U.S. patent application number 11/549064 was filed with the patent office on 2007-07-19 for magnetic resonance system and method to detect and confirm analytes.
This patent application is currently assigned to Menon & Associates, Inc.. Invention is credited to Steven C. Chan, Suresh M. Menon, David E. Newman.
Application Number | 20070166730 11/549064 |
Document ID | / |
Family ID | 38263633 |
Filed Date | 2007-07-19 |
United States Patent
Application |
20070166730 |
Kind Code |
A1 |
Menon; Suresh M. ; et
al. |
July 19, 2007 |
Magnetic resonance system and method to detect and confirm
analytes
Abstract
A system and method are provided to detect target analytes based
on magnetic resonance measurements. Magnetic structures produce
distinct magnetic field regions having a size comparable to the
analyte. When the analyte is bound in those regions, magnetic
resonance signals from the sample are changed, leading to detection
of the analyte.
Inventors: |
Menon; Suresh M.; (San
Diego, CA) ; Newman; David E.; (Fallbrook, CA)
; Chan; Steven C.; (San Diego, CA) |
Correspondence
Address: |
PROCOPIO, CORY, HARGREAVES & SAVITCH LLP
530 B STREET, SUITE 2100
SAN DIEGO
CA
92101
US
|
Assignee: |
Menon & Associates,
Inc.
|
Family ID: |
38263633 |
Appl. No.: |
11/549064 |
Filed: |
October 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60759788 |
Jan 19, 2006 |
|
|
|
60786033 |
Mar 27, 2006 |
|
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Current U.S.
Class: |
435/6.12 ;
435/287.2; 435/6.1; 977/924 |
Current CPC
Class: |
G01R 33/12 20130101;
G01N 24/084 20130101; G01R 33/307 20130101; G01R 33/1276 20130101;
Y10T 436/24 20150115; G01R 33/1269 20130101; B82Y 25/00 20130101;
G01R 33/465 20130101; G01R 33/448 20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 977/924 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 3/00 20060101 C12M003/00 |
Claims
1. A method, for detecting an analyte, comprising the steps of:
attaching nanoparticles to the analyte, thereby forming
nanoparticle-analyte complexes; applying a magnetic field to the
complexes in a known liquid, thereby magnetizing the nanoparticles;
then, allowing the magnetic field to exert forces on the
nanoparticles and allowing the nanoparticles to exert magnetic
forces on each other; then, allowing the complexes to undergo
motions responsive to the magnetic forces; allowing the complexes
to undergo interactions, which interactions are enhanced by said
motions; exciting magnetic resonance signals from a sample
comprising the complexes and the known liquid; determining the T2
of the sample by analyzing the magnetic resonance signals; and
determining whether the analyte is present in the sample by
analyzing the determined T2 and a predetermined value.
2. The method of claim 1 wherein the analytes are selected from the
group of molecules, molecular fragments, molecular complexes,
viruses, cells, and bacteria.
3. The method of claim 1 wherein the interactions are molecular
bonding reactions.
4. The method of claim 1 wherein the magnetic field is
substantially uniform in a region occupied by the complexes.
5. The method of claim 1 wherein the magnetic field is
substantially non-uniform in a region occupied by the complexes,
and wherein the magnetic field is strongest in a subvolume of said
region.
6. The method of claim 5 wherein the complexes have an initial
concentration in the fluid medium; and wherein the magnetic field,
in cooperation with the nanoparticles, urges the complexes to move
toward the subvolume, thereby causing the complexes to have a
concentration in the subvolume which is higher than the initial
concentration; and wherein the interactions are accelerated due to
the higher concentration of the complexes in the subvolume.
7. The method of claim 1 wherein pairs of the nanoparticles exert
mutual magnetic forces on each other; and wherein the mutual
magnetic forces urge the complexes to approach each other in
alignment with the magnetic field, thereby causing the interactions
to occur while the reactants are positioned in alignment with the
magnetic field.
8. The method of claim 1 wherein the interactions produce a product
comprising nanoparticles and analyte; and wherein the motions cause
a particular form of the product to be produced.
9. The method of claim 8 wherein the particular form of the product
is an extended and substantially linear chain.
10. The method of claim 1 wherein the interactions produce a
product comprising nanoparticles and analyte; and wherein the
motions suppress production of a particular form of the
product.
11. The method of claim 10 wherein the particular form of the
product is an agglomerate comprising a distributed accumulation of
nanoparticles and analyte.
12. The method of claim 1 wherein the presence of the analyte is
indicated by the determined T2 being greater than the predetermined
value.
13. The method of claim 1 wherein the predetermined value is the T2
of the combination of the known liquid with the nanoparticles.
14. The method of claim 1 1 wherein the presence of the analyte is
indicated by the determined T2 being less than the predetermined
value.
15. The method of claim 1 further comprising measuring T2 of the
known liquid and the nanoparticles.
16. The method of claim 1 wherein the T2 parameter is measured
before the interactions undergo and again after the interactions
undergo; and comparing the T2 values so obtained.
17. The method of claim 16 wherein an increase in T2 indicates that
extended substantially linear chain structures have formed; and
wherein a decrease in T2 indicates that distributed agglomerates
have formed.
18. A method to detect DNA in a sample, comprising the steps of:
binding nanoparticles to DNA probes having a sequence complementary
to the DNA to create nanoparticle probes; mixing the sample with
the nanoparticle probes in a fluid to form a fluid sample and to
promote binding between the nanoparticle probes and DNA; measuring
the magnetic resonance parameter T2 of the fluid sample; applying a
substantially non-uniform magnetic field to the fluid sample,
thereby magnetizing the nanoparticles, drawing the nanoparticles
into a sub-volume of the fluid sample where the magnetic field is
strongest; forming chain-like structures of nanoparticle probes and
DNA in the sub-volume; re-mixing the fluid sample; measuring the
magnetic resonance parameter T2 of the fluid sample; and
determining whether the DNA is present by comparing the T2
measurements.
19. A method to detect DNA in a sample, comprising the steps of:
binding nanoparticles to DNA probes having a sequence complementary
to the DNA to create nanoparticle probes; mixing the sample with
the nanoparticle probes in a fluid to form a fluid sample and to
promote binding between the nanoparticle probes and DNA; measuring
the magnetic resonance parameter T2 of the fluid sample; applying a
substantially uniform magnetic field to the fluid sample, thereby
magnetizing the nanoparticles, forming chain-like structures of
nanoparticle probes and DNA; measuring the magnetic resonance
parameter T2 of the fluid sample; and determining whether the DNA
is present by comparing the T2 measurements.
20. A method, for detecting an analyte, comprising the steps of:
binding analytes to paramagnetic nanoparticles to form
nanoparticle-analyte complexes; placing the complexes in a known
liquid in a non-uniform magnetic field thereby concentrating the
complexes in a region of the non-uniform magnetic field having the
strongest field; wherein the concentrating of the complexes
enhances the process of the complexes binding to each other;
exciting magnetic resonance signals from a sample comprising the
complexes and the known liquid; and determining whether the analyte
is present in the sample by analyzing one or more of the magnetic
resonance signals.
21. A method, for detecting an analyte, comprising the steps of:
binding analytes to paramagnetic nanoparticles to form
nanoparticle-analyte complexes; placing the complexes in a known
liquid in a uniform magnetic field thereby magnetizing the
nanoparticles and producing dipole-dipole forces between the
nanoparticles; wherein the forces enhance the process of the
complexes binding to each other; exciting magnetic resonance
signals from a sample comprising the complexes and the known
liquid; and determining whether the analyte is present in the
sample by analyzing one or more of the magnetic resonance
signals.
22. A portable magnetic resonance system comprising: a magnet
system having an upper permanent magnet and a lower permanent
magnet which generate a magnetic field in a sample area located
between the upper permanent magnet and the lower permanent magnet;
a pulse generator configured to produce electromagnetic pulses at a
selected frequency; a coil coupled to the pulse generator and
configured to transmit the electromagnetic pulses generated by the
pulse generator to the sample area and to receive responsive
magnetic resonance signals from the sample area; a receiver coupled
to the coil so as to receive the magnetic resonance signals from
the coil and configured to convert the magnetic resonance signals
into a digital form; and a controller in communication with the
pulse generator and the receiver, and configured to control the
operation of the pulse generator so as to cause the pulse generator
to produce electromagnetic pulses at a selected frequency; said
controller being further configured to receive the digital form of
the magnetic resonance signals from the receiver caused by the
electromagnetic pulses being transmitted to the sample area by the
coil in the presence of the magnetic field, and to analyze the
digital form of the magnetic resonance signals.
23. The system of claim 22 further comprising a concentrating
magnet system which generates a non-uniform magnetic field in an
area occupied by a sample.
24. The system of claim 22 further comprising a uniform magnet
system which generates dipole-dipole forces between paramagnetic
bodies placed within the uniform magnet system.
25. The system of claim 22 further comprising: a sample collector
having a concentrator to collect sample material, a mixer
configured to receive sample material from the concentrator and to
mix sample material with a liquid, a fluidic transport system in
communication with the mixer and extending into the sample area for
transporting the sample material mixed with the liquid to the
sample area.
26. A magnetic resonance system comprising: a concentrating magnet
system which generates a non-uniform magnetic field in an area
occupied by a sample; a magnetic resonance measurement device
having a sample area; and a fluidic sample delivery system having a
sample container and a delivery system for transporting liquid
samples to the concentrating magnet system and the sample area.
27. A magnetic resonance system comprising: a magnet system which
generates a magnetic field in an area occupied by a sample so as to
magnetize paramagnetic bodies placed in the system and to generate
dipole-dipole forces among those bodies; a magnetic resonance
measurement device having a sample area; and a fluidic sample
delivery system having a sample container and a delivery system for
transporting liquid samples to the concentrating magnet system and
the sample area.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
applications Ser. No. 60/759,788, filed Jan. 19, 2006 titled
MAGNETIC ENHANCEMENT OF NANOPARTICLE REACTIONS, and Ser. No.
60/786,033, filed Mar. 27, 2006 titled MAGNETIC CONCENTRATION OF
REAGENTS both of which are hereby incorporated by reference.
GOVERNMENT INTEREST
[0002] This invention was made with U.S. Government support under
one or more of the following contracts: Naval Air Warfare Center
n68335-02-c-3120, Department of Homeland Security contracts
NBCHC060017 and HSHQPA-05-9-0039. The U.S. Government has certain
rights in this invention.
BACKGROUND
[0003] 1. Field of the Invention
[0004] The present invention generally relates to the field of
analyte detection and additionally relates to detecting analytes
using magnetic resonance.
[0005] 2. Related Art
[0006] Detection technology for specific analytes spans a wide
range of laboratory instrumentation and techniques including liquid
and gas chromatography (LC and GC, respectively), mass spectrometry
(MS), nuclear magnetic resonance (NMR) spectroscopy, polymerase
chain reaction (PCR), optical spectroscopy and fluoroscopy, Fourier
transform infrared (FTIR) spectroscopy, and ion mobility
instruments. Today's chemical analysis instruments however, are
large and expensive, require a skilled operator, involve complex
sample preparation, and require substantial amounts of time for
analysis.
[0007] There is a critical need worldwide for improved detection of
specific chemicals and microbes. For example, in the area of
national security, a system is needed to detect biological agents,
toxins, and chemical weapons to provide early alert in case of a
terrorist attack. Such a detection capability could also be used to
search for clandestine sites where such weapons are under
development or in production, thus enabling action to prevent their
use. A system is also needed to scan mail and packages to detect a
terrorist attack.
[0008] Improved pathogen detection is also needed for medical
science. Sensitive detection of DNA or proteins associated with
avian flu, bovine spongiform encephalopathy (more commonly referred
to as "mad-cow disease"), or severe acute respiratory syndrome
(SARS) would enable intervention to avoid a pandemic. Broad
clinical use of such a system would assist in identifying ordinary
diseases or serious illnesses, greatly assisting physicians in
diagnosis.
[0009] Detection of various chemicals is also needed for industrial
applications to detect toxic industrial chemicals (TICs) and toxic
industrial materials (TIMs). Such a system would enable leak
detection, process control, detection of material degradation,
control of concentration, and a host of other process applications
in a wide range of industries.
[0010] Improved detection is also needed in agriculture and food
production, as well as a means to detect contamination, spoiling,
or poisoning of food. Food includes for example, items such as
drinking water and fruit juices. There is also a need in forensic
testing, including for example, searching for specific DNA
sequences in a sample at the search site.
[0011] Magnetic resonance detection techniques are under
development involving nanometer-scale paramagnetic particles
(nanoparticles) which have previously been used as MRI contrast
agents. The particles comprise a core of paramagnetic or
superparamagnetic (both generally referred to herein as
paramagnetic) material, coated by a shell of nonmagnetic material
which are adorned with reactant molecules to promote binding to
target cells such as pathogens, tumor cells, etc. Nanoparticles are
injected into a patient prior to MRI analysis. They bind to the
target cells, cause a local change in the MRI image properties, and
enable detection or localization of the target cells.
[0012] The nanoparticles have also been used in vitro. Dissolved or
suspended in a liquid medium, the nanoparticles bind to target
cells or molecules in the medium. The nanoparticles and analytes
may form aggregates incorporating dozens to thousands of
nanoparticles. Such aggregates are detectable by light scattering,
atomic-force microscopy, electron microscopy, and in some cases by
NMR effects. See, for example, U.S. Pat. No. 5,254,460 to Josephson
et al.
