] FIGS. 3A through 3D show alternative embodiments of the
spatially addressable arrangement of the cleavable signal elements.
In particular, FIG. 3A shows an address pattern 500 formed on a
substrate 70 of a circular disk to provide coded address
information, from which the location of a cleavable signal element
200 may be optically or fluorescently measured, and the attachment
of the cleavable signal element 200 to annular tracks 351 by
deposition. The circular disk has a central void 61 to engage a
rotational drive means. Adjacent annular tracks are connected with
each other by spiral-track bridges 352 and 353 to permit the sample
injected through a sample injection port 354 to uniformly and
outwardly spread by a centrifugal force generated as the disk
rotates. The address information of the disk can be obtained from
the address pattern 500, which is a regular pattern impressed at a
fixed location. Reference numeral 350 denotes a track-to-track
interval. FIG. 3A shows the deposition of the cleavable signal
element in an appropriate pattern to assay in parallel a single
sample for multiple analytes.
[0170] FIG. 3B shows an address pattern 401 formed on the substrate
70 of the circular disk to provide coded address information, from
which the location of the cleavable signal element 200 may be
optically or fluorescently measured, and the attachment of the
cleavable signal element 200 to radial tracks 351 by deposition.
The circular disk has a central void 61 to engage a rotational
drive means. FIG. 3B shows the deposition of the cleavable signal
element in an appropriate pattern to assay in parallel multiple
samples. A database associated with bioinfomatics required for
diagnosis and assay interpretation, and telephone numbers, web link
information, and software required for remote diagnosis may be
coded and stored in a central track 402. The embodiment of FIG. 3B
shows assay sectors with individual sample injection ports 354 and
segregated from one another, thereby permitting rotation of the
assay device without sample mixing. In FIG. 3B, reference numeral
444 denotes a sample flow channel along which a sample flows from
the sample injection port 354 to a corresponding assay sector 800.
If multiple sample injection ports 354 are interconnected with each
other, a single sample can be assayed for multiple analytes.
[0171] FIG. 3C shows the attachment of the cleavable signal element
200 to a radial track for each assay sector 800 by deposition. To
electrically measure whether the cleavable signal element 200 is
cleaved or not using the detector including a capacitance and
impedance measurement device described above, an electronic control
unit 63 and a circuit pattern 64 connecting each of the assay
sectors 800 to the electronic control unit 63 are mounted on the
substrate 70 of the circular disk. The electronic control unit 63
measures the capacitance and impedance with respect to each of the
assay sectors 800 by checking for their frequency response
characteristics, thereby providing information on whether the
cleavable signal element 200 is cleaved or not, or information on
the degree of cleavage. The frequency response characteristics
measured by the electronic control unit 63 is transmitted to an
external central controller (not shown) or storage device (not
shown) via a non-contact interface 107, for example, an infrared
interface or optical interface, designed on the disk. In FIG. 3C,
reference numeral 354 denotes a sample injection port.
[0172] FIG. 3D shows an address pattern 401 formed on the substrate
70 of the circular disk to provide coded address information, from
which the location of the cleavable signal element 200 may be
optically or fluorescently measured, and the attachment of the
cleavable signal element 200 at a constant interval over the entire
surface of the disk by deposition. The circular disk has a central
void 61 to engage a rotational drive means. The structure of FIG.
3D is suitable for a single-analyte assay with multiple samples or
for a multiple-analyte assay with a single sample. To end this,
samples may be injected through individual sample injection ports
arranged in an ink-jet array corresponding to the location of each
capture probes. Alternatively, a sample may be injected through a
single sample injection port and spread over the entire substrate
by a rotational force.
[0173] FIGS. 4A and 4B shows embodiments of a bio-driver, which is
a mechanical device for rotating the disk of the assay devices
described above. The electronic control device 63 transmits the
measured frequency response characteristics to an external central
controller 101 or storage device 111 through a non-contact
interface, for example, an infrared interface or optical interface,
which are located adjacent the central void 61 of the disk.
Reference numerals 106 and 107 denote reception and transmission
portions, respectively, of the non-contact interface. The reception
and transmission portions 106 and 107 of the non-contact interface
may be implemented with infrared sensors for infrared interfacing,
or photosensors for optical interfacing.
[0174] In FIGS. 4A and 4B, embodiments of the supply of power to
the electronic control unit 63 on the disk while it rotates are
also shown. Reference numeral 100 denotes a driver body for
supporting the bio-driver. A printed circuit board (PCB) 140 is
connected to the driver body 100 below the bio-driver, and the
central controller 101 for controlling the bio-driver and the
storage unit 111 are mounted on the PCB 104. The central controller
101 controls a motor 102 to rotate the disk or stop rotation of the
disk, controls movement of an optical device 103, and controls
upper and lower rotors 104 and 105 such that they rotate adjacent
the central void 651 of the disk upon rotation of the disk. The
central controller 101 transmits the information read from the disk
by the optical device 103 to the storage unit 111, or information
to be written to the optical device 103, and provides a number of
control signals required to read/write information to the other
elements.
[0175] FIG. 4A shows an embodiment of the supply of power to the
electronic control unit 63 on the disk by frictional contact
between the upper and lower rotors 104 and 105 and respective
brushes 108 and 109. In FIG. 4A, reference numeral 110 denotes a
power supply unit for supplying a DC power to the brushes 108 and
109, and reference numerals 227 and 228 denotes arms.
Alternatively, one of the upper and lower rotors 104 and 105 may be
used. In this case, two opposite nodes of the power supply unit 110
are connected to one brush.
[0176] FIGS. 4C and 4D show embodiments of the supply of power to
the electronic control unit 63 mounted on the disk by frictional
contact between the upper rotor 104 and the brush 108, and between
the lower rotor 105 and the brush 109, respectively. In particular,
in FIG. 4C, an annular electrode plate 223 mounted on a top plate
222 of the upper rotor 104 to frictionally contact the brush 108 is
shown. The two conductive arms 227 connected to the annular
electrode plate 223 act as connectors to engage holes 302, which
are described later, formed near the central void 61 of the disk.
The annular electrode plate 223 has a radius of r1. Reference
numeral 277 denotes a groove which supports the upper rotor 104
against the driver body 100. In FIG. 4C, an annular electrode plate
225 mounted on a bottom plate 224 of the lower rotor 105 to
frictionally contact the brush 109 is shown. The two conductive
arms 229 connected to the annular electrode plate 225 act as
connectors to engage holes 301, which are described later, formed
near the central void 61 of the disk.
[0177] FIG. 4E shows the holes 301 and 302 to engage the conductive
arms 227 and 229, respectively, formed in the central void 61 of
the disk. The central void 61 has a radius of r0. Reference numeral
333 denotes a hole in the central void 61. As the disk starts to
rotate, the conductive arms 227 and 228 rotate while being engaged
with the holes 301 and 302 as the upper rotor 104 and the lower
rotor 105 are pushed closer together. A negative (ground) voltage
is applied to the conductive arm 227 connected to the upper rotor
104, whereas a positive voltage is applied to the conductive arm
228 connected to the lower rotor 105. The holes 301 and 302 of the
disk, which are engaged with the conductive arms 227 and 228, are
connected to circuit patterns 303 and 304 to thereby supply power
to the electronic control unit 63. To make the holes 301 and 302
engage easier with the conductive arms 227 and 118 when the upper
rotor 104 and the lower rotor 105 are pushed closer together upon
rotation of the disk. The conductive arms 227 and 228 have a spring
226 at its one end connected to the respective annular electrode
plates 223 and 225.
[0178] FIG. 4B shows an embodiment of the supply of power to the
electronic control unit 63 where an AC voltage is induced to a
wound coil 152 on the disk and rectified by magnetic induction
between an electromagnet 150 attached to the driver body 100 and
the wound coil 152 to thereby supply power to the electronic
control unit 63 in a non-contact manner. In FIG. 4B, reference
numeral 110 denotes a power supply unit for supplying an AC current
to the electromagnet 150.
[0179] FIG. 4F shows an implementation of detection of
analyte-specific signals generated by the assay device of FIG. 3A,
3B, or 3D using the optical (or fluorescent) device 103. The
optical device 103 is provided with differentially reflective
(fluorescent) signals between the uncleaved signal element 66 and
the cleaved signal element 65 with respect to incident light, in
particular, incident laser light. The optical device 103 may
include a light source, an incident light emitting portion, and a
reflective light receiving portion.
[0180] FIGS. 5A through 5G show alternative embodiments of the
detection of analyte-specific signals generated by the assay device
of FIG. 3C using a capacitance and impedance measurement device
having interdigitated array electrodes. In particular, FIG. 5A
shows an embodiment of the capacitance and impedance measurement
device implemented by interdigitated array electrodes 702 and 703
and a plurality of cleavable signal elements. The cleavable signal
elements are attached to digits between the interdigitated array
electrodes 702 and 703. The sensitivity of the detector increases
with more digits.
[0181] Capacitance and impedance can be determined by measuring the
frequency characteristics of the sample with application of AC
signals having a predetermined bandwidth from the electronic
control unit 63 to two input ports 704 and 705 of the
interdigitated array electrodes 702 and 703.