[0013] Target-specific reactants can be mounted onto the
nanoparticles to provide analyte-specific selectivity. A
disadvantage is the need to form aggregations comprising a
plurality of nanoparticles and a plurality of target cells or
molecules, because aggregation occurs only when each nanoparticle
is bound to multiple analytes, and each analyte is bound to
multiple nanoparticles. Aggregation can be inhibited by geometrical
effects such as a variation in size among nanoparticles.
Substantial time may be required for the aggregations to form.
[0014] Prior studies on agglomeration were conducted on benchtop
relaxometers and high-field MR instruments. Manual sample
preparation and insertion into the NMR tube can be tedious.
Important events such as binding of the analyte to the
nanoparticles may be missed. A compact and automated instrument is
required to speed up measurements. Also, it is important to
understand the phenomena describing the changes seen in the
measurement from a basic physics and biochemistry standpoint.
[0015] Earlier studies did not model the change in T2 effects from
a physics standpoint. Simple agglomeration effects were observed
through optical means (microscopes) to establish the phenomena
relating change in T2. In addition, early studies did not take
advantage of stoichiometry control of the nanoparticles to adapt
the measured parameters for various applications leading to
specific NMR products.
[0016] Earlier studies used samples that were pure and not subject
to interferences such as dust, acids, etc. Moreover, there was no
requirement for fast measurements combined with no interference
from clutter and near neighbor molecules, cost of overall system,
low false alarms and high probability of detection. There was also
no defined range of analyte concentrations to be detected.
[0017] Earlier studies did not consider use of improved
paramagnetic materials such as compounds of iron, cobalt and nickel
leading to stronger magnetization and improved sensitivity.
[0018] Earlier studies did not consider use of magnetic fields to
influence interactions between nanoparticles or between molecules
attached to nanoparticles. Use of magnetic fields to control the
formation or geometrical configuration of structures comprising
nanoparticles and analytes has not been considered. Use of magnetic
fields to concentrate reactants so as to accelerate selected
interactions was not previously considered.
SUMMARY
[0019] A system and method are provided which can detect target
analytes based on magnetic resonance measurements. In one aspect
analytes are detected using specific nanoparticles in the form of
magnetic resonance nanoswitches. The reaction between the
nanoparticles and analytes is controlled through the application of
a magnetic field.
[0020] In one aspect, a system and method for detecting an analyte
include attaching nanoparticles to the analyte, thereby forming
nanoparticle-analyte complexes. A magnetic field is applied to the
complexes in a known liquid, thereby magnetizing the nanoparticles.
The magnetic field exerts forces on the nanoparticles, and the
nanoparticles exert magnetic forces on each other. The complexes
undergo motions responsive to the magnetic forces. The complexes
undergo interactions, which interactions are enhanced by the
motions. Magnetic resonance signals are excited from a sample
comprising the complexes and the known liquid. A magnetic resonance
parameter such as the T2 of the sample is determined from the
magnetic resonance signals. Then, the system determines whether the
analyte is present in the sample by analyzing the determined
parameter and a predetermined value.
[0021] In one aspect of the invention, systems and methods detect
targeted analytes with very high specificity, despite near-neighbor
interference, dirt, clutter, biological interferents such as mold
spores, proteinaceous interferents such as skim milk and ova
albumin, paramagnetic interferents such as hemoglobin and humic
acid (containing chelated iron), environmental interferents such as
the so-called Arizona dust, diesel soot, etc.
[0022] One aspect of the invention includes a system and method for
detecting analytes in a liquid medium. In another aspect analytes
may be introduced as aerosol, hydrosol, and in complex media such
as food.
[0023] The system includes a magnetic resonance system to detect
resonance signals from the liquid, a magnetic field passing through
that liquid, and a region within the liquid where the magnetic
field has a distinct property such as a particular value or
gradient. Liquid within that region produces magnetic resonance
signals which depend on the field property, and liquid outside that
region may also be influenced by the region due to diffusion. A
material having particular affinity for the analyte is adjacent to
the region. The analyte binds to or is held by the affinity
material and displaces liquid from that region, thus altering the
magnetic resonance signals and revealing the analyte.
[0024] A system for detecting an analyte comprises: a sample which
contains the analyte within a liquid medium, means for generating a
first magnetic field within the liquid, means for generating a
second magnetic field within a special region within the liquid,
means for holding the analyte within the special region, a magnetic
resonance instrument capable of measuring magnetic resonance
signals from the liquid, and means for analyzing those signals to
determine whether the liquid occupies the special region. The
second magnetic field is distinct from the first magnetic field.
Magnetic resonance signals from the liquid residing within the
special region respond to the second magnetic field, which causes
magnetic resonance signals which differ detectably from signals of
the liquid located exterior to the special region. In addition,
liquid may pass through the special region and then return to the
rest of the liquid, thereby influencing the magnetic resonance
signals of the remaining liquid. In addition, liquid in the special
region responds to the second magnetic field, for example by
becoming depolarized, and then communicates that depolarization to
the rest of the liquid through spin diffusion. When present, an
analyte displaces liquid from the special region. Thus if the
signals show that liquid occupies the special region, analyte must
be absent. If the signals show that the liquid is displaced from
the special region, then the analyte must be present, and is thus
detected.
[0025] The analyte can be any molecule, molecular complex, microbe,
chemical, or material which can be contained in the liquid medium,
and which displaces the liquid when so contained. Examples of
analytes include bio-molecules such as proteins, DNA, RNA, or
fragments or complexes thereof; enzymes, small molecules,
organisms, microbes such as whole or disrupted viruses or bacteria;
whole or disrupted cells from other species including humans,
non-biological chemicals such as chemical weapon molecules,
explosives, insecticides, pharmaceuticals, and industrial
chemicals.
[0026] In one embodiment the liquid contains the analyte. Here
"contains" means that the analyte is dissolved, suspended,
emulsified, or otherwise wholly enclosed in and dispersed within
the liquid. Also, the analyte displaces the liquid, meaning that
molecules of the analyte can not co-occupy space with molecules of
the liquid.
[0027] The liquid can be any fluid material that includes a nucleus
having non-zero spin. Only nuclei with non-zero spin give rise to
the NMR phenomena. The liquid includes such nuclei when molecules
comprising the liquid comprise a nucleus with non-zero spin, such
as hydrogen in the water molecule. Alternatively, the liquid may
include such nuclei as solutes or suspensions, such as a
fluoridated solute which generates magnetic resonance signals at
the .sup.19F Larmor frequency.
[0028] In a further aspect a system includes a first magnetic field
which passes through the liquid. The first magnetic field may be
produced by an electromagnet, permanent magnet, superconducting
coil, or any other source. Normally the first magnetic field is a
static and substantially uniform magnetic field that can be in the
range of 0.01 Tesla to 20 Tesla, and is a part of the magnetic
resonance system.
[0029] A second magnetic field is generated in a special region of
the sample. The second magnetic field is distinct from the first
magnetic field in some parameter that is detectable using magnetic
resonance. For example, the second magnetic field may differ from
the first magnetic field in magnitude, orientation, uniformity,
gradient, or any other detectable parameter. A second magnetic
field generator or means for generating the second magnetic field
may be a nanoparticle, which may be suspended in the liquid and
immersed in the first magnetic field or applied field. In one
embodiment the nanoparticle becomes magnetized and produces a
dipole-shaped field that adds vectorially to the applied field,
producing a net magnetic field. The special region is that volume
occupied by the distinct magnetic field. When the distinct magnetic
field is caused by a nanoparticle, the special region is that
nanometer-scale volume adjacent to but exterior to the surface of
the nanoparticle, where the net field differs substantially from
the applied magnetic field. Alternatively, the special magnetic
field region could be produced by paramagnetic ions such as
chelated iron or gadolinium instead of nanoparticles. An advantage
of this approach is that diffusion-limited reaction rates may be
increased due to the higher mobility of metal-ion chelates. Similar
ions are used in MRI (Gd-DTPA and Gd-DOTA.).
[0030] Alternatively, the special magnetic field region is produced
by particles or structures having a size larger than
nanometer-scale, provided that the magnetic resonance signals
differ detectably when analyte is present or absent. For example,
shaped magnetic structures may provide two specific values of the
magnetic field in two regions, and the analyte binding molecules
could be coupled to only one of the field regions. The detection
measurement is then a spectral analysis of the composite magnetic
resonance signal, which will exhibit two frequency peaks
corresponding to the two field regions when no analyte is present,
or only a single peak when analyte obscures one of the field
regions.
[0031] In one aspect, temperature cycling is used to accelerate
binding between the analyte and nanoparticle. This shortens the
binding event time by increasing the mobility of the analyte and/or
the nanoparticle. When an energy barrier inhibits binding, higher
temperatures improve the rate of binding. Temperature cycling may
include heating and cooling or vice versa. Then the sample is
measured in the magnetic resonance instrument.
[0032] In one aspect, the system includes a mechanism or binding
agent for holding the analyte in the special region, to displace
the liquid from the special region, leading to detection of the
analyte. Such a binding agent can include any material surface or
molecule for which the analyte has an affinity. Such holding may be
accomplished by hydrogen bonds, ionic forces, covalent bonds,
sulfide bridges, van der Waals forces, electrostatic forces, or any
other type of molecular or material attachment or affinity ligand.
The binding agent is positioned adjacent to the region of shaped
magnetic field so that the target molecule, when bound, occupies
that region and excludes the liquid therefrom. For example, the
binding agent may be an antibody raised against an analyte protein,
or DNA complementary to analyte DNA sequences. Preferably the
binding agent also has null affinity or negative affinity for all
solutes other than the analyte that may be present. In addition to
DNA, other holding means can be used such as aptamers, small
molecules, etc. Targets include, but are not limited to the
following: [0033] a. An antibody that recognizes and binds to an
antigen [0034] b. an oligonucleotide or DNA sequence complementary
to a DNA- or RNA-target [0035] c. a DNA- or RNA-aptamer that binds
to a target protein, bacteria, virus, yeast or fungus. [0036] d. a
protein or peptide that binds to a target protein, bacteria, virus,
yeast or fungus. [0037] e. a pseudopeptide composed of unnatural
amino acids with a stronger binding to a target or better
environmental stability. [0038] f. a small molecule or combination
of small molecules that can bind to a target. [0039] g.
monosacharides, polysacharides, carbohydates and sugars that can
bind to a target protein, bacteria, virus, yeast or fungus.
[0040] A further aspect includes a magnetic resonance instrument,
which is capable of exciting and detecting magnetic resonance
signals from the liquid medium. Existing magnetic resonance systems
may perform this function. More preferably, the instrument is a
simple, compact, automated, single-purpose magnetic resonance
system which can perform the detection measurement automatically.
The system measures signals related to the presence or absence of
liquid, affected by the second magnetic field in the special
region. For example, when the magnitude of magnetic field in the
special region differs from that in the rest of the liquid, then
the magnetic resonance system can measure the spectral content of
the magnetic resonance signals to determine the magnetic field from
which the signals emerged. Thus by analyzing for the Larmor
frequency of the liquid in the special region, the system
determines whether liquid occupies that region.
[0041] An alternative measurement is the spin-spin dephasing time
(T2) of the liquid. T2 is affected when the magnetic field in the
special region has strong gradients, and particularly when the
liquid diffuses through those gradient fields in times short
compared to the measurement. Thus the system can determine the
presence of analyte by measuring the T2 of the liquid to determine
if depolarization is occurring in the special region.
[0042] In one aspect, the compact magnetic resonance system can
measure either a positive or negative change in T2. Agglomeration
is described in the Josephson patent as the formation of a large
supermolecular assembly of molecules. In the case of agglomeration,
all measurements show a negative T2 change. Likewise, the parameter
defined as "positive 1/T2" in Josephson represents a negative
change in T2. Agglomeration is described by Josephson as a process
where several molecules attach to each other and they form
assemblies large enough to change the T2 of the water. In one
embodiment, the inventive system measures T2 changes due to the
analyte binding event, leading to positive and negative T2 changes
prior to agglomeration.
[0043] In a further aspect, a system includes a magnetic field to
control interactions involving nanoparticles and analytes. The
analytes bind to the nanoparticles, producing nanoparticle-analyte
binaries. A magnetic field is applied to the binaries. The magnetic
field magnetizes the nanoparticles, the magnetization direction
being substantially parallel to the magnetic field direction. The
magnetized nanoparticles exert magnetic forces called dipole-dipole
forces on each other. The forces can be mutually attractive,
repulsive, or torsional depending on the relative positions of the
nanoparticles and the magnetic field direction. When a line between
two nanoparticles is parallel to the field direction, the mutual
magnetic force is attractive. When a line between two nanoparticles
is perpendicular to the field direction, the force is repulsive. At
all other orientations, the nanoparticles exert mutual torsional
forces on each other, and the torsional forces are such as to drive
the nanoparticles into parallel alignment with the field.
[0044] In one aspect, the magnetic field is substantially uniform
throughout the sample volume. The magnetic field, through induced
dipole-dipole forces between nanoparticles, urges the
nanoparticle-analyte binaries into alignment with the magnetic
field. The nanoparticles or the analytes interact when so aligned,
producing for example a linear chain-like structure. The forces
also drive the nanoparticles away from the perpendicular
orientation. Nanoparticles are in the perpendicular orientation
when a line between the nanoparticles is perpendicular to the
magnetic field. The forces between nanoparticles thus suppress
interactions in the perpendicular orientation, leading to
suppression of three-dimensional aggregate structures.
[0045] In one aspect, the magnetic field is substantially
non-uniform in the sample volume. The strength or magnitude of the
non-uniform magnetic field varies throughout the sample volume.
Preferably the magnetic field has maximal strength in a small
subvolume of the sample volume. The magnetic field generates a
force on the nanoparticles in addition to the mutual dipole-dipole
forces between nanoparticles, the additional force being such as to
draw the nanoparticles toward the region having the highest
magnetic field magnitude. Responsive to that force, the
nanoparticles or binaries drift toward the subvolume, thereby
greatly increasing the concentration of the nanoparticles or
binaries in that subvolume and depleting the concentration in the
remainder of the sample volume. Since many chemical interactions
exhibit reaction rates which depend on the concentration of
reactants, interactions between the nanoparticles or analytes or
binaries can be accelerated in the subvolume, and inhibited in the
rest of the sample volume.
[0046] In one aspect, the compact magnetic resonance system
measures a baseline value of T2 using the nanoparticles or
nanoparticle solution prior to analyte-nanoparticle binding.
Analyte is then mixed with the nanoparticles or allowed to interact
with the nanoparticles, and then the T2 of the sample is measured
again to determine whether a change in T2 has occurred. The
baseline measurement ensures the correct concentration of
nanoparticles and consistent stoichiometry. Comparison of the
baseline and subsequent T2 measurements enables cancellation of
metering and mixing errors, variations in nanoparticle properties,
fluidic transport errors, etc.
[0047] In one embodiment the inventive system can detect analyte by
measuring magnetic resonance signals from the sample at a single
time. Alternatively, the system can perform a series of
measurements spanning a period of time and can compare or analyze
the measurements to improve the detection of analyte. For example,
the binding between analyte and nanoparticles may proceed during an
interval which is longer than the time required for a particular
measurement. Then the system can perform the measurements
repeatedly to observe the changes caused by the binding. As another
example, the analyte may first bind to nanoparticles to form
binaries, causing a positive shift in T2. Then the binaries may
combine to form agglomerates, causing a negative shift in T2. Such
data can greatly enhance the quality of the result by reducing the
false alarm rate, providing a lower detection threshold, and
enhancing the detection probability for a given quantity of
analyte.
[0048] In one aspect the system can derive parameters related to
reaction rates or kinetics from repeated measurements on the same
sample. For example, a rate of change of a measured parameter may
indicate a rate of binding or other interaction between analytes
and nanoparticles. A net change in a measured parameter may
indicate an accumulated reaction parameter such as the total
quantity of analyte bound to nanoparticles. These results can then
be used to guide additional measurements to confirm or clear the
initial indication. For example, if a sample exhibits a small but
suspicious T2 change soon after mixing, the system can initiate a
series of tests to determine the rate of change in T2 over a period
of time. Then, if those later results confirm that the analyte is
present, an alarm can be issued. If the follow-on measurements
indicate no analyte, then the initial suspicion may be cleared,
thereby averting a false alarm. Using a provisional re-scan
protocol, combined with a rate-magnitude analysis, the system
enhances both reliability and threshold sensitivity.
[0049] Based on experimental results and theoretical modeling, a
positive T2 change is due to analyte displacing water molecules
upon binding to the nanoparticles, and negative T2 change is due to
repeated dephasing of water molecules within a cage structure
formed by multiple nanoparticles. In addition, the positive or
negative T2 change can be promoted by processing and stoichiometry.
For example, the ratios of nanoparticles and the reagent can be
adjusted to provide negative or positive T2 changes.
[0050] In some circumstances it can be important to measure both
negative and positive T2 effects so as to detect analyte despite an
interferent present in the sample solution. For example, a test
sample contaminated with a paramagnetic ion, such as humic acid
with chelated iron, causes a reduction in the T2 of the mixture. If
the sample contains an analyte mixed with the humic acid, the
analyte can be detected despite the interferent in the following
manner. First, measure the sample prior to mixing the
nanoparticles, to generate a first T2 measurement value. Then, mix
the nanoparticles into the solution and perform a second T2
measurement, completing that measurement before the analyte has had
time to interact with the nanoparticles. Then allow the analyte to
interact with the nanoparticles, and then measure T2 a third time.
The initial measurement reveals the presence of the humic acid
interferent so that the resulting T2 effects may be accounted for.
The second T2 measurement, in comparison with the first value,
provides a check that the nanoparticle concentration and other
mixing parameters are correct. The third measurement reveals the
analyte as a change in T2 relative to the second measurement, the
change being due to the analyte-nanoparticle interactions.
[0051] Alternatively, in some circumstances a separate baseline
measurement cannot be taken, or it may not be known when the
analyte interacts with the nanoparticles. However many
interferents, including humic acid, cause a negative shift in T2.
In that case the nanoparticles can be mixed so as to generate a
positive T2 shift upon binding to the analyte. When the positive T2
shift is larger than the negative shift from the interferent, the
analyte may thus be detected.
[0052] In one aspect the invention includes nanoparticle
multiplexed mixtures which detect any of a plurality of different,
but specific, analytes. "Nanoparticle multiplexed mixtures" are
nanoparticle preparations sensitized to multiple analytes. There
are two multiplexing scenarios. In the first scenario, each
nanoparticle in the mixture is sensitized to a single analyte.
Nanoparticles sensitized to different analytes are then mixed
together in the solution. In the second scenario, each nanoparticle
is sensitized to multiple analytes.
[0053] In one embodiment, an automated air monitoring system
includes inlets to admit an airborne sample along with air, a
collector that gathers the sample material and concentrates it into
a liquid form, called a raw sample, and a fluidics system. The
fluidics system holds the raw sample, for example in a container
and provides consistent metering of the raw sample, for example via
an outlet tube using a pump such as a peristaltic pump. Metered
sample is mixed with selected nanoparticles which may be in water,
for example, drawn from reservoirs via an outlet by a pump. As soon
as the analyte-nanoparticle reaction takes place the fluidics
system moves the sample into the sample area of the magnetic
resonance system for measurement, for example, via a tube driven by
a pump. Alternatively, sample mixing and processing may take place
within the magnetic resonance system. The fluidics system may
include means for cell lysing wherein the fluidics system may lyse
or disrupt cells or viruses in the sample to release proteins, RNA,
or DNA of the target cell. The fluidics system may also have a
temperature control built in to speed up the binding event.
Fluidics system also may have an overall system cleaning solvent to
eliminate contamination. The cleaning solvent or rinsing agent can
be drawn from a reservoir and pumped through the tube which
delivers the samples to the sample area. The fluidics system also
allows positive and negative control tests to ensure the overall
system is functional, and performs calibration tests using
calibration standards.
[0054] In one embodiment, chelates are used in place of
nanoparticles to generate the distinct magnetic field region and to
bind to analyte. An advantage of using chelated ions is that it
allows faster diffusion through the liquid medium to speed up
diffusion-limited processes. On the other hand, with nanoparticles
one can tailor the affinity molecules to select the analyte
desired, whereas chelates occur only in specific molecular forms.
Nanoparticles have more area to attach the affinity molecules
compared to chelates. As an alternative, nanoparticles can be
decorated with chelates for binding to analytes, explosives and
chemicals.
[0055] In one aspect, the second magnetic field region, being
generated by paramagnetic cores or chelates or other magnetic
structures, has a size comparable to the size of the analyte, so
that the bound analyte just fills the second magnetic field region,
excluding the liquid from that region, thus providing highest
signal and highest sensitivity. For example, when the analyte is a
relatively small molecule such as an explosive vapor molecule or a
chemical weapon molecule, then the size of the second magnetic
field region is preferably chosen to be in the range of 1 to 10 nm.
To detect a larger analyte, such as a toxin or DNA or virus
particle, then the size of the second magnetic field region would
be 10 to 100 nm. When the analyte is an even larger objects such as
a bacterium, the size of the second magnetic field region may be
100 to 1000 nm or larger as needed to match the analyte.
[0056] The nanoparticles may include structures that provide an
optical signature. For example, fluorescent dyes or centers may be
attached to or included within the nanoparticles, and may be
exposed to photons of sufficient energy to excite fluorescence,
causing emission of fluorescence photons having an energy different
from, and usually lower than, the excitation photons. The
excitation and fluorescence photons may be in the ultraviolet,
visible, or infrared range. Detection of the fluorescence photons
provides a measure of the nanoparticle concentration. In addition,
the structures may be modified when analyte binds to the
nanoparticle, and such action may result in a detectable change in
the fluorescence such as a change in the intensity or energy of the
fluorescence photons. Detection of this change would provide an
indication, independent of magnetic resonance measurements, that
analyte binding has occurred and thus that analyte is present in
the sample.
[0057] Other features and advantages of the invention will be
apparent from the following detailed description, the claims and
the appended drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] The details of the present invention, both as to its
structure and operation, may be gleaned in part by study of the
accompanying drawings, in which like reference numerals refer to
like parts, and in which:
[0059] FIG. 1 is a schematic representation of a nanoparticle
showing the applied magnetic field and the second magnetic field
around the nanoparticle.
[0060] FIG. 2 is a graph of the net magnetic field surrounding the
nanoparticle of FIG. 1.
[0061] FIG. 3 is a plot of the magnitude of the magnetic field
gradient around the nanoparticle.
[0062] FIG. 4 is a plot of the field gradient magnitude along the
axis of the particle.
[0063] FIG. 5 is a schematic representation of the mutual forces
between nanoparticles in a magnetic field.
[0064] FIG. 6 is a schematic representation of the formation of a
chain structure from nanoparticles and analyte.
[0065] FIG. 7 which is a functional block diagram of a magnetic
resonance system.
[0066] FIGS. 8a-d is a representation of four configurations of the
antenna.
[0067] FIG. 9 is a schematic representation of one embodiment of a
magnet.
[0068] FIG. 10 is a circuit diagram of a buffered oscillator.
[0069] FIG. 11 is a schematic illustration of an installation
having one controller and multiple sensor units.
[0070] FIG. 12 is a schematic depiction of an analyzer system
suitable for use with an HVAC system.
[0071] FIG. 13 is a representation of a concentrator magnet
system.
[0072] FIG. 14 is a representation of an alternative concentrator
magnet system.
[0073] FIG. 15 is a graph of magnetic resonance data with and
without magnetic processing.
[0074] FIGS. 16a-e depict an embodiment of a fixed installation
system and three collector intakes.
[0075] FIG. 17 is a front perspective view of a hand-portable
system.
[0076] FIG. 18 is a block diagram of a system adapted to a medical
diagnostic application.
DETAILED DESCRIPTION
[0077] After reading this description it will become apparent to
one skilled in the art how to implement the invention in various
alternative embodiments and alternative applications. However,
although various embodiments of the present invention will be
described herein, it is understood that these embodiments are
presented by way of example only, and not limitation. As such, this
detailed description of various alternative embodiments should not
be construed to limit the scope or breadth of the present invention
as set forth in the appended claims.
[0078] Magnetic Resonance
[0079] A brief summary of the technical elements used in certain
embodiments is provided herein. The analyte or target molecule is
contained in a medium, preferably a liquid such as water, which
includes an atomic nucleus that has a non-zero spin, such as
hydrogen. As is well known, (see for example, Pulse Methods in 1D
& 2D Liquid-Phase NMR, Wallace S. Brey, Academic Press 1988),
that the magnetic component of such a nucleus becomes polarized or
spatially oriented in a magnetic field, and may be induced into
magnetic resonance precession at a frequency given by:
f.sub.Larmor=.gamma.B/2.pi.
where B is the magnetic field strength at the position of the
nucleus, .gamma. is the magnetogyric ratio of the nucleus, and
f.sub.Larmor is the resonance frequency or Larmor frequency
(.gamma.=2.675.times.10.sup.8 Tesla.sup.-1 sec.sup.-1 for the
hydrogen nucleus). The magnetic components, or magnetic moments, of
the nuclei are vector quantities and add to give a resultant bulk
magnetization vector that is the NMR signal measured by NMR
spectrometers.
[0080] Following a perturbation such as that employed in recording
NMR signals (see below), the bulk magnetization vector recovers to
its original steady state over time; this process is referred to as
nuclear magnetic relaxation. Two fundamental time constants are
used to describe this relaxation in terms of a single-exponential
process. Recovery of the bulk magnetization along the direction of
the first magnetic field is described by the spin-lattice
relaxation time or longitudinal relaxation time, designated as T1.
Typically, T1 is of order milliseconds to seconds. The
single-exponential decay of bulk magnetization in the plane
perpendicular to the direction of the first magnetic field is
described by the spin-spin relaxation time, or transverse
relaxation time, designated as T2. For liquid signals, T2 is
generally in the range of 100 milliseconds or more. Solid samples
on the other hand, generally have T2 values in the range of 1 to
100 microseconds.
[0081] A magnetic resonance measurement is performed by applying
one or more RF (radio frequency) energy pulses to the sample and
measuring the bulk magnetization that becomes reoriented by the
pulse. The RF pulses have a frequency equal to the Larmor
frequency, and duration sufficient to cause the bulk magnetization
vector to reorient into a plane perpendicular to the first magnetic
field, where the bulk magnetization vector (the NMR signal) can be
recorded over time. The RF pulses therefore, are usually multiples
of 90 degrees.