[0182] FIG. 5B shows a state where the uncleaved signal element 34
remains between the interdigitated array electrodes 702 and 703 on
the surface of a substrate 701. FIG. 5C shows a state where only a
cleaved residue 38b remains after most of the cleavable signal
element is has been detached. The electronic control unit 63 is
provided with the differential frequency response characteristics
between the uncleaved signal element 34 and the cleaved signal
element 38b.
[0183] FIGS. 5D and 5E are for illustrating an embodiment of the
capacitance and impedance measurement device implemented with
interdigitated array electrodes and cleavable signal elements
having one end labeled with a label 40 such as a metal microsphere,
conducting polymer (e.g., polyaniline), or a fluorescent label.
FIG. 5D shows a state where the uncleaved signal element 34 remains
on the surface of the substrate 701 after cleavage of the cleavable
signal elements and washing. FIG. 5E shows a state where the
cleavable signal element has been detached.
[0184] FIGS. 5F and 5G is for illustrating an embodiment of the
capacitance and impedance measurement device implemented with
interdigitated array electrodes and a "label-attached uncleaved
probe" structure formed through additional contact between the
uncleaved signal element 34 and a label 39 after cleavage and wash
steps. FIG. 5F shows a state where the uncleaved signal element 34
remains on the surface of the substrate 701 being labeled with the
label 39. FIG. 5G shows a state where the cleavable signal element
has been detached.
[0185] FIG. 5H shows an embodiment of arrangement of a plurality of
assay sectors 800 on the disk, each including a pair of
interdigitated array electrodes 702 and 703. Each of the assay
sectors 800 may be constructed by combination of multiple pairs of
interdigitated array electrodes 702 and 703 for multiple-analyte
assay.
[0186] To enable the detector including the capacitance and
impedance measurement device constructed with the interdigitated
array electrodes 702 and 703 to electrically measure whether the
cleavable signal element is cleaved or not, circuit patterns 64
which connect the electronic control unit 63 to each of the
detectors arranged in the assay sectors 800, are imprinted in the
substrate 70 of the circular disk. The electronic control unit 63
measures the capacitance and impedance with respect to each of the
assay sectors 800 by checking for the frequency response
characteristics from the assay sectors 800 and thereby obtains
information on whether the cleavable signal element is cleaved or
not or information on the degree of cleavage. In FIG. 5H, reference
numeral 354 denotes a sample injection port, and reference numeral
444 denotes a sample inflow channel. Although multiple sample
injection ports 354 are illustrated in FIG. 5H, only one simple
injection port may be formed to assay a signal sample for multiple
analytes.
[0187] FIG. 5I shows an embodiment of the capacitance and impedance
measurement device in which a plurality of assay sectors 800, each
including the interdigitated array electrodes 702 and 703, are
arranged on a solid support 71 of a common shape. The electronic
control unit 63 and circuit patterns 64 which connect the
electronic control unit 63 to each of the detectors including the
capacitance and impedance measurement device and arranged in the
assay sectors 800, are mounted in the solid support 71, so that
whether the cleavable signal element is cleaved or not can be
measured using the converter. The electronic control unit 63
measures the capacitance and impedance with respect to each of the
assay sector 800 by checking for the frequency response
characteristics from the assay sectors 800 and thereby obtains
information on whether the cleavable signal element is cleaved or
not or information on the degree of cleavage. In FIG. 51, reference
numeral 354 denotes a sample injection port, reference numeral 444
denotes a sample inflow channel, reference numeral 356 denotes a
sample exhaust port, and reference numeral 445 denotes a sample
exhaust channel.
[0188] FIGS. 6A and 6B show alternative embodiments of
implementation of the differential reflection between a cleaved
signal element and an uncleaved signal element. Referring to FIG.
6A, a gold layer 22 and a self-assembled monolayer (SAM) 32 are
sequentially formed on a substrate, and a cleavable signal element
34 is immobilized on the SAM 32. Reference numeral 65 denotes a
cleaved signal element having a cleaved residue 38b left after the
cleavable signal element has been detached. Reference numeral 66
denotes an uncleaved signal element. FIG. 6B illustrates the
application of a label 39, such as a metal microsphere, conducting
poymer, or fluorescent label, to increase the sensitivity of the
detector. As shown in FIG. 6B, the gold layer 22 and the SAM 32 are
sequentially formed on the substrate, and a cleavable signal
element 34 is immobilized on the SAM 32. Reference numeral 65
denotes a cleaved signal element having a cleaved residue 38b left
after the cleavable signal element has been detached. Reference
numeral 66 denotes an uncleaved signal element.
[0189] FIGS. 6C through 6E show alternative embodiments of
implementation of the differential conductance (impedance or
capacitance) between the cleaved signal element and the uncleaved
signal element using interdigitated array electrodes. Referring to
FIG. 6C, the gold layer 22 and the SAM 32 for immobilization of the
cleavable signal element 34 are formed on a substrate 20. The gold
layer 22 constitutes the interdigitated array electrodes. A
protective layer 33 is formed to protect the gold layer 22 from the
cleavable signal element 34 adhering to the gold layer 22. As shown
in FIG. 6C, which is a partial cross-sectional view of the assay
sector 800 of FIG. 5A, only a cleaved residue 38b of a cleaved
signal element 65 remains after the cleavable signal element has
been detached. Reference numeral 66 denotes an uncleaved signal
element.
[0190] FIG. 6D shows an embodiment of labeling one free end of the
cleavable capture probe, which constitutes a cleavable signal
element whose the other end is attached to the substrate, with a
label 40 such as a metal microsphere, conducting polymer, or
fluorescent label to increase the sensitivity of the detector. FIG.
6E shows an embodiment of formation of a "label-attached uncleaved
probe" structure after washing by additional labeling of the
uncleaved signal element 66 with a label 39, such as a metal
microsphere, conducting polymer, or fluorescent label. As shown in
FIGS. 6D and 6E, which are partial cross-sectional views of the
assay sector 800 of FIG. 5A, only a cleaved residue 38b of a
cleaved signal element 65 remains after the cleavable signal
element has been detached. Reference numeral 66 denotes an
uncleaved signal element labeled with the label 40 or 39.
[0191] FIG. 7A illustrates the arrangement of four separate assay
sectors in an assay device, each containing a different cleavable
signal element 200, to assay in parallel a single sample for four
kinds of anlytes. The single sample injected through the sample
injection port 354 is supplied to each of the assay sectors through
the sample inflow channel 444. On each of the assay sectors 800,
the cleavable signal element 200 having a capture probe
complementary to a different analyte is deposited. Preferably, the
cleavable signal element 200 is fluorescently detected. In this
case, a fluorescent label is applied to the end of the cleavable
signal element 200, as illustrated in FIG. 2A. The assay device of
FIG. 7A includes a sample exhaust port 356 and a sample exhaust
channel 445. Alternatively, different kinds of cleavable signal
elements 200 that are complementary to a plurality of discrete
analytes may be deposited within one assay sector to enable
multi-analyte assay in a single assay sector.
[0192] FIG. 7B shows an embodiment of the assay device according to
the present invention, in which a plurality of assay devices of
FIG. 7A are radially arranged on a disk.
[0193] FIG. 8 shows an embodiment of a remote diagnostic system
according to the present invention, in which the information read
from the assay device is digitalized as computer software and
mutually transmitted to and received by a patient 151 and a doctor
125 through an existing communication network 133. In FIG. 8,
reference numeral 120 denotes a detector including an optical
device, an electrochemical device, a mass measurement device, or a
capacitance and impedance measurement device, as described above,
to detect the presence or absence of the cleavable signal element
on the solid support (substrate). The detector 120 may be a
bio-driver including a central controller and an assay device in
the form of disk, bio-CD, or bio-DVD where analyte-specific
cleavable signal elements are spatially and addressibly arranged in
a variety of ways. Reference numeral 127 denotes a
software-installed hard disk driver (HDD) or memory. The software
may include assay interpretive algorithms, bioinformatics
information, and self-diagnostics related information. The software
may further include software capable of uploading the diagnostic
information to remote locations and device drivers. The software
may include educational information for patients on clinical
assays, and may be modified for chosen audiences. The software may
include a variety of wet sites and links, for example, enabling a
patient to communicate with a doctor or hospital based on his/her
diagnosis result. Reference numerals 121 and 123 denote a camera
and a microphone for viewing a patient's face and listening to
his/her voice, respectively. Reference numeral 15i denotes a
patient. A hospital 124, a doctor 125, and a nurse 126, which
provide remote diagnosis services, are also shown in FIG. 8.
[0194] <Method of Applying Sample>
[0195] A cleavable signal element according to the present
invention is suitable for detecting, in particular, a nucleic acid
amplified to a limited size through an amplification scheme using a
variety of polymerase chain reactions (PCRs), ligase chain
reactions (LCRs), and T7 and SP6RNA polymerases.
[0196] In an assay method according to the present invention, a
sample to be tested is first introduced. After a dilute fluid
sample is applied near the center of the substrate (solid support)
of a circular, disk-type assay device, the assay device is rotated.
The fluid sample evenly diffuses over and uniformly covers the
surface of the substrate by a centrifugal fore generated by the
rotation of the assay device.
[0197] In this method of applying the sample, 100 .mu.L of the test
sample is diluted to about 1 mL. This dilute sample is dropwise
added near the center of the disk. The assay sites and the surface
of the disk are hydrophilic, and the fluid sample forms a thin
fluid film on the rotating disk. The thickness of the fluid film
can be adjusted by the frequency of the dropwise addition and the
frequency of disk rotation. Preferably, the thickness of the fluid
film is less than 10 .mu.m to permit all molecules in the fluid
sample to react with the cleavable signal element. About 10 .mu.L
of the fluid sample is needed to fully cover the surface of the
disk. This sample apply method is suitable for, in particular, the
assay devices of FIGS. 3A through 3D and FIG. 5F.
[0198] Another sample apply method is available with the cleavable
signal element and assay device according to the present invention,
in particular, the assay devices of FIGS. 3B, 3C, and 5H, each of
which includes 8 separate assay sectors 800 and is suitable to
apply a single sample to each assay sector.
[0199] In other aspects of the present invention, separate samples
may be applied to discrete sites of the disk-type assay device. In
view of this, the assay device according to present invention can
assay approximately one thousand different samples. In addition, to
increase the sensitivity of the detector, approximately one million
gold microspheres, conducting polymers, or fluorescent labels can
be applied to label assay sites.
[0200] As an embodiment, the assay device of FIG. 3D, which has at
the assay sites on the disk a plurality of cleavable signal
elements with identical capture probes conferring identical analyte
specificity, may be designed to concurrently assay 1024 patient
samples. In other words, the assay device of FIG. 3D may include
1024 cleavable signal elements on the disk. In such an embodiment,
each of the capture probes on the disk may be identical, so as to
assay for the same analyte. Capture probes at particular sites on
the disk have the same oligonucleotide sequence as those at other
sites on the disk. This application is particularly useful in mass
analysis conducted in clinical laboratories where a large number of
patient samples are analyzed at the same time for the presence or
absence of a single analyte.
[0201] Patient samples may be applied to particular assay sites on
the disk by a known method, such as ink jet printing, micropippet
arrays with disposable tips, or a combination thereof.
[0202] Alternatively, the assay device of FIG. 3D may be applied to
assay a single sample for multiple analytes by using a plurality of
diverse, cleavable signal elements specific to different analytes
for each assay device.
[0203] <Hybridization>
[0204] In a nucleic acid hybridization assay according to the
present invention, after the sample injection, rotation of the disk
is halted, and the disk is incubated in a stationary state at room
temperature for hybridization reaction between the capture probe
and the complementary target nucleic acid in the sample.
[0205] <First Wash Step>
[0206] The nucleic acid hybridization assay according to the
present invention involves first and second wash steps. After the
even application of the sample over the disk surface and an
appropriate incubation period for the hybridization, a first wash
step is necessary. For example, in nucleic acid hybridization
assays, at a lower salt concentration of the wash solution, washing
is smooth, thus reducing mismatch as between analyte (target
nucleic acid) and capture probes. In contrast, at a higher salt
concentration, washing is not smooth, thereby permitting mismatch
to occur. Adjusting the stringency of wash in nucleic acid
hybridization assays, in terms of salt concentration, is well
within the skill in the art.
[0207] In one aspect according to the present invention, the
surface of the circular, disk-type assay device may be washed by
adding a wash solution near the center of the rotating disk. The
sample solution is removed as it pushes out from the periphery of
the disk and is collected. Because of the rotation of the disk, the
wash step may be eliminated if the fluid sample is adequately
removed from the disk by centrifugal force. This centrifugal force
is strong enough to mechanically denature mismatching
oligonucleotides.
[0208] Alternatively, mismatching oligonucleotides may be removed
with application of an external electric field. Due to the nature
of its phosphate backbone which is negatively charged, the sample
oligonucleotides hybridized to the cleavable signal element with a
weak binding force can be denatured by applying an external
negative electric field.
[0209] As is shown in FIG. 9, the external electric field is
applied with an electrode plate 133, disposed directly above the
assay device, and an external voltage source 221. FIG. 9 shows an
embodiment of washing away mismatching oligonucleotides 333 from
the assay device of FIG. 6D with application of an external
electric field.
[0210] <DNA Extension Step>
[0211] When a restriction enzyme specifically responsive to a
particular sequence of a double stand is used according to the
present invention, DNA extension is needed after the hybridization
and first wash step. In DNA extension, a single-stranded
restriction probe is double-stranded using the target nucleic acid
previously hybridized to the capture probe as a primer with
addition of a DNA polymerization solution containing four dNTPs and
a polymerase.
[0212] Prior to addition of the DNA polymerization solution, it is
preferable to contact the restriction probe with a 3'-5'
exonuclease solution. As is shown in FIG. 12, a step of hydrolytic
cleaving a single-straned target nucleic acid portion 79, which is
unbound to the capture probe, by addition of the 3'-5' exonuclease
solution, may be further included before the DNA extension. The
result is readily applied to DNA extension using the target nucleic
acid previously hybridized to the capture probe as a primer.
[0213] <Cleavage Step>
[0214] After the first wash step (or DNA extension), a solution
containing a restriction enzyme or cleavage enzyme (DNAse or
nuclease) is added and distributed over the surface of the disk.
The disk is incubated in a stationary state at room temperature,
and the complementary double strand or single strand resulting from
the hybridization is specifically cleaved. This enzymetic cleavage
is maintained for a few seconds.
[0215] <Second Wash Step>
[0216] After the enzymatic cleavage step, a second wash step is
needed to remove the cleaved signal elements. In this second wash
step, differential wash stringencies are provided to permit
variation in the specificity and sensitivity of the nucleic acid
hybridization assay.
[0217] The cleaved signal elements may be removed by rotating the
assay device, with or without addition of wash solution, or by
applying an external electric field. In this aspect, four
parameters may be varied to provide differential wash stringency:
label particle size (such as metal microspheres, conducting
polymers, or fluorescent labels), rotational speed, the valency of
capture probe attachment, and the intensity of external electric
field.
[0218] Gold microspheres suitable for use in the cleavable signal
element and assay device of the present invention are readily
available in varying diameters from Aldrich Chemical Company,
British BioCell International, Nanoprobes, Inc., ranging from 1 nm
to and including 0.5-5 micrometers in diameter. Gold microspheres
of lesser or greater diameter may be formed as needed in the
present invention. At a given rotational speed, the largest gold
microspheres experience larger centrifugal and drag forces and are
removed before smaller micsrospheres with equal bonding. This
provides a basis for differential stringency of wash, and also of
quantitative analysis.
[0219] The centrifugal force affecting the gold microspheres may
also be adjusted by rotation frequency so that the loose and weakly
bound gold microspheres are removed. Only the capture probes which
have bound to a complementary molecule from the sample will
continue to bind the gold microspheres to the substrate.
[0220] Furthermore, while the above embodiments of the invention
have been described with a single metal microsphere attached to the
end of a single cleavable signal element, it should be appreciated
that when gold microspheres are used in a preferred embodiment of
the invention, thousands of cleavable signal elements may bind to
one gold microsphere, depending upon its diameter. Thus, the
stringency of the assay wash may be adjusted, at any given
rotational speed, by varying the diameter of the gold microsphere,
and by varying additionally the relative density of cleavable
signal elements to gold microspheres. Thus, if virtually all
cleavable signal elements under a certain gold sphere are connected
by complementary molecules, the binding is very strong. If the
cleavable signal elements are fixated only partially under a
certain gold microsphere, the microsphere may remain or be removed
depending on the radius of the microsphere and the frequency of
rotation. Alternatively, ferromagnetic microspheres, gold-coated
iron beads, or an iron alloy beads may be used instead of the gold
microspheres. In this case, those probes detached through cleavage
may be removed with application of a magnetic field.
[0221] <Detection Step>
[0222] After removal of cleaved signal elements through the second
wash step, the disk may be read directly. Alternatively, the disk
may be covered by an optically transparent plastic coating to
prevent the further removal of the gold microspheres through spin
coating with a polymerizable lacquer that is polymerized with
UV-light. Spin coating of compact disks is well established in the
art. The assay device disk is expected to have a shelf-life of over
ten years.
[0223] Subsequently, the disk can be scanned by a laser reader
which will detect, through reflection, the presence of a
microsphere or other reflective elements at the various spatially
predetermined locations. Based on the distance of the microsphere
from the axis of rotation of the disk and the angular distance from
an address line forming a radial line on the disk, the location of
a particular metal microsphere can be specifically determined.
Based on that specific location and the predetermined locations of
specific binding pairs as compared to a master distribution map,
the identity of the bound material can be identified. Thus, in the
foregoing manner it is possible in one fluid sample to analyze for
thousands, or even greater numbers, of analytes simultaneously.
[0224] <Additional Labeling Step>
[0225] In the case of forming the "label-attached uncleaved probe"
structure, an additional step of labeling the cleavable signal
element or uncleaved probe is included before sample injection or
after cleavage. In particular, the additional labeling before
sample injection follows the synthesis of the capture probe or
capture probe attachment to the substrate (solid support). The
additional labeling after cleavage requires a preceding sample
washing step.
[0226] The nucleic acid hybridization assay according to the
present invention involves the sample injection, hybridization,
first wash and cleavage, additional labeling reaction, and second
wash steps described above.