[0082] Spin-spin relaxation is typically measured by a series of RF
pulses to give rise to spin echo signals. A spin echo is generated
by a 90-degree pulse followed by a small delay time (typically
designated as .tau.), followed by a 180-degree pulse
(90.degree.-.tau.-180.degree.). A second .tau., identical in time
to the first, is used before the bulk magnetization vector is
recorded. The series of RF pulses and time delays is used to first
dephase the nuclear magnetic moments comprising the bulk
magnetization in the plane perpendicular to the first magnetic
field during the first .tau., and refocus the remaining bulk
magnetization in this plane during the second .tau.. This latter
refocusing creates an echo signal, which can be recorded. The most
common method to measure spin-spin relaxation is that originally
described by Carr and Purcell (Carr, H. Y. and Purcell, E. M.:
Effects of Diffusion on Free Precession in Nuclear Magnetic
Resonance Experiments, Physical Review 94, no. 3 (1954): 630-638),
a modification of the method described earlier by Meiboom and Gill
(Meiboom, S. and Gill, D.: Modified Spin-Echo Method for Measuring
Nuclear Relaxation Times, The Review of Scientific Instruments 29,
no. 8 (1958): 688-691). The Carr-Purcell modified Meiboom-Gill
(CPMG) method uses a series of small time delays followed by
180-degree pulses after the initial 90.degree.-.tau.-180.degree.
sequence described above. This in turn is followed by the resultant
bulk magnetization vector
[90.sub.x.degree.-(.tau.-180.sub.y.degree.-.tau.-record).sub.n].
The amplitudes of the spin echo signals are proportional to the
bulk magnetization remaining at the time of the echo, which becomes
successively smaller as the number of sequences increases (as the
value of n increases). Therefore, measuring the amplitude of the
bulk magnetization vector after various values of n and fitting the
data to a single exponential decay with T2 as the relaxation time
provides a direct measure of T2.
[0083] Paramagnetic Nanoparticle Fields
[0084] In a preferred embodiment, nanoparticles are employed to
influence the magnetic field in a region close to the
nanoparticles. The paramagnetic or superparamagnetic core of the
nanoparticle becomes magnetized when an external magnetic field is
applied to it. Superparamagnetism is related to ferromagnetism in
which the size of the magnetized body is too small to form a
magnetic domain. The superparamagnetic core exhibits a high
permeability and fairly high saturation field comparable to iron,
but little or no hysteresis (H.sub.c.about.0). When placed in a
magnetic field, the core becomes strongly magnetized parallel to
the direction of the applied field. When the external field is
removed, the core loses essentially all of its magnetization.
Disregarding anisotropy and shape effects, the induced magnetic
moment of the core is given by:
m.sub.core=(4.pi./3)(r.sub.core.sup.3)(B.sub.0)
where m.sub.core is the dipole moment of the core, r.sub.core is
its radius, B.sub.0 is the applied field, and is the
susceptibility. Normally .apprxeq.0 for nonmagnetic materials,
>.apprxeq.1 for superparamagnetic materials when B.sub.0 is
below a saturation field, and 1.ltoreq..ltoreq.0 for B.sub.0 above
saturation. For example, magnetite (Fe.sub.3O.sub.4) is
superparamagnetic with a susceptibility of about 1 for fields below
saturation of about 0.5 Tesla.
[0085] The magnetized core produces a magnetic field which usually
approximates a dipole field, or the magnetic field produced by an
ideal magnetic dipole located at the center of the paramagnetic
core of the nanoparticle. At locations outside the nanoparticle
core, the dipole magnetic field is parameterized as follows:
B.sub.r=2 m.sub.core cos .theta./r.sup.3
B.sub..theta.=-m.sub.core sin .theta./r.sup.3
[0086] Here B.sub.r is the radial component of the dipole field,
B.sub..theta. is the circumferential component, r is the distance
from the center of the core, .theta. is the polar angle relative to
the applied field, and m.sub.core is the dipole moment.
[0087] The dipole field adds linearly to the applied field (as
vectors), resulting in the net magnetic field. The Larmor frequency
is determined by the net magnetic field experienced by the
polarized nucleus. Components of the dipole field orthogonal to the
applied field cause primarily a field rotation, whereas the dipole
components parallel to the applied field directly change the
magnitude of the net field and therefore change the Larmor
frequency, relative to the undistorted applied field. The net field
B.sub.net, disregarding second order terms, and for
r>>r.sub.core, is as follows:
B.sub.net=B.sub.0(1+4.pi./3(r.sub.core/r).sup.3(2 cos.sup.2
.theta.-sin.sup.2 .theta.))
[0088] In some embodiments the magnitude of the gradient of the net
magnetic field is also important. The field gradient is given
by:
.gradient.B.sub.net=B.sub.08.pi.(r.sub.core.sup.3/r.sup.4)(-{r}
cos.sup.2 .theta.+{.theta.} cos .theta. sin .theta.)
where curly brackets denote unit vectors in the r or .theta.
directions.
[0089] Diffusion in a Liquid
[0090] Some embodiments include a liquid medium. The liquid
contains the analyte and the nucleus that emits the magnetic
resonance signals. Those signals are influenced by diffusion,
particularly the diffusion of the molecules of the liquid through
the liquid, or molecular self-diffusion. Diffusion is formulated as
follows:
.sigma..sub.walk=(2 D.sub.molec t).sup.1/2
where .sigma..sub.walk is the average distance traveled in an
isotropic three-dimensional random walk in time t, and D.sub.molec
is the translational diffusion coefficient. For example,
D.sub.molec=1.5.times.10.sup.-9 m.sup.2/s for water at room
temperature.
[0091] Magnetic resonance measurements are also influenced by spin
diffusion, a phenomenon in which the spin or polarization of a
nucleus is interchanged with that of a nearby nucleus of the same
type. Spin diffusion can distribute spin-dependent effects, such as
depolarization, throughout the sample. For example, if a small
fraction of the hydrogen nuclei in water experience a depolarizing
force, spin diffusion can cause all of the hydrogen in the sample
to assume an averaged polarization value.
[0092] A Model
[0093] This model addresses spin-dependent interactions between
nanoparticles and solvent, and provides a useful framework for
quantifying the observed T2 effects. It is used in some embodiments
as the basis for measuring and detecting analytes. A simplified
nanoparticle is assumed to consist of a spherical core of
superparamagnetic material, surrounded by a spherical shell of
non-magnetic material, all in water. However, the model can be
applied or modified for use with nanoparticles of other shapes and
for use with other solvents. The model suggests the following
mechanisms for the observed T2 changes:
[0094] (1) Nanoparticles in solution reduce T2 relative to plain
water. The model suggests that depolarization is due to a dipole
magnetic field produced by the magnetized core. The field
distortion causes spins to precess at different frequencies,
leading to destructive interference. Although CPMG normally
refocuses static field-nonuniformity effects, the Brownian motion
of the water molecules causes them to enter and exit the field
distortions in a time shorter than the echo interval, thereby
making the spin dispersion time-dependent and breaking the CPMG
refocusing effect.
[0095] (2) When nanoparticles react with analyte, but do not
agglomerate, the T2 increases. This may be due to the analyte
molecules occupying part of the distorted-field region around the
nanoparticle, thereby excluding water from that region, thus
reducing the spin dispersion and increasing T2. Similarly, when
chain or string like structures of nanoparticles and analyte are
formed, T2 increases. Formation of the chains is described below in
connection with the use of magnetic fields.
[0096] (3) T2 decreases when nanoparticles and analyte agglomerate.
This may be due to the formation of a water-filled cage-like
structure in which water molecules undergo repeated spin-dispersion
collisions with the surrounding nanoparticles. Sufficient
repetition of incremental depolarization would reduce T2, despite
the analyte occluding portions of the non-uniform field
regions.
[0097] (4) A single exponential usually fits the polarization decay
curve. This is despite the fact that hydrogens close to
nanoparticles are strongly dephased, while the general solvent sees
only a uniform field, a two-population system. However, the spin
populations are rapidly equilibrated across the sample by spin
diffusion via homonuclear flip-flop interactions, resulting in a
single averaged T2.
[0098] The model nanoparticle is depicted in cross section in FIG.
1 in the presence of an applied magnetic field indicated by arrow
101. The nanoparticle comprises a magnetizable core 102, a
non-magnetic shell 103, and binding molecules 104. The core 102
preferably is paramagnetic and more preferably is
superparamagnetic. The induced local dipole-shaped field 105 of the
nanoparticle is represented by the dashed lines. The radius of the
core 102 should be large enough to produce a significant magnetic
field distortion in a large enough region to produce a change in T2
of the liquid in that region. The radius of the core 102 should be
small enough that the core 102 does not become ferromagnetic.
Typically the core radius is about 1 to 20 nm. Desirable properties
of the core 102 include high susceptibility at the applied magnetic
field strength, high saturation field preferably in excess of the
applied magnetic field strength, chemical compatibility with the
liquid medium, and very low remnant field. The last feature is
desirable to prevent nanoparticles from clumping together due to
magnetic attraction. The core material may be any magnetizable
material such as iron oxide, cobalt, and nickel compounds.
Nanoparticles can be non-toxic and biodegradable if an iron core is
used. The core is coated by one or more shells 103 of non-magnetic
material, for example, dextran or silica. Silica coatings are
stable and robust, and may avoid the need for refrigeration. Other
polymeric coatings may be considered such as polystyrene,
polyacrylic acid, polyacrylamide and polyvinyl alcohol.
[0099] The net field magnitude at location (r,.theta.) around the
nanoparticle has both positive and negative variations relative to
a uniform field. This is shown in the graph of FIG. 2.
[0100] While the CPMG procedure refocuses static field
non-uniformities, those water molecules that move from one field
region to another, in the time between refocusing pulses, are not
refocused and produce T2 effects. Thus, T2 changes are related to
the gradient of the net field.
[0101] To consider a specific example, the core is Fe.sub.3O.sub.4,
with a 4-8 nm diameter, and the rest of the particle is a dextran
shell, with an overall 50 nm diameter. The susceptibility and
saturation field depend on the composition, crystal structure, and
core diameter. Values of the saturation field range from 0.2 to 0.5
T, and susceptibility ranges from 0.2 to 2. A numerical simulation
was prepared using 0.5T saturation and 0.5 for susceptibility. The
net field in the vicinity of this nanoparticle is shown in FIG. 2.
Strong field enhancements at the two "poles" of the particle are
seen, relative to the field reduction around the "equator". The
field within the shell is of no interest and is not calculated; it
is plotted as B.sub.0.
[0102] The magnetic field gradient is shown in FIGS. 3 and 4. FIG.
3 is a plot of the magnitude of the field gradient around the
nanoparticle. FIG. 4 is a plot of the field gradient magnitude
along the axis of the particle. Again, fields inside the particle
are not analyzed.
[0103] For an echo interval of T.sub.Echo=4 msec, the average walk
distance is about 3.5 microns. This is much larger than the length
scale of the distorted-field regions; hence it is safe to assume
that the water molecule has enough time to enter and exit the
distorted-field region between refocusing pulses.
[0104] The spin dephasing produced by the water molecule passing
through the distorted field region can be estimated as follows. The
instantaneous precession frequency is proportional to the net
magnetic field at the water molecule's location. For simplicity we
assume that the molecule random-walks through the distorted-field
region of one nanoparticle, during one echo interval, starting and
ending in the solvent exterior to the distorted-field region. Thus
the molecule trajectory begins and ends in the applied field of
B.sub.0 but passes through the distorted-field region between CPMG
echoes. While the molecule is within the distorted field, it
accumulates extra precession compared to molecules in the rest of
the solvent. That portion of the phase advance due to the B.sub.0
field is then refocused as usual by the 180 pulses, but the extra
precession phase, accumulated during the time spent in the
distorted field, will not be refocused. The unrefocused phase
increment due to traversal of a field distortion is the integral of
the field experienced by the particle, minus that in the applied
field alone:
d.sub.phase=.intg..gamma.(B.sub.net(r)-B.sub.0)dt
where d.sub.phase is the accumulated phase difference between a
hydrogen which passes through B.sub.net (here an explicit function
of space) versus one remaining in the uniform field B.sub.0,
.gamma. is again the Larmor coefficient and the integral is over
the time between refocusing pulses. To obtain a rough estimate of
the phase shift, the previous equation may be simplified by
assuming that the molecule resides in a constant field for a time
needed to diffuse through the distorted field region, resulting in
the following approximation:
d.sub.phase=[x.sub.dis.sup.2/(2
D.sub.molec)][B.sub.net-B.sub.0].gamma.
[0105] Using the nanoparticle sizes and field assumptions discussed
above, the net magnetic field deviates from the applied field by
typically 20 mT. The spins within that field will precess about 850
kHz faster than in the undistorted field. A typical length scale
for this distortion is x.sub.dis=20 nm. The time needed to diffuse
20 nm is 133 nsec. During that time, the spins precess an extra
d.sub.phase=0.1 radians. This represents a substantial dephasing in
a single echo interval by a single molecular traversal, which if
not refocused by CPMG will result in a short T2. In the sample,
many water molecules will be interacting with the nonuniform field
continuously, and each will experience a positive or negative phase
shift depending on the specific path. In the ensemble, the extra
spin dispersion causes destructive interference and overall
depolarization.
[0106] The spin diffusion coefficient in water is in the range of
D.sub.spin.apprxeq.10.sup.-15 to 10.sup.-16 m.sup.2/s, depending on
temperature and other factors. Although spin diffusion is slower
than molecular diffusion, it is sufficient to spread the
depolarization among many water molecules in a few msec.
Interestingly, solid-state spin diffusion rates tend to be much
higher, of order 10.sup.-9 m.sup.2/s which is comparable to the
molecular diffusion in free water. If the shell exhibits rapid spin
diffusion, it could serve as a conduit for distributing
polarization among all of the water molecules contacting the
nanoparticle surface.