[0227] <Synthesis of Cleavable Signal Element and Attachment to
Glass Substrate>
[0228] FIGS. 10A through 10C show alternative embodiments of the
synthesis of cleavable signal elements and attachment to glass
substrates.
[0229] 1. Cleaning of Glass Substrate
[0230] A detergent (Alconox) is first dissolved in distilled water,
and glass substrates are sonicated in the detergent solution for
approximately 50 minutes. The glass substrates are rinsed with
distilled water to remove any sticking detergent. The rinsed glass
substrates are boiled or sonicated in a piranha solution (a 3:7
mixture of H.sub.2O.sub.2 and H.sub.2SO.sub.4) for 30 minutes. For
glass substrates coated with, for example, gold, the glass
substrates are socked in the piranha solution for washing, without
sonication. Next, the glass substrates are removed from the piranha
solution and rinsed copiously with distilled water to completely
remove the piranha solution from the glass substrate surface (Steps
10a-1, 10b-1, and 10c-1 of FIGS. 10A, 10B, and 10C).
[0231] 2. Reaction for Oligonucleotide Attachment
[0232] (a) Attachment of Oligonucleotide Using
Amine-Ologinucleotide
[0233] FIG. 10A shows the procedure of attachment of
oligonucleotide 34 on a cleaned glass substrate 24 using
amino-oligonucleotide. The cleaned glass substrate 24 is reacted
with a silanazation material, for example,
(10-carbomethoxydecyl)dimethylchlorosilane (ClSi(C
H.sub.3).sub.2--(CH.sub.2).sub.n--COOH), with a
carboxyl-convertible functional group.
[0234] For the reaction, the cleaned glass substrate 24 is dried in
a vacuum and reacted in a solution of the silanization material of
about 0.5 mL in 20 mL of toluene for about 24 hours in an argon gas
atmosphere. Next, the glass substrate is washed with toluene and
then acetone, and dried in a vacuum or by flowing gas. The glass
substrate is socked and reacted in a 1 M HCl solution at 50.degree.
C. for 5 hours, thereby resulting in a carboxyl-substituted glass
substrate (Step 10a-2). The glass substrate with the carboxyl group
is washed with acetone and dried. The glass substrate is reacted in
an aqueous solution of 0.2M N-hydroxysuccinimide (NHS), 0.2M
1-(3-dimethylaminopropyl)-3-ethylcarbodi- imide hydrochloride
(EDCl), and amine-oligonucleotide (H.sub.2N-oligo) of 0.01-0.1
mg/mL for 20 hours, followed by surface washing. The result is an
oligonucletide-attached glass substrate (Step 10a-3).
[0235] (b) Attachment of Oligonucleotide Using Amine-Oligo-Thiol
Group
[0236] FIG. 10B shows the procedure of attachment of
oligonucleotide on the cleaned glass substrate 24 using an
amine-oligo-thiol group. The cleaned glass substrate 24 is reacted
with a silanazation material, for example,
(10-carbomethoxydecyl)dimethylchlorosilane
(ClSi(CH.sub.3).sub.2--(CH.sub.2).sub.n--COOH), with a
carboxyl-convertible functional group.
[0237] For the reaction, the cleaned glass substrate 24 is dried in
a vacuum and reacted in a solution of the silanization material of
about 0.5 mL in 20 mL of toluene for about 24 hours in an argon gas
atmosphere. Next, the glass substrate is washed with toluene and
then acetone, and dried in a vacuum or by flowing gas. The glass
substrate is socked and reacted in a 1M HCl solution at 50.degree.c
for 5 hours, thereby resulting in a carboxyl-substituted glass
substrate (Step 10b-2). The glass substrate with the carboxyl group
is washed with acetone and dried. The glass substrate is reacted in
an aqueous solution of 0.2M NHS, 0.2M EDCl, and an
amine-oligonucleotid-thiol group (H.sub.2N-oligo-SH) of 0.01-0.1
mg/mL for 20 hours, followed by surface washing. The result is an
oligonucletide-attached glass substrate (Step 10b-3).
[0238] (c) Attachment of Oligonucleotide Using Biotin-Avidin
Reaction
[0239] FIG. 10C shows the procedure of attachment of
oligonucleotide on the cleaned glass substrate 24 using
biotin-avidin reaction. The cleaned glass substrate 24 is reacted
with a silanazation material, for example,
(10-carbomethoxydecyl)dimethylchlorosi lane
(ClSi(CH.sub.3).sub.2--(CH.su- b.2).sub.n--COOH), with a
carboxyl-convertible functional group.
[0240] For the reaction, the cleaned glass substrate 24 is dried in
a vacuum and reacted in a solution of the silanization material of
about 0.5 mL in 20 mL of toluene for about 24 hours in an argon gas
atmosphere. Next, the glass substrate is washed with toluene and
then acetone, and dried in a vacuum or by flowing gas. The glass
substrate is socked and reacted in a 1M HCl solution at 50.degree.
C. for 5 hours, thereby resulting in a carboxyl-substituted glass
substrate (Step 10c-2). The glass substrate with the carboxyl group
is washed with acetone and dried. The glass substrate is reacted in
an aqueous solution of 0.2M NHS, 0.2M EDCl, and avidin (denoted by
reference numeral 51) of 11 mg/mL for 20 hours, followed by surface
washing with buffer (10 mN Tris, pH 7.2).
[0241] Next, the glass substrate surface is reacted with a solution
of biotin (denoted by reference numeral 50)-oligonucleotide in 1
XTBE for 8 hours and washed with buffer (Step 10c-4).
Alternatively, the glass substrate is reacted with a solution of
biotin-oligo-SH in 1 XTBE and washed with buffer (Step 10c-5).
[0242] <Synthesis of Cleavable Signal. Element and Attachment to
Gold Substrate>
[0243] FIGS. 10D through 10H show alternative embodiments of the
synthesis of cleavable signal elements and attachment to gold
substrates.
[0244] 1. Cleaning of Glod Substrate
[0245] Gold substrates are soaked in a saturated KOH solution for 1
hour and rinsed copiously with distilled water. The gold substrates
are then soaked in sulfuric acid for approximately 2 hours,
followed by rinsing with distilled water (Steps 10d-1, 10e-1,
10g-1, and 10h.sup.-1).
[0246] 2. Reaction for Oligonucleotide Attachment
[0247] (a) Attachment of Oligonucleotide Using Thiol Group
[0248] FIG. 10D shows the procedure of attachment of the
oligonucleotide 34 on a cleaned gold substrate 22 using a thiol
group. The entire surface of the gold substrate 22 is spray-coated
with a buffer solution into which HS--(CH.sub.2).sub.n-oligo has
been dissolved, tightly sealed to prevent coating evaporation, and
reacted for approximately 5 hours. Following washing with buffer
(Step 10d-2), a buffer solution containing HS--(CH.sub.2).sub.n--OH
is applied to the gold substrate surface to space out the
oligonucleotides immobilized on the surface (Step 10d-3).
[0249] FIG. 10E shows another embodiment of the attachment of the
oligonucleotide 34 on the cleaned gold substrate 22 using the thiol
group. The entire surface of the gold substrate 22 is spray-coated
with a buffer solution containing HS--(CH.sub.2).sub.n--COOH, for
example, HS--(CH.sub.2).sub.6--COOH in 0.2M aqueous
mercaptohexanoic acid solution, tightly sealed to prevent coating
evaporation, and reacted for approximately 10 hours. Following
washing with distilled water and drying (Step 10e-2), the resulting
gold substrate is reacted in an aqueous solution of 0.2M NHS, 0.2M
EDCl, and amine-oligonucleotide-thiol (H.sub.2N-oligo-SH) of
0.01-0.1 mg/mL for 10 hours (Step 10e-3).
[0250] (b) Attachment of Oligonucleotide Using Biotin-Avidin
Reaction
[0251] FIG. 10F shows the procedure of attachment of the
oligonucleotide 34 on the cleaned gold substrate 22 using
biotin-avidin reaction. The entire surface of the gold substrate 22
is coated with a solution of biotin disulfide N-hydroxysuccinimide
(prepared by dissolving 1 g of biotin disulfide
N-hydroxysuccinimide in 200 .mu.L of dimethylformamide (DMF) and
diluting the solution with addition of 800 .mu.L of distilled
water), tightly sealed to prevent coating evaporation, and reacted
for approximately 5 hours. Following washing with distilled water
(Step 10f-2), the resulting gold substrate is spray-coated with an
avidin solution, tightly sealed, and reacted for 10 hours (Step
10f-3, reference numeral 51 denotes an avidin molecule. Next, the
glass substrate surface is reacted with a solution of biotin
(denoted by reference numeral 50)-oligonucleotide in 1 XTBE for
approximately 8 hours and washed with buffer (Step 10f-4).
[0252] FIG. 10G shows another embodiment of the attachment of the
oligonucleotide 34 on the cleaned gold substrate 22 using the
biotin-avidin reaction. The surface of the gold substrate 22 is
coated with a solution of avidin (denoted by reference numeral 51)
in 1 XTBE, tightly sealed to prevent coating evaporation, and
reacted for approximately 5 hours (Step 10g-2). Next, the glass
substrate surface is reacted with a solution of biotin (denoted by
reference numeral 50)-oligonucleotide in 1 XTBE for approximately 8
hours and washed with buffer (Step 10g-3).
[0253] (c) Attachment of Oligonucleotide-Biotin Using Thiol
Group
[0254] FIG. 10H shows the procedure of attachment of
ologonucleotide-biotin on the cleaned gold substrate 22 using the
thiol group. The entire surface of the cleaned gold substrate 22 is
spray-coated with a buffer solution into which
HS--(CH.sub.2).sub.n-oligo- -biotin has been dissolved, tightly
sealed to prevent coating evaporation, and reacted for
approximately 5 hours. Following washing with buffer (Step 10h-2),
a buffer solution containing HS--(CH.sub.2).sub.n--OH is applied to
the gold substrate surface to space out the oligonucleotides
immobilized on the surface (Step 10h-3).
[0255] <Synthesis of Cleavable Signal Element and Attachment to
Plastic Substrate>
[0256] FIGS. 10I through 10K shows alternative embodiments of the
synthesis of cleavable signal elements and attachment to plastic
substrates.
[0257] 1. Cleaning of Plastic Substrate
[0258] Gold substrates are soaked and sonicated in a Alconox
solution for about 30 minutes and rinsed copiously with distilled
water.
[0259] 2. Reaction for Oligonucleotide Attachment
[0260] FIG. 10I shows an embodiment of the attachment of the
ologonucleotides 34 on a cleaned plastic substrate 20. The surface
of the plastic substrate 20 is aminated by ammonia plasma (Step
10i-1) and completely spray-coated with a buffer solution in which
--COOH--R--COOH, where R is any amine-reactive formula, for
example, 3,3-diimethylgutaric acid
(--HOOC--CH.sub.2--C(CH.sub.3).sub.2--CH.sub.2--COOH), has been
dissolved (Step 10i-2). Preferably, R is alkane or other functional
groups. The plastic substrate 20 is tightly sealed and reacted for
approximately 10 hours. Following washing with distilled water and
drying, the plastic substrate is reacted in an aqueous solution of
0.2M NHS, 0.2M EDCl, and amine-oligonucleotide (H.sub.2N-oligo) of
0.01-0.1 mg/mL for 10 hours (Step 10i-3).
[0261] FIG. 10J shows another embodiment of the attachment of the
oligonucleotides on the cleaned plastic substrate 20. The surface
of the plastic substrate 20 is aminated by ammonia plasma (Step
10j-1) and completely spray-coated with a buffer solution in which
--COOH--R--COOH has been dissolved (Step 10j-2). The plastic
substrate 20 is tightly sealed and reacted for approximately 10
hours. Following washing with distilled water and drying, the
plastic substrate is reacted in an aqueous solution of 0.2M NHS,
0.2M EDCl, and amine-oligonucleotide-thiol (H.sub.2N-oligo-SH) of
0.01-0.1 mg/mL for 10 hours (Step 10j-3).
[0262] FIG. 10K shows still another embodiment of the attachment of
the oligonucleotides to the cleaned plastic substrate 20. The
surface of the plastic substrate 20 is aminated by ammonia plasma
(Step 10k-1) and completely spray-coated with a solution of
succinimidyl 4-maleimido butyrate (SMB), a heterobifunctional
crosslinker, in a 1:10 mixture of DMF and sodium bicarbonate buffer
(50 mM, pH 8.5) (Step 10k-2: monolayer formation). The resulting
plastic substrate is tightly sealed and reacted for approximately 3
hours. Following washing with distilled water and drying, the
plastic substrate is reacted with HS-oligonucleotide-biotin in
HEPES buffer (10 mM, pH 6.6, 5.0 mM EDTA) for 3 hours (Step
10k-3).
[0263] <Method to Increase Detector Sensitivity>
[0264] In the nucleic acid hybridization assay according to the
present invention, to increase the sensitivity of the detector
including an optical device, an electrochemical device, a mass
measurement device, or a capacitance and impedance measurement
device, after Steps 10b-3, 10c-5, 10e-3, and 10j-e, or after the
first wash step and cleavage, a metal microsphere suspension is
spread over the substrate and reacted at room temperature for about
0.5 hours to form a metal microsphere-attached cleavable signal
element or a "label-attached uncleaved probe" structure.
[0265] In the nucleic acid hybridization assay according to the
present invention, to increase the sensitivity of the detector
including an optical device, an electrochemical device, a mass
measurement device, or a capacitance and impedance measurement
device, alternatively, after Steps 10a-3, 10c-4, 10d-3, 10f-4,
10g-4, and 10i-3, or after the first wash step and cleavage, a
conducting polymer solution is spread over the substrate and
reacted at room temperature for about 5 hours to form a conducting
polymer-attached cleavable signal element or a "label-attached
uncleaved probe" structure.
[0266] In the nucleic acid hybridization assay method according to
the present invention, to increase the sensitivity of the detector
including an optical device, an electrochemical device, a mass
measurement device, or a capacitance and impedance measurement
device, alternatively, after Steps 10a-3, 10c-4, 10d-3, 10f-4,
10g-4, and 10i-3, or after the first wash step and cleavage, an
aqueous solution of fluoreceine isothiocyanate of 0.1 mg/mL, a
fluorescer, is spread over the substrate and left in a dark room
for about 5 hours to form a fluorescent-labeled cleavable signal
element or a "label-attached uncleaved probe" structure.
[0267] For label attachment, the amine group of the
oligonucleotides should extend towards the reaction solution.
[0268] In the nucleic acid hybridization assay method according to
the present invention, to increase the sensitivity of the detector
including an optical device, an electrochemical device, a mass
measurement device, or a capacitance and impedance measurement
device, alternatively, after Steps 10h4 and 10k-3 or after the
first wash step and cleavage, a suspension of streptavidin labeled
microbeads 40 or streptavidin-labeled magnetic microbeads is spread
over the substrate and reacted at room temperature for about 5
hours to form a streptavidin-labeled microbead-attached (or
magnetic microbead attached) cleavable signal element" or a
"label-attached uncleaved probe" structure.
[0269] <Gold Particles as Signal Responsive Moieties>
[0270] In preferred embodiments of the present invention, particles
that reflect or scatter light are used as signal responsive
moieties. A light reflecting and/or scattering particle is a
molecule or a material that causes incident light to be reflected
or scattered without absorbing the light energy. Such light
reflecting and/or scattering particles include, for example, metal
particles, colloidal metal such as colloidal gold, colloidal
non-metal labels such as colloidal selenium, dyed plastic particles
made of latex, polystyrene, polymethylacrylate, polycarbonate or
similar materials.
[0271] The size of such particles ranges from 1 nm to 10 .mu.m,
preferably from 500 nm to 5 .mu.m, and most preferably from 1 to 3
.mu.m. The larger the particle, the greater the light scattering
effect. Metal microspheres 1 nm to 10 .mu.m in diameter, preferably
0.5-5 .mu.m, most preferably 1-3 .mu.m in diameter, are presently
preferred in the light reflecting/light scattering embodiment of
the present invention. Metal microspheres provide a convenient
signal responsive moiety for detection of the presence of an
uncleaved signal element bound to the disk. Typical materials are
gold, silver, nickel, chromium, platinum, copper, and the like, or
alloys thereof, with gold being most preferred. The metal
microspheres may be solid metal or may be formed of plastic, or
glass beads or the like, upon which a coating of metal has been
deposited. Metal microspheres may also be alloys.
[0272] Gold spheres suitable for use in the cleavable reflective
signal element and assay device of the present invention are
readily available in varying diameters from Aldrich Chemical
Company, British BioCell International, Nanoprobes, Inc., and
others, ranging from 1 nm to and including 0.5 .mu.m (500 nm)-5
.mu.m in diameter. It is within the skill in the art to create gold
microspheres of lesser or greater diameter as needed in the present
invention. Much smaller spheres can be used advantageously when
reading is performed with optical microscopy, UV-light, electron
beam or scanning probe microscopy. Smaller spheres are preferred
because more cleavable signal elements can be discriminated in a
given area of a substrate.
[0273] Although spherical particles are preferred, non-spherical
particles are also useful for some embodiments. In biological
applications, the signal responsive moiety--particularly gold or
latex microspheres--will preferably be coated with detergents or
derivatized so that they have a surface charge. This is done to
prevent the attachment of these particles nonspecifically with
surfaces or with each other.
[0274] The preferred gold microspheres bind directly to the thiol
group of the end of the cleavable signal element, yielding a very
strong bond.
[0275] Furthermore, while the above embodiments of the invention
have been described with a single metal microsphere attached to the
end of a single cleavable signal element, it should be appreciated
that when gold is used in a preferred embodiment of the invention,
thousands of cleavable signal elements may bind one gold
microsphere, depending upon its diameter. It is estimated that one
sphere of 1-3 .mu.m may be bound by approximately 1,000-10,000
cleavable signal elements.
[0276] As a result, the stringency of the assay wash may be
adjusted to give higher assay reliability, at any given rotational
speed, by varying not only the diameter of the gold sphere, but
also the relative density of cleavable signal elements to gold
microspheres.