[0107] Several experiments have demonstrated a T2 increase of 20 to
200 msec. The model suggests that this is due to the analyte
molecules obstructing the surface of the nanoparticle, effectively
preventing water molecules from sampling the distorted-field
regions at the surface of the nanoparticle.
[0108] When analyte molecules attach to the surface of a
nanoparticle, a portion of the surface is occluded. The global
depolarization rate goes down and T2 increases. The change in decay
rate is roughly proportional to the fraction of the distorted-field
volume occupied by the analyte. If multiple analyte molecules are
attached, they all contribute a similar T2 change on average. If
the analyte spends only part of its time covering up the surface of
the nanoparticle, then the T2 change scales proportionately.
[0109] A decrease in T2 may also be observed by changing the ratio
of the nanoparticles to antibodies. Here antibody is used as an
example of the connection to the analyte. This is defined as
stoichiometry control. Depending on the level of detection of
analyte one can adjust the stoichiometry to allow rapid detection
of analyte.
[0110] The reagents and processing conditions may be adjusted to
cause a decrease in T2. Formation of extended aggregates of
nanoparticles and analytes is correlated with such a T2 decrease.
The model posits that the aggregates are open, cage-like structures
through which water molecules may pass easily. This is not
explained in earlier studies. In one embodiment, spin information
diffuses in and out of the agglomerate structure rapidly, so that
the depolarization occurring within the cage is equilibrated
throughout the sample.
[0111] The model suggests that the T2 decrease for agglomerates is
due to repeated dephasing when water molecules within the cage
repeatedly encounter depolarizing fields. Such repeated dephasing
represents a more effective polarization sink than isolated
nanoparticles in the free liquid because the caged water molecule
remains in close proximity to numerous nanoparticle surfaces. While
portions of the nanoparticle's distorted-field volumes are occluded
by analyte, the water molecule could spend a significant fraction
of its time sampling fields that differ from the main field, and
thus would become totally dephased in a time short compared to the
echo interval. Then, by trading polarization with neighboring
molecules including those outside the agglomerate, a uniformly
reduced T2 would result.
[0112] The model has utility because it leads to new measurements
and new ways of performing measurements related to analyte in the
sample. The model explains how the analyte interactions with
nanoparticles produce both increases and decreases in T2, and
suggests ways to control the effects by adjusting reagent
concentrations. Noting that speed of detection is a critical
parameter for many applications, the model suggests that the T2
increase method due to analyte-nanoparticle binding will provide
the signals faster than the T2 decrease from aggregation, because
binding must occur before the agglomerations. The model also guides
the development of more sensitive nanoparticles using
higher-susceptibility core material and thinner non-magnetic
shells. The model also leads to steps for canceling systematic
errors, such as measuring the T2 of the nanoparticle solution and
the sample separately, before mixing, to better quantify any T2
changes from the binding. The model also explains how thermal
effects and diffusion effects participate, and can be exploited to
accelerate the detection or confirm analyte reactions. The model
also guides the development of products exploiting the inventive
methods by quantifying signal and noise versus sample size and
other design parameters.
[0113] Method Description
[0114] In one embodiment a method for detecting one or more
analytes includes: preparing a liquid sample mixture, which may
contain the analyte and other materials; applying a first magnetic
field to the liquid; preparing a second and distinct magnetic field
within a special region of the liquid; maintaining the analyte, if
any is present, within the special region (for example, by
providing means for holding the analyte, securing that binding
agent adjacent to the special region and allowing the analyte to
interact with the binding agent); exciting magnetic resonance
signals from the mixture while the analyte is maintained within the
special region; analyzing the signals to determine whether analyte
occupies the special region; and then concluding that analyte is
present when the signals indicate that the liquid is displaced from
the special region. In one embodiment nanoparticles having a
binding agent for the analyte of interest are used to create the
special region and to hold analyte within the special region.
[0115] In one embodiment preparing the liquid sample mixture
includes the use of a liquid which contains an atom with a nucleus
having non-zero spin. The atoms may be an intrinsic part of the
liquid, or they may be added as solute. The step of preparing a
liquid sample can include mixing or stirring to ensure that analyte
reaches the nanoparticles. Mixing can be achieved in numerous ways,
including by driving the sample fluids through convoluted tubes
using a pump, and such motion may be unidirectional or reciprocal
to produce the desired level of mixing. Alternatively, the
nanoparticles and the analyte may be contained in the same type of
liquid, so that when the nanoparticles and analyte are placed in
the same container, they spontaneously become mixed without the
need for physical stirring. For example, the nanoparticles and the
sample material may be dissolved in water and then intermingled by
diffusion in the measurement container. Unassisted mixing may also
be arranged by use of highly miscible solvents, such as alcohol and
water, for the various ingredients.
[0116] The method can also include using a magnetic field to
enhance the reactions between the nanoparticles and the analyte.
The magnetic field to enhance reactions may be the same field as
that used for magnetic resonance measurements, or the two magnetic
fields may be different. In one embodiment the steps of a method
for enhancing reactions between the nanoparticles and the analyte
are: (1) place the analyte and nanoparticles in a fluid medium and
allow the nanoparticles to bind to the analyte to form complexes;
(2) apply a magnetic field to the complexes, thereby magnetizing
the nanoparticles; (3) then allow the magnetic field to exert
forces on the nanoparticles and allow the magnetized nanoparticles
to exert magnetic forces on each other; and (4) allow the complexes
to move responsive to those forces. For example, if the applied
magnetic field is non-uniform, the complexes are drawn into a
region where the magnetic field is strongest, and are concentrated
in that region. The interactions are then accelerated due to the
increased concentration of the complexes.
[0117] In different embodiments, steps (1), (2), and (3) occur in
various orders and simultaneously. The nanoparticles and analyte
may first be placed in a fluid medium and then bind with the
analyte to form complexes, or the complexes may be formed elsewhere
and then added to the fluid medium. The attachment of the reactants
to nanoparticles can be any association sufficiently strong so that
the reactant can be carried along with the nanoparticle when the
nanoparticle moves through the fluid medium under influence of
magnetic forces. The magnetic field may be applied before or after
the analyte binds to the nanoparticles. This method can also be
combined with the described mixing and/or temperature cycling.
[0118] The magnetic field to enhance reactions may be substantially
uniform or a highly non-uniform field, and may have a particular
shape or direction, and may be generated by external means, and may
be generated or shaped in cooperation with the reactants or the
nanoparticles or paramagnetic beads other magnetic entities. The
magnetic field to enhance reactions may be generated by
electromagnets, permanent magnets, superconducting magnets, or any
source of magnetic field. The strength of the magnetic field is
sufficient to magnetize the nanoparticles, which usually falls in
the range of about 0.01 to 20 Tesla. The field may be on all the
time, as with a permanent magnet, or it may be transient, as with a
pulsed electromagnet. Magnetization of the nanoparticles is
essentially instantaneous when they enter the magnetic field.
[0119] The magnetic field is produced by a magnet, which may be an
electromagnet, a permanent magnet, a superconducting magnet, or any
other source of magnetic field. The preferred magnet type depends
on the sample size. For small sample volumes of order 1 milliliter
or less, permanent magnets are preferred because they require no
electrical power, do not generate ohmic heat, and do not require a
cryostat. A wide variety of strong permanent magnet forms are
available, including NdFeB (neodymium iron boron) which forms
provide a strength (field-energy product) of 30 to 55 MGOe at
moderate cost. When mounted in a suitable magnetic circuit, these
permanent magnets are capable of generating fields exceeding the
saturation field of ferrite, which is the paramagnetic component in
many nanoparticles, and of producing strong field gradients of 1
Tesla/cm or higher. Such fields and field gradients are sufficient
for many magnetic separation applications including concentration
of nanoparticles, magnetizing paramagnetic beads, and chain
formation.
[0120] In step (3), the magnetic field to enhance reactions exerts
forces on the nanoparticles when it is non-uniform, and urges them
in the direction of increasing field strength. The nanoparticles
exert forces on each other, urging neighboring nanoparticles into
alignment with the applied magnetic field, drawing them together
when so aligned, and repelling them when positioned perpendicular
to the field. The various forces occur continuously and essentially
instantaneously.
[0121] In step (4) The analyte and nanoparticles move in the same
way because they are sufficiently strongly attached, that the
magnetic forces do not detach the analyte. Often the net motion is
to bring the analytes closer together, thereby promoting
interactions between them. That is the case when the applied field
is non-uniform, thus concentrating the complexes into the strongest
field region. For example if the non-uniform magnetic field draws
the complexes into a sub-volume comprising one tenth of the volume
of the initial mixture, then the average distance between reactants
is reduced by a factor of 2.16 which, for diffusion-limited
processes, increases the reaction rate by a factor of 4.7. The net
effect of mutual magnetic forces between nanoparticles is also
primarily to cause complexes to come together, when the complexes
are free to move, because those forces first realign the complexes
with the field, and then draw them together.
[0122] Alternatively, the magnetic field of steps (3) and (4) may
be substantially uniform. The nanoparticles exert mutual magnetic
forces on each other, due to the dipole-shaped magnetic fields
generated by the magnetized cores of the nanoparticles. These
dipole-dipole forces cause the nanoparticles to move in various
ways, and the motions influence the interactions of the
nanoparticles and of the attached analytes. The forces between
magnetized nanoparticles are illustrated in FIG. 5. The direction
of the applied magnetic field to enhance reactions is given by the
arrow 501. A particular nanoparticle 502, and neighboring
nanoparticles 503, 504, and 505 are shown. All of the nanoparticles
in FIG. 5 are magnetized in the same direction, as indicated by the
small white arrows. The nanoparticles 502 and 503 are aligned with
the applied field, and thus attract each other. The force exerted
on nanoparticle 503 by nanoparticle 502 is shown by a gray arrow
506, which points toward nanoparticle 502 signifying that
nanoparticle 503 is attracted toward nanoparticle 502. An equal an
opposite force exerts on nanoparticle 502, but for graphical
clarity is not shown.
[0123] Also in FIG. 5, another nanoparticle 504 is in parallel
alignment with 502 but on the other side of nanoparticle 502.
Nanoparticle 504 is also attracted toward 502 as shown by arrow
507.
[0124] Nanoparticle 505 is perpendicularly oriented relative to 502
and the field. Correspondingly, the dipole-dipole force exerted on
505 is repulsive, as shown by arrow 508. Not shown are additional
forces which the peripheral nanoparticles 503, 504, and 505 exert
on each other. In an actual mixture, all of the nanoparticles exert
forces on each other continuously.
[0125] Dipole-dipole forces tend to produce linear chain-like
structures. As an example, the nanoparticles can include a bonding
means represented as A with the nanoparticle represented as N. The
bonding means represented by A may be polyclonal, or able to bond
to multiple nanoparticles. Accordingly, in step (1) the reactants
bond to nanoparticles forming complexes symbolized as A-N. Then the
interactions of step (4) may produce structures of the form N-A-A-N
when the reactants bond as identical partners, or of the form
N-A-N-A when the reactants bond to a nanoparticle. Further
complexes may be added to form long chains under either scenario.
Alternatively, two different bonding means of type A (a 3' probe)
and C (a 5' probe) may be attached to the same nanoparticle. Then
the complexes are of the form C-N-A-B, and the chains are then of
the form C-N-A-B-C-N-A-B-C-N-A-B-C-N.
[0126] FIG. 6 is a schematic representation of the formation of a
chain structure from nanoparticles and analyte. The example chain
structure depicted in FIG. 6 can be formed using the methods and
systems described above with a uniform or non-uniform field. The
type of chain formation formed depends on the type of nanoparticle
employed. A nanoparticle 601 and an analyte 602 are in a magnetic
field with a direction indicated by arrow 603. The nanoparticle 601
is of the type that can attach to multiple reactants, that is, for
example, a polyclonal nanoparticle. The analyte 602 is of the type
that can form bonds to multiple nanoparticles. An example is
protein G which can attach to two nanoparticles treated to receive
such reactants. In step (1) the nanoparticle 601 and reactant 602
are attached, for example by mixing protein G with suitable
nanoparticles in water and incubating them for 4 hours at 37 C.
This produces a nanoparticle-reactant complex 604. Multiple such
complexes come together in the magnetic field 603 and form a chain
605 of the general type N-A-N-A-N-A-N etc.
[0127] The method can also include temperature cycling wherein a
sample may be heated or cooled at a fixed location, or the sample
may be moved between locations maintained at high or low
temperatures. The method can include taking measurements before,
during, and after such temperature changes. For example, a
measurement for T2 may be taken immediately upon mixing the sample,
and again after a period of heating and cooling when the sample
comes to equilibrium temperature. Comparison of the T2 values
before and after thermal processing will reveal reactions, such as
analyte binding to nanoparticles, which occurred during thermal
processing.
[0128] The method can include the steps of changing the temperature
of the sample and then measuring the T2 parameter. Temperature
affects the nanoparticle interactions and the magnetic resonance
measurement. Selective binding between the analyte and the affinity
molecules on the nanoparticles may be accelerated by raising the
temperature, particularly for diffusion-limited reactions. Thus the
method may include measuring the T2 of a mixture of nanoparticles
and unknowns within the liquid at a first temperature, preferably a
sufficiently low temperature that the analyte has not reacted with
the nanoparticles when the measurement is made. The method may then
include the step of heating the sample to a second temperature
sufficient to promote analyte-nanoparticle interactions. The method
may include measuring the T2 at the second temperature to observe
effects of the binding. The method may include a further
temperature change, such as return to the first temperature, and
further T2 measurements to confirm that the T2 of the sample after
the various temperature changes differs from the T2 of the sample
before the temperature changes. The steps provide many advantages,
including improved discrimination against interferents,
demonstration that the T2 change is due to analyte-specific
binding, and a check for instrumental errors.