[0277] Accordingly, if virtually all captures probes under a
certain gold microsphere are connected by complementary molecules,
the binding is very strong. If the capture probes are fixated only
partially under a certain gold microsphere, the microsphere may
remain or be removed depending on the radius of the microsphere and
the frequency of the rotation.
[0278] In another preferred embodiment of the present invention,
since the metal microsphere increases conductivity, it can improve
the sensitivity of a detector constructed of the capacitance and
impedance measurement device.
[0279] In still another preferred embodiment of the present
invention, conducting polymers or fluorescent labels may be used
instead of the metal microsphere. The conducting polymer or
fluorescent label acts as a light reflecting (light diverging) and
scattering particle or a conductivity-increasing particle, so it
can improve detection sensitivity when used with a photodetector
(fluorescent detector) or a detector constructed of the capacitance
and impedance measurement device.
[0280] <Other Light-Responsive Signal Responsive
Moieties>
[0281] In other embodiments of the cleavable signal element and
assay device of the present invention, a light-absorbing rather
than light-reflective material can be used as a signal responsive
moiety. The approach is analogous to that used in recordable
compact disks.
[0282] Although similar in concept and compatible with CD readers,
information is recorded differently in a recordable compact disk
(CD-R) as compared to the encoding of information in a standard CD.
In CD-R, the data layer is separate from the polycarbonate
substrate. The polycarbonate substrate instead has impressed upon
it a continuous spiral groove as a reference alignment guide for
the incident laser. An organic dye is used to form the data layer.
Although cyanine was the first organic dye used for these disks, a
metal-stabilized cyanine compound is generally used instead of
"raw" cyanine. An alternative material is phthalocyanine. One such
metallophthalocyanine compound is described in U.S. Pat. No.
5,580,696.
[0283] In CD-R, the organic dye layer is sandwiched between the
polycarbonate substrate and the metalized reflective layer, usually
24 carat gold, but alternatively silver, of the media. Information
is recorded by a recording laser of appropriate preselected
wavelength that selectively melts "pits" into the dye layer, it
simply melts it slightly, causing it to become non-translucent so
that the reading laser beam is refracted rather than reflected back
to the reader's sensors. As in a standard CD, a lacquer coating
protects the information layers.
[0284] A greater number of light-absorbing dyes may be used in this
embodiment of the present invention than may be used in CD-R.
Light-absorbing dyes are any compounds that absorb energy from the
electromagnetic spectrum, ideally at wavelength(s) that correspond
to the wavelength(s) of the light source. As is known in the art,
dyes generally consist of conjugated heterocyclic structures,
exemplified by the following classes of dyes: azo dyes, diazo dyes,
triazine dyes, food colorings or biological stains. Specific dyes
include: Coomasie Brilliant Blue R-250 Dye (Biorad Labs, Richmond,
Calif.); Reactive Red 2 (Sigma Chemical Company, St. Lois, Mo.),
bromophenol blue (Sigma); xylene cyanol (Sigma); and
phenolphthalein (Sigma). The Sigma-Aldrich Handbook of Stains, Dyes
and Indicators by Floyd J. Green, published by Aldrich Chemical
Company, Inc., (Milwaukee, Wis.) provides a wealth of data for
other dyes. With these data, dyes with the appropriate light
absorption properties can be selected to coincide with the
wavelengths emitted by the light source.
[0285] In other embodiments, the signal responsive moiety may be a
fluorescer, such as fluorescein, propidium iodide or phycoerythrin,
or a chemiluminescer, such as luciferin, which responds to incident
light, or an indicator enzyme that cleaves soluble fluorescent
substrates into insoluble form. Other fluorescent dyes useful in
this embodiment include texas red, rhodamine, green fluorescent
protein, and the like. Fluorescent dyes will prove particularly
useful when blue lasers become widely available.
[0286] The present invention preferentially employs a circular
assay device as the substrate for the patterned deposition of
light-reflective, light-scattering, light-absorptive, or
fluorescent cleavable signal elements. In a preferred embodiment,
the assay device is compatible with existing optical disk readers,
such as a compact disk (CD) reader or a digital versatile disk
(DVD) reader, and is therefore preferentially a disk of about 120
mm in diameter and about 1.2 mm in thickness. It will be
appreciated, however, that the cleavable signal elements of the
present invention may be deposited in spatially-addressable
patterns on substrates that are not circular but rectangular.
[0287] The maximum number of cleavable signal elements that can be
spatially discriminated on an optical disk is a function of the
wavelength and the numerical aperture of the objective lens. One
known way to increase memory capacity in all sorts of optical
memory disk, such as CD-ROMs, WORM (Write Once Read Many) disks,
and magneto-optical disks, is to decrease the wavelength of the
light emitted by the diode laser which illuminates the data tracks
of the optical memory disks. Smaller wavelength permits
discrimination of smaller data spots on the disk, that is, higher
resolution, and thus enhanced data densities. Current CD-ROMs
employ a laser with a wavelength of 780 nanometers (nm). Current
DVD readers employ a laser with a wavelength between 635 and 650
nm. New diode lasers which emit, for example, blue light (around
481 nm) would increase the number of signal elements that could be
spatially addressed on a single assay device disk of the present
invention. Another way to achieve blue radiation is use of a second
harmonic generator (SHG) that achieves frequency doubling of
infrared laser by non-linear optical material.
[0288] Current CD-ROM readers employ both reflection reading and
transmission reading. Both data access methods are compatible with
the present invention. Gold particles are especially suitable for
use as a signal responsive moiety for reflection type CD-ROM
readers. Light-absorbing dyes are more suitable for transmission
type readers such as the ones discussed in U.S. Pat. No.
4,037,257.
[0289] <Other Signal Responsive Moieties>
[0290] It will be apparent to those skilled in the art that signal
responsive moieties suitable for adaptation to the cleavable signal
element of the present invention are not limited to
light-reflecting or light-absorbing metal particles or dyes.
Suitable signal responsive moieties include, but are not limited
to, any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
In preferred embodiments, suitable signal responsive moieties
include calorimetric labels such as colloidal gold or colored glass
or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads,
biotin-bound beads with labeled streptavidin conjugate, magnetic
beads (e.g., DynabeadS.TM.), radiolabels (e.g., .sup.3H, .sup.125I,
.sup.35S, .sup.14C, or .sup.32P), and enzymes (e.g., horse radish
peroxidase (HRP), alkaline phosphatase, etc.).
[0291] It will be apparent to those skilled in the art that
numerous variations of signal responsive moieties may be adapted to
the cleavable signal elements of the present invention. A number of
patents, for example, provide ari extensive teaching of a variety
of techniques for producing detectible signals in biological
assays. Such signal responsive moieties are generally suitable for
use in some embodiments of the present invention. As a non-limiting
illustration, the following is a list of U.S. patents teach the
several signal responsive moieties suitable for embodiments of the
present invention: U.S. Pat. No. 3,646,346, radioactive signal
generating means; U.S. Pat. Nos. 3,654,090, 3,791,932 and
3,817,838, enzyme-linked signal generating means; U.S. Pat. No.
3,996,345, fluorescer-quencher related signal generating means;
U.S. Pat. No. 4,062,733, fluorescer or enzyme signal generating
means; U.S. Pat. No. 4,104,029, chemiluminescent signal generating
means; U.S. Pat. No. 4,160,645, non-enzymatic catalyst generating
means; U.S. Pat. No. 4,233,402, enzyme pair signal generating
means; U.S. Pat. No. 4,287,300, enzyme anionic charge label. All
above-cited U.S. patents are incorporated herein by reference for
all purposes.
[0292] Other signal generating means are also known in the art, for
example, U.S. Pat. Nos. 5,021,236 and 4,472,509, both incorporated
herein by reference for all purposes. A metal chelate complex may
be employed to attach signal generating means to the cleavable
signal elements. In other embodiments, magnetic spheres may be used
in place of reflective spheres, and magnetic poles may be
vertically aligned by treating the disk with a magnetic field that
is of sufficient strength. Since the empty sites will not have any
magnetic material present, the presence or absence of a target
nucleic acid in the test sample can be identified. The location of
the uncleaved signal element can be detected using an optomagnetic
sensor widely used in existing optomagnetic disks based on the Kerr
effect or a magneto resistance (MR) sensor.
[0293] Paramagnetic ions might be used as a signal generating
means, for example, ions such as chromium (III), manganese (II),
iron (III), iron (II), cobalt (II), nickel (II), copper (II),
neodymium (III), samarium (III), ytterbium (III), gadolinium (III),
vanadium (II), terbium (III), dysprosium (III), holmium (III) and
erbium (III), with gadolinium being particularly preferred. Ions
useful in other contexts, such as X-ray imaging, include but are
not limited to lanthanum (III), gold (III), lead (II), and
especially bismuth (III).
[0294] Means of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
light. Enzymatic labels are typically detected by providing the
enzyme with a substrate and detecting the reaction product produced
by the action of the enzyme on the substrate, and calorimetric
labels are detected by simply visualizing the colored label.
Colloidal gold label can be detected by measuring scattered light.
A preferred non-reflective signal generating means is biotin, which
may be detected using an avidin or streptavidin compound. The use
of such labels is well known to those of skill in the art and is
described, for example, in U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each
incorporated herein by reference for all purposes.
[0295] <Patterned Deposition of Cleavable Signal Elements on
Plastic Substrate>
[0296] A photoresist may also profitably be used to pattern the
deposition of cleavable signal elements. The resist is partially
depolymerized by incident laser light during fabrication and can be
dissolved from these areas. The exposed or metalized portion of the
plastic substrate is treated chemically, for example, aminated by
ammonia plasma. After the resist is removed, the cleavable signal
elements are attached to the substrate. The use of photoresists for
the patterning of master disks is well known in the compact disk
fabrication arts.
[0297] Alternatively, instead of using a resist, a solid mask
containing small holes can be used during ammonia plasma treatment.
Holes have a diameter of about 1 to 3 micrometers. The holes are
located circularly in the mask, forming a spiral track or a pattern
that is a combination of spiral and circular paths. The mask can be
metal or plastic. Several metals, such as aluminum, nickel or gold
can be used. Polycarbonate is a preferred plastic, because it will
retain shape well. Plastics are reactive with the ammonia plasma,
however, and a preferred method for using plastic masks therefore
involves depositing a metal layer on the plastic, by evaporation,
sputtering, or other methods known in the art. Holes may be made in
the mask by laser. Those with skill in the art will appreciate that
it is possible to create 1000 1 .mu.-sized holes in one second in a
thin metal or plastic plate. Alternatively, the holes can be etched
by using conventional methods known in the semiconductor industry.
In the mask approach to patterning the deposition of cleavable
signal elements, the mask is pressed against the substrate and
subjected to amination by ammonia plasma. The mask may be used
repeatedly.
[0298] <SNP Detection in Nucleic Acid Hybridization Assay using
Cleavable Signal Element>
[0299] In a nucleic acid hybridization assay according to the
present invention, the capture probe of the cleavable signal
element is oligonucleotides designed to hybridized to a
complementary sequence of a target nucleic acid to be detected in
the sample. For many applications of this methodology,
cross-reactivity with sample oligonucleotides having even a single
mismatched nucleotide should be minimized. In particular, nucleic
acid hybridization assays adapted to use the cleavable signal
element of the present invention for detection of point mutations,
as, e.g., for detection of point mutations in the BRCA1 and BRCA2
genes that predispose to breast and ovarian cancers, must be able
to discriminate between nucleic acid samples containing a single
mismatched nucleotide, i.e., must be able to detect SNP.
[0300] The longer the oligonucleotides of the capture probe--and
thus the longer the sequence that is complementary between the
oligonucleotides and the nucleic acid sample--the greater the
possibility of erroneously recognizing a mismatched sample, since
the strength of hybridization, even given the presence of a
mismatch, will be reasonably high.
[0301] Thus, one way to reduce erroneous recognition of mismatched
nucleic acid sequences is to reduce the length of the
oligonucleotides. Specificity is increased by shortening the length
of the oligonucleotides to 15-20-mers. In this case, the mismatched
oligonucleotides would use fewer nucleotides for pairing and will
form highly unstable binding at room temperature. This unstable
binding is denatured during the first wash step and removed.
However, multiple SNP detections at a plurality of assay sites are
required for diagnosing a certain disease.
[0302] FIGS. 11A through 11B show alternative embodiments of an
assay device using the cleavable signal element according to the
present invention capable of both single nucleotide polymorphism
(SNP) detection and gene expression profile determination for
diagnosis. Reference numeral 61 denotes a center void of the
substrate (disk) 70. An assay sector with shorter capture probes
for SNP detection and an assay sector with longer capture probes
(cDNA) for gene expression profile analysis are arranged separate
on the substrate 70. The assay devices shown in FIGS. 11A through
11B can be modified in a variety of ways and forms according to the
arrangements shown in FIGS. 3A through 3D, to concurrently measure
SNP and expression profile.
[0303] FIG. 11A shows that the assay sectors for SNP detection and
expression profile analysis are arranged separate in an angular
direction. FIG. 11B shows that the assay sectors for SNP detection
and expression profile analysis are arranged separate in a radial
direction. The concurrent determination of the DNP and expression
profile doubles diagnostic reliability and reduces assay sites by
appropriate combination of assay sites.
BEST MODE FOR CARRYING OUT THE INVENTION
[0304] The present invention will be described in greater detail
with reference to the following examples. The following examples
are for illustrative purposes and are not intended to limit the
scope of the invention.
EXAMPLE 1
Detection of HIV-1
[0305] HIV-1 proviral DNA from clinical samples is amplified as
follows, essentially as described in U.S. Pat. No. 5,599,662,
incorporated herein by reference.
[0306] Peripheral blood monocytes are isolated by standard
Ficoll-Hypaque density gradient methods. Following isolation of the
cells, the DNA is extracted as described in Butcher and Spadoro,
Clin. Immunol. Newsletter 12:73-76 (1992), incorporated herein by
reference. Polymerase chain reaction (PCR) is performed in a 100
.mu.L reaction volume, of which 50 .mu.L is contributed by the
sample. The PCR contained the following reagents at the following
initial concentrations:
1 .10 mM Tris-HCl (pH 8.4) 50 mM KCl 200 .mu.M each dATP, dCTP,
dGTP, and dUTP 25 pmoles of Primer 1 (sequence: 5'-TGA GAC ACC AGG
AAT TAG ATA TCA GTA CAA TGT-3') 25 pmoles of Primer 2 (sequence:
5'-CTA AAT CAG ATC CTA CAT ATA AGT CAT CCA TGT-3') 3.0 mM MgC.sub.2
10% glycerol 2.0 units of Taq DNA polymerase (Perkin-Elmer) 2.0
units UNG (Perkin-Elmer)
[0307] Amplification is carried out in a TC9600 DNA Thermal Cycler
(Perkin Elmer, Norwal, Conn.) using the following temperature
profile: (1) pre-incubation--50.degree. C. for 2 minutes; (2)
initial cycle--denature at 94.degree. C. for 30 seconds, anneal at
50.degree. C. 30 seconds, extend at 72.degree. C. for 30 seconds;
(3) cycles 2 to 4--denature at 94.degree. C. for 30 seconds, anneal
for 30 seconds, extend at 72.degree. C. for 30 seconds, with the
annealing temperature increasing in 2.degree. C. increments (from
52.degree. C. to 58C); (4) cycles 5 to 39--denature at 90.degree.
C. for 30 seconds, anneal at 60.degree. C. for 30 seconds, extend
at 72.degree. C. for 30 seconds.
[0308] Following the temperature cycling, the reaction mixture is
heated to 90.degree. C. for 2 minutes and diluted to 1 mL.
Alternatively, the sample is stored at -20.degree. C., and after
thawing, heated to 90.degree. C. for 2 minutes then diluted to 1
mL.
[0309] The cleavable signal elements are attached in a uniform
density to a derivatized 120-mm polycarbonate disk substrate, as
described above.
2 First capture probe 5'-TAG ATA TCA GTA CAA-3' portion: Second
capture probe 5'-TAT TCA GTA GGT ACA-3' portion: First restriction
probe 5'-CCCGGG-3' portion: Second restriction probe 5'-CCCGGG-3'
portion:
[0310] A suspension of gold microspheres, 1-3 .mu.m in diameter, is
added dropwise to the disk, which is gently rotated to distribute
the gold particles. Gold particles are added until the cleavable
signal elements are saturated with the gold particles. Cleavable
signal elements labeled with the gold particles at its end are
attached in a uniform density to a derivatized 120-mm polycarbonate
disk substrate, as described above.
[0311] Sample is applied at room temperature dropwise near the
center of the stationary assay device, and the assay device is
rotated. Rotation is halted after the sample reaches the outer edge
of the disk, and the disk is incubated in a stationary state at
room temperature for 3-5 minutes (Hybridization reaction).
[0312] One mL of buffer is added dropwise as a washing solution
while the disk is rotated, to distribute the buffer by disk
rotation. The disk was incubated in a stationary state for 1-2
minutes, then 5 ml of buffer is added dropwise during vigorous
rotation of the disk to wash the disk, with or without the
application of an external electric field vertically through the
disk (First Wash Step).
[0313] About 0.1 L of a DNA polymerase solution (e.g., PCR Core
Systems I, Promega Corporation) containing a mixed solution of the
four dNTPs listed above and a DNA polymerase is added dropwise
while the disk is rotated, to distribute the DNA polymerase
solution. The disk is incubated in a stationary state for 1-2
minutes (DNA extension).
[0314] A restriction enzyme solution (e.g., sma 1, Promega
Corporation) that recognizes the sequence CCCGGG of the restriction
probe is added dropwise and distributed by disk rotation. The
restriction enzyme cleaves between C and G of the restriction probe
sequence. The disk is incubated in a stationary state for 1-2
minutes (Cleavage Step).
[0315] Five mL of buffer was added dropwise during vigorous
rotation of the disk with or without the application of an external
electric field (Second Wash Step). An appropriate restriction
solution has a reaction temperature of 37.degree. C. and to contain
10 mM Tris-HCl (pH7.4), 300 mM KCl, 0.1 mM EDTA (Ethylene Diamine
Tetra Acetic acid), 1 mM DTT (DiThioTreitol), 0.5 mg/mL BSA (Bovine
Serum Albumine), and 50% glycerol.