[0129] The method may include heating the sample to a temperature
sufficient to disrupt the analyte-nanoparticle aggregations, thus
producing a solution of analyte-nanoparticle binaries, with a
corresponding T2 change. The temperature may be raised further
until the analyte is disbonded from the nanoparticles, thus
releasing analyte back into the solution and causing a further T2
change. The temperature may then be lowered until binding or
aggregation is restored, with corresponding reversion of T2 to the
earlier value. This behavior in T2 versus temperature would
strongly discriminate against interferents or instrumental errors,
and would confirm the presence of analyte.
[0130] The method may include the step of measuring the T2 of the
sample material prior to mixing with nanoparticles. This would
reveal a sample material which causes a shift in T2, such as a
high-viscosity solution or chelated iron in the sample. When the
sample material causes only a small T2 shift, the measurement may
proceed as usual, but in analysis the T2 of the processed sample
may be compared to that initially observed in the raw sample to
determine whether analyte is present. When the sample produces a
large T2 shift, it may be advantageous to dilute the sample until
its effects are low enough to permit the magnetic resonance
measurements. Analyte in the diluted sample may then be detected as
described. When the sample produces such a large T2 shift that
magnetic resonance measurements are prohibited, the invention can
flag that sample as un-testable, thereby avoiding a false alarm, or
it can archive the sample for further analysis.
[0131] The method can include preparing a magnetic field in a
particular way. The field may be prepared by first generating a
substantially uniform first magnetic field with sufficient
intensity to permit magnetic resonance measurements, and then
perturbing that field locally to produce a second magnetic field,
distinct from the first, within a special region. The second field
is distinct from the first when the magnetic resonance signals of
the liquid outside the special region are influenced by or can be
distinguished from signals of liquid inside the special region. For
example, the second field can be created by mixing or dissolving
paramagnetic particles, for example, those nanoparticles described
above, in the liquid. The nanoparticles then spontaneously generate
the second magnetic field, in a region closely exterior to the
nanoparticles, as a result of magnetization of the nanoparticles by
the first magnetic filed. Alternatively, paramagnetic ions such as
chelated iron or gadolinium could be employed instead of
nanoparticles. An advantage of this approach is that
diffusion-limited reaction rates may be increased due to the higher
mobility of metal-ion chelates. Similar ions are used in MRI
imaging (Gd-DTPA and Gd-DOTA.).
[0132] Holding the analyte within the special region can be
accomplished by reacting or binding or otherwise attracting the
analyte to a material surface or molecule for which the analyte has
particular affinity. Such holding may be accomplished by hydrogen
bonds, ionic forces, covalent bonds, van der Waals forces,
electrostatic forces, or any other type of molecular or material
attachment. For example, the holding mechanism may be an antibody
raised against an analyte protein, or DNA complementary to analyte
DNA sequences, and can include any material surface or molecule for
which the analyte has an affinity. Preferably the holding mechanism
also has null affinity or negative affinity for all solutes other
than the analyte which may be present. Preferably, the holding
mechanism is secured proximate to the special region, so that the
analyte will be held within the special region. For example, when
the special region is exterior to a nanoparticle, antibodies to the
analyte, or the other holding mechanisms mentioned above, may be
attached to the surface of the nanoparticle, so that the analyte
will be held adjacent to the nanoparticle within that region and
the liquid will be excluded. Optionally, the nanoparticle may
include multiple antibodies, or complimentary DNA, or other binding
agents so as to interact with a number of different, but selected,
analytes. For example, the nanoparticle could be adorned with
complementary DNA for anthrax, antibodies for ricin, and
complementary DNA sequences for smallpox, thereby enabling
detection of any of these analytes in a single mixture.
[0133] The magnetic resonance measurements and analysis to
determine whether the analyte occupies the special region can
include analyzing the magnetic resonance signals by spectral
analysis to seek a frequency component characteristic of the
special region. That frequency component, if present, indicates
that the liquid is in the special region, and therefore the analyte
is not present. Alternatively the step could include applying the
CPMG procedure, and analyzing the signals to determine the T2 of
the liquid. The T2 distribution may be a single exponential
component, or it may include a multitude of components, depending
on the spin diffusion rate. In either case, however, a T2 which is
longer than the T2 of the baseline case (liquid with the
nanoparticles and no analyte) indicates the presence of the
analyte.
[0134] A variation of the method includes forming an aggregate
comprising a plurality of analyte entities. Then, a reduction in T2
(compared to the baseline) indicates the presence of the analyte.
For example, an aggregate of nanoparticles with attachment
mechanisms and analyte molecules may form when both nanoparticles
and analyte molecules have multiple attachment points. Since the
aggregation results in a decrease in T2, whereas binding of analyte
to nanoparticles results in an increase in T2, it is important to
previously calibrate the signals, so that the expected sign of T2
change is known in advance. Nanoparticle stoichiometry can be
adjusted to prevent agglomeration or to cause agglomeration
depending on the measurement process to be used.
[0135] In one embodiment, analyte causes nanoparticles to form
extended aggregates. Membrane filters are used to separate those
aggregates from the liquid medium. The pore size of the filter is
preferably larger than the size of the nanoparticles or of the
analyte, but smaller than the aggregates. When an agglomerated
sample is filtered, the filtrate has a reduced concentration of
both nanoparticles and analyte, which are thus both greatly
concentrated as a filter cake. When secondary analysis means are
desired, for example to confirm detection of a microbe, the filter
cake is used for that secondary analysis. Likewise, the filtrate
liquid may be re-measured using the inventive system as an
additional check, since the T2 of the filtrate should be much
longer than of the initial nanoparticle solution when most of the
nanoparticles have been filtered out.
[0136] The method may include the steps of measuring the T2 value
of a standard. Here a standard is any material which has a known
T2. Preferably the T2 of the standard is unchanging in time and is
known from prior calibration measurements. For example the standard
may be a solution of nanoparticles or of copper sulfate with a
concentration adjusted to provide a particular value of T2.
Standards enable detection and correction of instrumentation
drifts. The standard may be a liquid which is not a solution, such
as an oil selected to have a T2 in the desired range. The standard
may be arranged to have a T2 substantially equal to that of an
analyte-free sample, in which case it is called a negative
comparator. The standard may have a T2 close to that produced by
the analyte, a positive comparator. The method may include
measuring the T2 of multiple standards with different T2
values.
[0137] The method can include the step of testing a positive and/or
a negative control. A positive control can be a benign analyte,
such as bacillus subtilis along with nanoparticles sensitized to
it. The positive control may be analyzed at any time, and should be
detected in the same way as a threat analyte. Preferably the T2
change produced by the positive control is known from prior
calibration, and testing the positive control should always produce
the expected T2 change, and failure to do so would reveal a
malfunction in the system. A negative control is a benign analyte
along with nanoparticles sensitized to some other materials, for
example bacillus subtilis combined with nanoparticles sensitized to
anthrax. The negative control should never produce a T2 change
because the analyte and the nanoparticles are not matched. If a
negative control produces a T2 change, it would reveal a
malfunction of the system. An advantage of running positive and
negative controls is that the entire sample collection, fluidics,
sample processing, and detection stages are tested realistically.
For comparison, the positive and negative comparator standards
discussed in the previous paragraph test only the magnetic
resonance portion of the system, not the sample processing
stages.
[0138] The method can include the steps of producing both an
increase and a decrease in T2 of the sample. The increase or
decrease in T2 depends on the properties of the nanoparticles,
ratios of other reagents such as antibodies, and on other
processing parameters. Thus a sample may be tested for a T2
increase using processing steps to generate a T2 increase when
analyte is present, and then the same sample may be tested for a T2
decrease by adding the ingredients or processing steps which
produce a T2 decrease. Observation of both increasing and
decreasing T2 values would enhance the reliability of the analysis
and reduce the false alarm rate. Alternatively, two aliquots drawn
from the same sample may be processed to generate a T2 increase in
one and a decrease in the other.
[0139] Interferents are materials which, if present in a sample,
cause a change in T2 mimicking that of the target analyte. Most
interferents produce a shorter T2, including materials containing
chelated iron and materials causing an increase in viscosity of the
liquid. Thus the effects of analyte and interferents may be
discriminated by processing the sample so that the analyte will
produce a T2 increase. For even greater analyte-interferent
discrimination, both increases and decreases in T2 may be arranged,
either by sequential processing of the same sample or by comparison
of parallel aliquots.
[0140] The method can include the step of measuring the T2 of a
nanoparticle mixture prior to adding the sample material to that
mixture. The advantage of this step is that any errors in the
nanoparticle concentration or properties would be revealed before
the sample material is used. If the nanoparticle solution exhibits
an unexpected value of T2 (for example due to a high or low
nanoparticle concentration from a metering error) then the
nanoparticle solution may be dumped and a new nanoparticle solution
may be prepared. If the nanoparticle solution exhibits a value of
T2 close to that expected, then the nanoparticle solution may be
employed. Preferably the measured value of T2 is then used in the
analysis for comparison against the T2 of the mixed and reacted
sample, thereby negating errors due to nanoparticle concentration
and also improving reproducibility.
[0141] The method may include the steps of mixing the sample
material and nanoparticles in the liquid, then measuring the T2 of
the mixture, then promoting reactions between analyte and
nanoparticles, and then measuring the T2 after such reactions. For
example, the sample may be shaken or heated to promote the
reactions. Simultaneous mixing and heating may be used to
accelerate reactions. Comparison of the T2 of the mixture before
and after the reactions reveals the analyte. An advantage of these
steps is that any errors in the volumes of sample and nanoparticles
would be detected and negated.
[0142] In one embodiment hazardous chemicals are generally not
required. For example, analytes can be tested using only water,
salts, nanoparticles, and harmless proteinaceous reagents such as
antibodies.
[0143] System Description
[0144] One embodiment of a system which can carry out or implement
the measurement or detection techniques described above will now be
described with reference to FIG. 7 which is a functional block
diagram of a magnetic resonance system generally indicated as 700.
The system includes magnet or magnet system 712. In one embodiment
the magnet 712 is a permanent magnet configured to produce a 0.5
Tesla magnetic field with 0.01% uniformity within a sample area or
volume 714 of 1 ml. Alternatively, the magnet system may include an
electromagnet, a superconducting coil, or any other source of
magnetic field. A coil or antenna 716 is located adjacent to the
sample volume. In one embodiment the coil encircles the sample
volume 714. A pulse generator 718 is coupled to the coil 716 to
provide electromagnetic pulses at the desired Larmor frequency to
the sample volume 714. An amplifier 719 may be placed between the
pulse generator and the antenna to amplify the signal from the
pulse generator. A receiver 720 is also coupled to the coil 716 so
as to receive signals picked up by the coil. A preamplifier 721 may
be placed between the receiver and the antenna to amplify the
antenna signals. The receiver 720 converts the received signals
into a digital form. A controller 722 is in communication with the
pulse generator 718 and the receiver 720. The controller controls
the operation of receiver and the pulse generator. The controller
also receives the signals received by the receiver after they have
been converted into the digital form. The controller 722 can be a
general purpose processor, a digital signal processor (DSP), an
application specific integrated circuit (ASIC), a field
programmable gate array (FPGA) or other programmable logic device,
discrete gate or transistor logic, discrete hardware components, or
any combination thereof designed to perform the functions described
herein. Alternatively, the functions of the controller, pulse
generator, receiver, and user interface may be combined into a
single unit such as an ASIC or FPGA, or a board integrating such
circuits. A user interface system 724 is coupled with the
controller 722. The user interface system 724 provides a mechanism
for interaction between a user and the system 700. The interface
system can include, for example, a display such as a liquid crystal
screen, indicator lights, a key board, a mouse, an audio speaker, a
microphone, switches, or a touch screen.
[0145] In an alternative embodiment, a concentrating magnet 704 is
provided which includes a field-concentrating pole piece 705 made
of, for example, steel. The magnet 704 can be a permanent NdFeB
magnet and is magnetized as indicated by the white arrow 706. The
magnet 704 and pole piece 705 produce a magnetic field passing
through the sample volume 714. The shape of the pole piece 705 is
selected so that the magnetic field is highly non-uniform and
produces a strong gradient throughout the sample volume 714. The
region where the magnetic field is strongest in the sample volume
is in the region closest to the pole piece 705. The concentrating
magnet 704 can be mounted within the magnet 712 or can comprise a
portion of the magnet 712. For example, the magnet 712 can produce
a uniform magnetic field in a first region, and a nonuniform field
in a second region. Then the sample may be moved between regions of
the magnet 712 to perform different tasks. For example the sample
may be moved so as to concentrate reactants using the nonuniform
field region, and then moved to the uniform field region for
measurements.
[0146] Alternatively, the concentrating magnet can be located away
from the magnet 712. In that embodiment, the sample can be exposed
to the field of the concentrating magnet, and can then be placed in
the coil 716.
[0147] In one embodiment, the field gradient produced by the
concentrating magnet acts on the magnetization of the complexes
(analyte plus nanoparticle) to draw them into a sub-volume of the
sample volume. In that sub-volume, the local concentration of
complexes is increased, and continues to increase as further
complexes continue to arrive from the rest of the sample. For
reactions which are limited by diffusion of the reactants,
increasing the concentration of the reactants reduces the average
diffusion distance between reactant partners and accelerates the
reaction rate correspondingly. For reactions limited by a reaction
barrier, the reaction rate is enhanced due to attractive magnetic
forces between the complexes.