[0316] The disk is dried, then read directly in a detector
programmed to assay each predetermined site upon which cleavable
signal elements are deposited, which includes an optical device, an
electrochemical device, a mass measurement device, or a capacitance
and impedance measurement device.
[0317] The diagnostic data and a prescription are displayed on a
computer monitor, the computer automatically or manually accesses
the Internet to transmit the diagnostic data to a specialist at a
remote location through the Internet. The patient waits for a
prescription from the specialist.
EXAMPLE 2
Detection of HIV-1
[0318] HIV-1 proviral DNA from clinical samples is amplified as
follows, essentially as described in U.S. Pat. No. 5,599,662,
incorporated herein by reference.
[0319] Peripheral blood monocytes is isolated by standard
Ficoll-Hypaque density gradient methods. Following isolation of the
cells, the DNA is extracted as described in Butcher and Spadoro,
Clin. Immunol. Newsletter 12:73-76 (1992), incorporated herein by
reference. PCR was performed in a 100 .mu.L reaction volume, of
which 50 .mu.L is contributed by the sample. The PCR contains the
following reagents at the following initial concentrations:
3 10 mM Tris-HCl (pH 8.4) 50 mM KCl 200 .mu.M each dATP, dCTP,
dGTP, and dUTP 25 pmoles of Primer 1 (sequence: 5'-TGA GAC ACC AGG
AAT TAG ATA TCA GTA CAA TGT-3') 25 pmoles of Primer 2 (sequence:
5'-CTA AAT CAG ATC CTA CAT ATA AGT CAT CCA TGT-3') 3.0 mM MgC.sub.2
10% glycerol 2.0 units of Taq DNA polymerase (Perkin-Elmer) 2.0
units UNG (Perkin-Elmer)
[0320] Amplification is carried out in a TC9600 DNA Thermal Cycler
(Perkin Elmer, Norwal, Conn.) using the following temperature
profile: (1) pre-incubation--50.degree. C. for 2 minutes; (2)
initial cycle--denature at 94.degree. C. for 30 seconds, anneal at
50.degree. C. 30 seconds, extend at 72.degree. C. for 30 seconds;
(3) cycles 2 to 4--denature at 94.degree. C. for 30 seconds, anneal
for 30 seconds, extend at 72.degree. C. for 30 seconds, with the
annealing temperature increasing in 2.degree. C. increments (from
52.degree. C. to 58C); (4) cycles 5 to 39--denature at 90.degree.
C. for 30 seconds, anneal at 60.degree. C. for 30 seconds, extend
at 72.degree. C. for 30 seconds.
[0321] Following the temperature cycling, the reaction mixture is
heated to 90.degree. C. for 2 minutes and diluted to 1 mL.
Alternatively, the sample is stored at -20.degree. C., and after
thawing, heated to 90.degree. C. for 2 minutes then diluted to 1
mL.
[0322] Following cleaning a polycarbonate disk substrate, the
surface of the disk substrate is aminated by ammonia plasma and
completely spray-coated with a solution of succinimidyl 4-maleimido
butyrate (SMB), a heterobifunctional crosslinker, in a 1:10 mixture
of DMF and sodium bicarbonate buffer (50 mM, pH 8.5). The resulting
polycarbonate substrate is tightly sealed and reacted for
approximately 3 hours. Following washing with distilled water and
drying, a HEPES buffer (10 mM, pH 6.6, 5.0 mM EDTA) containing
HS-oligonucleotide-biotin is applied to the derivatized surface of
the polycarbonate substrate to attach the HS-oligonucleotide-biotin
in a uniform density, thereby constructing an assay device. The
cleavable signal elements attached have the following
sequences:
4 First capture probe 5'-TAG ATA TCA GTA CAA-3' (oligonucleotide)
portion: Second capture probe 5'-TAT TCA GTA GGT ACA-3'
portion:
[0323] Sample is applied dropwise near the center of the stationary
assay device, and the assay device is rotated. Rotation is halted
after the sample reaches the outer edge of the disk, and the disk
is incubated in a stationary state at room temperature for 3-5
minutes (Hybridization reaction).
[0324] One mL of buffer is added dropwise as a washing solution
while the disk is rotated, to distribute the buffer by disk
rotation. The disk is incubated in a stationary state for 1-2
minutes, then 5 ml of buffer is added dropwise during vigorous
rotation of the disk to wash the disk, with or without the
application of an external electric field vertically through the
disk (First Wash Step).
[0325] A DNAse or nuclease solution is added dropwise and
distributed by disk rotation. The disk is incubated in a stationary
state for 1-2 minutes (Cleavage Step).
[0326] Following buffer addition, disk rotation, and a simple
washing with the application of an external field, a suspension of
streptoavidin-labeld gold microspheres is added drowpwise to the
disk surface, and the disk is gently rotated to evenly distribute
the gold particles (label-attached uncleaved probe structure
formation), thereby resulting in a biotin-avidin binding structure.
Next, distilled water is added dropwise during vigorous rotation of
the disk to wash the disk, with or without the application of an
external electric field (Second Wash Step).
[0327] The disk is dried, then read directly in a detector
programmed to assay each predetermined site upon which cleavable
signal elements are deposited, which includes an optical device, an
electrochemical device, a mass measurement device, or a capacitance
and impedance measurement device.
[0328] The diagnostic data and a prescription are displayed on a
computer monitor, the computer automatically or manually accesses
the Internet to transmit the diagnostic data to a specialist at a
remote location through the Internet. The patient waits for a
prescription from the specialist.
EXPERIMENTAL EXAMPLE 1
Optical Measurement
[0329] Following the hybridization reaction, first wash step,
cleavage step, and second wash step according to Example 2, whether
hybridization to a target nucleic acid had occurred or not was
determined by atomic force microscopy (AFM). As a result, the
topography images by AFM are shown in FIGS. 13A and 13B.
[0330] FIG. 13A is a topography image taken after hybridization of
biotin-attached cleavable signal elements to an oligonucleotide
sample of a complementary sequence, in which after the
hydbridization, the substrate was reacted with a mung bean-derived
nuclease, washed, and additionally labeled with streptavidin-coated
40-nm metal microspheres. Since the cleavable signal elements are
double-stranded, the cleavable signal elements are not cleaved by
the nuclease, and the biotin-attached cleavable signal elements
remain on the substrate and form a "label-attached uncleaved probe"
structure by additionally contacting the streptoavidin-coated metal
micrispheres, thereby increasing optical selectivity. Due to the
streptoavidin-to-biotin coupling, the sensitivity of the detector
is increased.
[0331] FIG. 13B is a topography image taken after reacting the
biotin-attached cleavable signal elements with an oligonucleotide
sample of a non-complementary sequence, in which after the
reaction, the substrate was reacted with a mung bean-derived
nuclease, washed, and additionally labeled with streptavidin-coated
40-nm metal microspheres. Since the cleavable signal elements
remain as single strands, the cleavable signal elements are cleaved
by the nuclease, and the biotin-attached cleavable signal elements
are removed from the substrate. As a result, even after
additionally contacting the streptoavidin-coated metal
micrispheres, the cleavable signal elements do not form a
"label-attached uncleaved probe" structure.
[0332] Apparently, the substrate is mostly covered with the metal
microspheres in FIG. 13A, whereas few metal microspheres are shown
in FIG. 13B. Differential signals from the metal microspheres are
provided to the detector.
EXPERIMENTAL EXAMPLE 2
Impedance Measurement
[0333] Following the hybridization reaction, first wash step,
cleavage step, and second wash step according to Example 2, whether
hybridization to a target nucleic acid had occurred or not was
determined by measuring the impedance characteristics with respect
to frequency. The result is shown in FIG. 14.
[0334] As can be inferred form FIG. 13, differential impedance
signals between the uncleaved signal element (low impedance) and
the cleaved signal element (high impedance) are provided to the
detector.
[0335] While this invention has been particularly shown and
described with reference to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims.
INDUSTRIAL APPLICABILITY
[0336] As described above, the present invention provides a
cleavage technique specifically responsive to a complementary
double strand or single strand of nucleic acids, a nucleic acid
hybridization assay method and device using the cleavable
technique, and a diagnostic method and system capable of more
accurately diagnosing many kinds of diseases through single
nucleotide polymorphism (SNP) detection and expression profile
determination that can be concurrently determined. In a preferred
embodiment according to the present invention, the diagnostic
system using the nucleic acid hybridization assay method and device
based on the cleavage technique specifically responsive to the
complementary double strand or single strand of nucleic acids can
be modified for detection with standard laser-based detection
systems, including CD-ROM readers and DVD readers, which enables
self-diagnosis by patients at home without the need to go to a
hospital. In addition, the present invention provides an assay
device and method for detecting analytes using the nucleic acid
hybridization assay device according to the present invention. This
analyte assay device and method are useful in assaying for a number
of discrete analytes with a test sample and a single analyte with
multiple samples. The present invention also provides a remote
diagnostic system providing convenience to both patients and
doctors, in which information read from the assay device is
digitized as software and transmitted to and received by patient
and doctor through an existing communication network, such as the
Internet.
* * * * *