[0148] The RF coil can be made small enough to interrogate volumes
of micro liter size. The coil can be made large enough to
accommodate liters of sample. FIGS. 8a-d are a representation of
four configurations of the antenna, each in perspective view. In
part a of the figure, a solenoidal coil is shown having a density
of windings which is constant along the length of the coil. The
sample is placed inside the coil for measurement. The coil acts as
an antenna to couple RF energy into the sample nuclei, and also to
couple the magnetic resonance signal from the nuclei out to the
rest of the system.
[0149] In part b of the figure, a solenoidal coil having a variable
winding density is shown. The winding densities are higher at the
ends of the coil than at the middle. An advantage of using a
variable winding density is that the RF magnetic field generated by
the coil may be made more uniform than that of a coil of the same
size with constant winding density.
[0150] In part c of the figure, a two-turn single-sided coil is
shown. An advantage of this configuration is that an elongated
container such as a tube may be inserted and removed without
disconnecting either the coil or the tube.
[0151] In part d of the figure, a coil configuration is shown
wherein four loops cooperatively generate a transverse RF magnetic
field. Elongated samples may be inserted without disconnecting the
coil or the tube.
[0152] The specific user interface and output of the system are
highly application-dependent, but will typically include
transmission of information dependent on detection of analyte. For
example, such communication may involve recording or archiving test
results, displaying a threat alert message, illuminating an alarm
or beacon, or activating an acoustical alarm. Communicating data
also includes sending signals to other devices, such as
automatically shutting off an HVAC system or sequestering a test
sample responsive to detection of selected analytes. The
communication via the user interface can include electronic,
optical, infrared, radio, microwave, mechanical, or acoustical
means, or any other means for transmitting data or commands
responsive to analyte test results. Additionally, the user
interface can include remote communication interfaces such as a
network interface card and a wireless access card which are in
communication with the controller. These can allow an operator or
another device to communicate with the system, to relay commands or
retrieve data or convey an alarm. The communication may include
transmitting information by the internet, by a local network, or by
direct electronic or wireless link.
[0153] In one embodiment, the system is configured in two separate
chassis, one with the magnet 712, the pulse generator 718 and the
receiver 720. The other chassis has the controller 722 and the user
interface 724. The two chassis exchange information such as
commands and data by an electronic communication link, for example,
cables, a wireless link, or a fiber optic link. In a preferred
embodiment, the communication link comprises a USB interface
employing standard USB connections on each chassis.
[0154] The magnetic resonance system 700 can excite magnetic
resonance signals from the hydrogen nuclei in water in the sample
volume 714 by applying electromagnetic pulses, for example radio
frequency (RF) pulses, generated by the pulse generator 718 via the
coil. The system detects the magnetic resonance signals from the
hydrogen nuclei in the water by inductively picking up the signals
in the coil 716. The receiver processes the received signals using
amplifiers, mixers, and analog-to-digital converters.
[0155] In one embodiment the system 700 measures the T2 of the
water by the CPMG procedure or technique under the control of the
controller 722. The measurement includes a 90-degree RF pulse
generated by the pulse generator followed by a 2 msec delay, and
then a string of 2000 180-degree pulses at 4 msec intervals. The
phase of the 180-degree pulses is orthogonal to that of the
90-degree pulse. The procedure generates spin echoes in the 4 msec
intervals which are received by the receiver 720. In one embodiment
the controller 722 performs an analysis routine which determines
and records the spin echoes, performs FFT analysis to obtain
spectral peaks, finds the maximum value of the peaks, and fits the
peak values to a formula with three variables: the amplitude and
decay time of an exponential, plus a time-independent background.
The observed T2 value is the best-fit exponential decay time.
[0156] The analysis performed by the controller includes a
comparison between the observed T2 value and a previously
calibrated or measured T2 value. The analyte is detected by the
system when the observed T2 value of the sample differs from that
of an analyte-free sample. The previously calibrated T2 value can
be determined by measuring a solution of water with the same
concentration of nanoparticles as is used for the measurement of
the analyte. The T2 of the water is influenced by the concentration
of nanoparticles. The T2 is also influenced by analyte binding to
the nanoparticles and occupying the high-gradient region around the
nanoparticles. In the preferred embodiment, the nanoparticle
concentration is controlled by formulation of the solution. The T2
values of the solution without analyte, and with various
concentrations of the analyte, are also known by prior
calibration.
[0157] In one embodiment the nanoparticles are dissolved or
suspended in a water medium. The nanoparticles have a
superparamagnetic magnetite core with a diameter of 8 nm,
surrounded by a shell with a diameter of 50 nm. Antibody molecules
(or other binding or attracting mechanism as described herein)
specific to the analyte are bound to the shell. When the
nanoparticles are in the sample 714, the core is magnetized by the
field applied by the magnet 712. The magnetized core produces a
local dipole field which adds to the applied field. The resulting
net field includes spatial gradients of up to 0.1 T/nm, within a
region extending radially from the surface of the nanoparticle to
about 20 nm from the surface. The nanoparticles are most effective
for detection and measurement purposes in low concentrations of
about 1:10000 in water. That results in very little consumption of
the nanoparticles per test. In one embodiment the magnet 712 of the
magnetic resonance system 700 uses a permanent magnet for this
purpose. The permanent magnet requires no power, may be made
arbitrarily compact, and is economical. Most prior magnetic
resonance systems employed electromagnets or superconducting coils
to generate the magnetic field. It is not feasible to arbitrarily
reduce the size of electromagnets or superconducting magnets. If an
electromagnet is scaled down in size, the magnetic field scales
proportionately. If the field is held constant, then the current
density in the electromagnet coils must be increased. Current
density can not be increased arbitrarily because of a fundamental
limit, the conductivity of copper. Above a certain current density
limit, roughly 100 amps/cm.sup.2, the coils must be water-cooled.
Above a second limit, roughly 200 amps/cm.sup.2, the coils
self-destruct. Small, high-field, steady-state copper coils are not
feasible.
[0158] It is likewise not feasible to reduce the size of
superconducting magnets arbitrarily. Superconducting coils may be
made much smaller and more powerful than nonsuperconducting coils,
and can carry high current densities. However, superconducting
coils must be surrounded by a vacuum-insulated cryostat, usually
having multiple shells maintained at different cryogenic
temperatures. Also, the various shells are mechanically and
thermally interconnected by support struts. It is not possible to
make the cryostat arbitrarily thin because of the thermal
conductivity of support members. The cryostat limits the
miniaturization feasible in superconducting magnets.
[0159] Permanent magnets have neither of these defects. A given
magnet design using permanent magnets will scale precisely, with no
change in geometry or field or field quality, to arbitrarily large
or small dimensions. The only fundamental limitation is the
ferromagnetic domain size, about 1 micron. By designing permanent
magnet systems, the magnets may be scaled to a size determined by
the sample volume, the RF coil properties, or other parameters of
the system, rather than forcing the other parameters to comply with
the magnet scale. As a result, it is feasible to miniaturize the
entire electromagnetic system. This leads to improved detection
sensitivity in smaller sample volumes, reduced cost and weight of
the sensor portion of the system, and reduced RF power
required.
[0160] One embodiment of the magnet 712 is depicted schematically
in cross section in FIG. 9. The magnet includes a frame 910, such
as a hollow steel frame. In one embodiment, the height H of the
frame is less than 50 cm and may be less than 5 cm. The width W can
also be less than 50 cm and can be less than 5 cm. An upper
permanent disk magnet 914 is attached to an upper section of the
frame, and a lower permanent disk magnet 916, located opposite the
upper permanent magnet, is attached to a lower section of the
frame. For example, the disks can be mechanically attached using
screws or bolts and/or they can be attached with an adhesive. A
disk shaped upper pole piece 918 is located atop the upper
permanent magnet and opposite a disk shaped lower pole piece 920
located atop the lower pole piece. Around the periphery of each
pole piece are eight fine-threaded holes with adjustment bolts,
which may be varied to improve the uniformity of the field. The
magnet is assembled by bolting the frame together, sliding the
permanent magnet disks into position, sliding the pole pieces into
position, and then shimming. The permanent magnet disks are very
strongly attracted to the steel frame, and the pole pieces are very
strongly attracted to the permanent magnet disks. The attractions,
and resulting friction among the various contacting members,
provide mechanical stability to hold the assembly together. Further
robustness may be obtained by applying clamps or adhesives to the
magnet disks or pole pieces, preferably not interfering with field
shimming or magnetic resonance measurements. Forces on permanent
magnet components are strong and potentially dangerous. Not shown
are jigs and tools used to control the assembly process in view of
the strong forces involved.
[0161] Shimming is the process of adjusting a magnet, such as
magnet 712, to produce the necessary uniformity. As built, most
magnets provide insufficient uniformity due to manufacturing
tolerances. Shimming consists of measuring the field distribution,
adjusting built-in parameters of the magnet, and repeating until
the desired uniformity is achieved. In one embodiment a simple
shimming design is utilized which focuses on the most important
field parameters, rather than providing an exhaustive set of
parameters of which most are never needed.
[0162] First, the magnetization of the two permanent magnet disks
is equalized. Based on the observed axial gradient, one or more
thin ferromagnetic sheets are affixed by magnetic attraction
circumferentially around only the stronger of the two magnets.
Iterative adjustment of the number and thickness of the sheets
results in near-perfect negation of the axial gradient. The sheets
may then be secured by clamps or adhesives.
[0163] Then, one or more of the miniature bolts, for example bolt
922, in the periphery of the pole pieces are adjusted. These bolts
press against the permanent magnet disks to slightly rock the pole
pieces as needed to negate transverse field gradients. Either or
both pole pieces may be adjusted, depending on the details of the
observed field. Final adjustment of the various bolts results in
near-perfect negation of transverse gradients.
[0164] Typically the shape of the pole pieces need not be altered,
although they can be demounted and their shape revised if needed to
achieve the desired field. Alternatively, the spacing between the
pole pieces may be reduced slightly by tightening all of the bolts
around both pole pieces. Such an adjustment is almost equivalent,
magnetically, to adjusting the depth of the pole piece relief
step.
[0165] To fabricate the magnet parts, powdered metals such as iron
or steel can be placed inside a mold of desired shape. Then in the
press pressure and heat are applied to generate the final part.
While only small parts can be made by this technique, mass
manufacturing can be achieved. Alternatively machining can be used
to make the individual parts.
[0166] The pole pieces can be designed to provide the highest field
uniformity and field volume for sample testing, with the constraint
that the gap be sufficient for inserting and tuning the magnetic
resonance sample coil. Design constraints include the maximum field
in the pole pieces to limit saturation, minimum number of shimming
parameters to achieve target field uniformity, and use of low-cost
commercial permanent magnet components where possible.
[0167] The permanent magnet material provides very high
magnetization density, but is temperature sensitive. In
applications where the frequency may be adjusted to the field,
thermal drift of the magnetic field is not a problem. For precision
T2 measurements, however, it is necessary to stabilize the magnetic
field. A temperature-controlled enclosure can be used. In one
embodiment, the enclosure can be built using foam insulation and a
pair of patch heaters. A thermocouple sensor and controller
complete the arrangement.
[0168] Precise determination of T2 using the CPMG procedure is
enhanced with an extremely stable local oscillator with minimal
phase noise on a time scale of at least the spin echo spacing. Even
high-cost crystal oscillators usually do not provide sufficient
stability due to the noisy computer power lines. Sufficient
stability can be obtained using inexpensive integrated crystal
oscillators by buffering both the DC power input, and the RF clock
output. Such an arrangement is depicted schematically in FIG. 10.
In one embodiment the oscillator shown in FIG. 10 is used in the
pulse generator 718 of FIG. 7. In general, the DC (direct current)
power input is buffered by wiring two or more voltage regulators in
series. The circuit depicted in FIG. 10 includes a first voltage
regulator 1002 (for example an 8 volt regulator which receives a
+12V input). A second voltage regulator 1004 receives the output of
the first voltage regulator and provides its output to the
oscillator 1006 (for example, a 5 volt regulator, receiving the
output of the 8 volt regulator). A third voltage regulator 1008
(for example, a 5 volt regulator) can also receive the output of
the first voltage regulator and can provide its output to a digital
logic gate 1010 with high speed and high source isolation, such as
the 74F3037 line driver NAND (available from Philips Semiconductors
and others). The digital logic gate 1010 buffers the output of the
oscillator.
[0169] The magnetic resonance system 700 (FIG. 7) interacts with
the sample using the antenna 716 which, in operation, is
electromagnetically coupled to the precessing nuclei of the sample.
In one embodiment the coil is mounted in a modular, interchangeable
platform to enable changing the sample size, replacing the coil in
case of contamination, or other changes needed.
[0170] The antenna may be encapsulated in a contamination-resistant
material. Contamination is a serious issue when multiple samples
bearing multiple diseases or toxins are to be tested. Prior
antennas are difficult to clean because they are highly convoluted
geometrically and include non-hygienic insulator and conductor
materials. Encapsulation of the antenna can resolve this issue. For
example, the antenna could be a copper coil embedded in a hollow
cylindrical Teflon form so that any contamination coming from the
sample container would encounter only a Teflon surface, never the
actual conductor. Since Teflon is non-absorbing and relatively easy
to clean up, contamination issues are greatly reduced. Also, the
encapsulated antenna would be more stable and mechanically rugged
than a freely mounted coil. Magnetic resonance signals from an
element in the encapsulant, such as deuterium or fluorine, may be
used to control a frequency or a magnetic field.
[0171] Cancellation of noise, interference signals, baseline
offsets and other background effects can be improved by performing
magnetic resonance measurements multiple times with various RF
phases alternated. This can be implemented under the control of the
controller. For example, the excitation may be alternated between
positive and negative phase rotation of the spins during RF pulses.
During signal processing by the controller, the phase of the
receiver oscillator can also be rotated by 90 degrees or its
multiple. Analysis software in the controller controlling these
phase alternations also performs the corresponding addition or
subtraction of the digitized data to accumulate the desired signal
while canceling backgrounds.
[0172] Various user interfaces can be provided with the system. For
example, the system 700 depicted in FIG. 7 can carry out
measurements to detect a selected analyte or analytes and report
the results by issuing an alarm if detected or provide a visual
indication or report via the user interface 724. In one version,
the operator inserts a mixed sample into the system and presses a
single button on the user interface to initiate a previously
prepared series of instructions for the controller to carry out and
analyze the sample. If more than one analyte is to be searched for,
the instructions automatically direct the mixing of nanoparticles
sensitized to each analyte and carries out the measurements
sequentially. In another version of the instrument, a mechanical or
optical switch senses the insertion of the sample into the magnetic
resonance system, and automatically initiates the measurement
sequence.
[0173] In one embodiment, a T2 change is the primary indicator that
analyte is present. To check for drifts or errors which could
affect the T2 measurement, the system can compare the measured T2
of the sample, with that of a sealed calibration sample having a
previously measured T2 value. The sealed sample may contain copper
sulfate in water, mineral oil, or other liquid having a stable T2
for comparison. Alternatively, the sealed calibration sample can be
periodically measured.
[0174] A wide diversity of mechanisms for presenting the sample
into the magnetic resonance system can be used. The sample,
comprising liquid medium, analyte, and nanoparticles, can be mixed
in a container such as a glass NMR tube, a plastic tube or vial, a
disposable container such as a plastic microcentrifuge tube or
flask, or other suitable container. An advantageous polymer is PEEK
(polyetheretherketone) due to its toughness, intertness, and
machinability. The container may be coated with a material to
prevent nanoparticles from adhering to the walls, clumping, or
precipitating out of the mixture. For example, the coating may be a
protein such as BSA (bovine serum albumin). The container including
the sample may be inserted, manually or by a mechanical feeder,
into the magnetic resonance system. Alternatively, a fixed
container in the magnetic resonance system may be used for multiple
sample measurements by inserting sample liquids into the container,
for example by pumping the sample or its ingredients through tubes
into the container. After the measurements, the sample is then
drawn from the fixed container using pumps, tubes, valves, and
related fluid flow devices. A washing or rinsing step can be
carried out between samples. Ultraviolet treatment of reservoirs
holding distilled water and nanoparticles can be carried out to
prevent bacteria formation. Alternatively, a fungicide such as
sodium azide can be mixed in the distilled water in trace
quantities to prevent growth of bacteria and algae in the
water.
[0175] In one embodiment depicted schematically in FIG. 11,
multiple sensor units are connected to a single controller. For
example, an automated, fixed-site system may consist of one central
controller 1102 with power supplies and a pulse generator or
transmitter, connected by cables to multiple remote sensor heads
1106 a and b. Though only two sensor heads are depicted, more can
be used. Each head 1106 includes a sample preparation apparatus
along with selected nanoparticles, a magnetic resonance magnet, a
preamplifier and a coil, for example as were described in
connection with FIG. 7. RF power pulses are routed to the sensor
units through an output multiplexer 1108 which is controlled by the
controller 1102. Signals from the sensor units are routed to the
receiver 1110 through the input multiplexer 1112, also controlled
by the controller. Interconnects are preferably by coaxial cable.
Alternatively, each sensor unit may include an RF amplifier. When
the RF amplifier is located at the sensor unit, the interconnects
do not carry high power RF pulses and thus may be wireless, fiber
optics, or other communication means as well as coaxial cable. The
elements of the system depicted in FIG. 11 operate in the manner
described above.
[0176] In one embodiment, particulate matter suspended in air may
be drawn from free air, HVAC ducts, interior spaces such as
shopping malls, subway trains and other mass transit areas, or any
other air system to test for diseases or terrorist attack. (HVAC
stands for heating, ventilation, and air conditioning.) Collection
preferably includes drawing particulate matter into the system or
concentrating particles from the air into the liquid medium. FIG.
12 shows a schematic of such a monitor system. The collector 1202
can be situated within a duct or in any other area to be monitored,
and can include a shroud (not shown) to exclude dirt and insects.
The collector 1202 can include an electrostatic concentrator to
attract analyte or sample material. A fluidics system 1204
transports the analyte from the collector 1202 to the concentrator
magnet system 1205 where the sample is exposed to a non-uniform
magnetic field as has been described above. Alternatively, the
concentrator magnet system can produce a uniform field. The fluidic
system then transports the sample from the concentrator magnet to
the sample area of the magnetic resonance analyzer or system 1206.
The magnetic resonance system 1206 can be the system described in
connection with FIG. 7. The fluidics system 1204 can include an
automated microfluidic mixer to mix analyte with a liquid, such as
the water medium and with nanoparticles configured for the one or
more analytes to be detected. A reservoir of the nanoparticles and
the water 1208 can also be part of the fluidics system. The mixed
sample is then transferred by the fluidics system to the
concentrator magnet system 1205 where the sample is exposed to a
non-uniform magnetic field. The fluidic system then transports the
sample to the sample area of the magnetic resonance system where
measurements are made. In one embodiment a fluidic transport system
is in communication with the mixer and extends into the sample
area. Depending on the measurement results, the sample may be
dumped into a waste container, stored as archive material, or sent
to secondary analysis systems. The waste water may be recycled to
be used again by passing through a filter.
[0177] FIGS. 13 and 14 are schematic representations of magnetic
concentrator systems. The system depicted in FIG. 14 is one
embodiment of a system which can be used as magnetic concentrator
705 to carry out the methods described herein. The embodiment shown
in the figure is a flow cell vessel including an external-type
magnetic separation system. A flow cell 1301 contains the
nanoparticle-analyte mixture (represented by the light stipple).
Tubes 1302 carry the mixture into and out of the flow cell 1301. A
permanent magnet 1303 and a flux concentrator 1304 for example, a
field-concentrating pole piece, are located in proximity to the
flow cell 1301. The magnet 1303 has an associated magnetic field
with a direction indicated by the arrow. The flux concentrator 1304
conveys magnetic flux from the magnet 1303 into the flow cell 1301
and produces a non-uniform magnetic field and a strong field
gradient throughout the flow cell 1301. A high-field region 1305
(dark stipple) where the field strength is highest is created in
the flow cell adjacent to the flux concentrator 1304. The flux
concentrator 1304 is shaped in this embodiment so as to produce a
roughly linear high-field region 1305 extending along the surface
of the flow cell 1301. Nanoparticle-analyte complexes in the fluid
mixture are drawn toward the high-field region 1305, thereby
increasing the concentration of complexes and enhancing the
interaction rate.
[0178] FIG. 14 is a combined overhead plan view and an elevation
cross-sectional view of a magnetic concentrator system which can be
used with the analyzer systems described herein. Centrifuge tubes
1412 and 1413 containing the nanoparticle-analyte mixture (shown in
stipple) are placed in the magnet assembly 1411. The magnet
assembly 1411 includes a disk-shaped permanent magnet 1414 which is
a NdFeB permanent magnet disk with a strength of 42 MGOe and a
magnetization direction as indicated by the arrow. Topping the
permanent magnet 1414 is a pole piece 1415 which is a steel disk
from which six semi-circular notches 1416 have been cut. The pole
piece 1415 in cooperation with the permanent magnet 1414 generates
a strong, highly non-uniform magnetic field 1417 in the space
between the circular notches, and in which the centrifuge tubes
1412 and 1413 are placed. The purpose of the shaped pole piece 1415
is to redirect magnetic flux from the permanent magnet 1414, and
emit that flux radially as close to the centrifuge tubes 1412 and
1413 as possible. The outer shell of the magnet assembly 1411 may
be steel, to confine the processing region and carry flux back to
the permanent magnet 1414. The embodiment shown produces a magnetic
field of 0.7 Tesla in the sample volume closest to the pole piece
1415, and falling to about 0.2 Tesla at the opposite side of the
centrifuge tube 1412. It will magnetically process six samples
simultaneously, converting raw nanoparticle-analyte complexes in
solution, to a concentrated deposit of reacted chains, in a few
minutes.
[0179] FIG. 15 is a graph of magnetic resonance data with and
without magnetic processing. The data was collected as part of an
experiment to detect biological threat material by measuring the
magnetic resonance T2 parameter. The graph shows the change in T2,
relative to the initial value, over a 20 hour period for various
conditions. The bold (upper) line shows the average of three
measurements in which anthrax (bacillus anthraces DNA, plasmid
px01, concentration 20 ng/mL) interacts with nanoparticles via
selective binding to suitable probes attached to the nanoparticles,
and with magnetic processing according to the inventive method.
Here the sample mixture was exposed to a nonuniform magnetic field
ranging from about 0.8 Tesla at one side of the mixture, to about
0.2 Tesla at the opposite side of the sample, for the duration of
the test except when the T2 measurement was actually being made.
Also shown as x's are the individual measurements. A substantial
signal or T2 change of about 23 milliseconds was observed for the
sample having both analyte and magnetic processing. The solid fine
line shows the results for the same analyte but with no magnetic
processing. Negligible T2 effect is observed. The dotted and dashed
lines show the same measurement for control samples having no
analyte, with and without magnetic processing. The conclusion from
this experiment is that the inventive magnetic processing greatly
enhances the detectable magnetic resonance signal from low
concentrations of anthrax DNA.
[0180] FIGS. 16a-e depict an embodiment of a fixed installation
system as described in connection with FIG. 12 and three collector
intakes. FIG. 16a is a perspective view and FIG. 16b is a elevation
view of the system showing the intake 1602 and a display 1604. The
other elements depicted in FIG. 12a are contained within the
casing. FIGS. 16c-e depict three inlet options for the system. Once
started the controller causes the system to collect samples
periodically for analysis. The system can also be operated
manually. A user interface may be provided through buttons or a
touch screen. The display 1604 can show the status of operation.
User access can be controlled through, for example, biometric
identification, such as fingerprint identification, or a
password.
[0181] FIG. 17 is a front perspective view of a hand portable
system. The system can operate in a single button autonomous
operation mode. A sample can be introduced via vials and tubes. A
sample in a container 1702 can be introduced into the system
through a receptacle or opening 1704 at the top. Inside the system
the fluidics system will handle the sample mixing and moving into
the NMR system in the manner described above in connection with
FIG. 10a. The user interface 1706 can include "biohazard" and
"safe" lighted areas on a display screen. To start operation, a
start button is provided on the touch screen. The status of system
operation is indicated on the screen.
[0182] One embodiment of a system which can carry out or implement
the measurement or detection techniques described above for medical
diagnostic purposes will now be described with reference to FIG. 18
which is a functional block diagram of an automated sample testing
system. For clinical applications, the sample comprises a specimen
of material from a patient. The material may include living or dead
cellular material such as skin, blood, prions, marrow, hair, biopsy
samples, or other tissue; or non-cellular biological material such
as saliva, mucous, sputum, intravenous fluid, urine, feces, pus,
spinal fluid, and contents of the stomach or intestines; or any
other sample material obtained from a human or animal body.
Collecting that material comprises a patient or subject producing
the material, a clinician extracting the material from the body of
a patient or subject, an investigator retrieving sample material
from a crime scene or accident, or any other steps resulting in the
accumulation of biological material for testing.
[0183] First, the fluidic system 1802 draws a patient's specimen
1804, or a portion thereof, or a solution thereof, into a mixer
which mixes the sample material with a solvent, for example stored
in a solvent reservoir 1806, and one or more types of nanoparticles
stored in reservoirs 1808a-c. Each type of nanoparticle can be
sensitized for one or more chemicals or analytes related to
diseases or medical conditions. FIG. 18 shows three nanoparticle
types, but any number of nanoparticle types, each sensitized to one
or more analytes related to one or more medical conditions can be
used. Diseases include communicable pathogens such as viruses and
bacteria, and non-communicable diseases such as cancer or
hypercholestremia. Chemicals include enzymes or other markers
produced by the body, toxins, and drugs. In one embodiment, the
user selects the types of nanoparticles to be used in testing a
particular patient's specimen. For example, a physician may extract
a sample of a patient's blood to check the concentration of a
medication so as to control dosage, or guards at an airport or
border crossing may take tissue samples of live or dead chickens to
check for avian flu. Additional processing steps may include lysing
the sample to release DNA or RNA or other components of the sample,
heating or cooling the sample, adjusting the pH of the sample, or
other steps needed to promote selective reaction between the
nanoparticles and the analyte. The mixed sample, or an aliquot
thereof, is then transferred into the magnetic resonance system
1810, such as the system depicted in FIG. 7. The system depicted in
FIG. 18 can also include a concentrator magnet as was described in
connection with FIGS. 7 and 14 above with the samples being exposed
to the non-uniform magnetic field while in the magnetic resonance
system or prior to the magnetic resonance system. The sample may
alternatively be mixed with nanoparticles within a container which
is within the magnetic resonance instrument, thereby avoiding the
step of transferring the mixed sample, and additional processing
steps may be taken while the sample is within the magnetic
resonance instrument.
[0184] The magnetic resonance instrument then measures signals from
the sample, such as the T2 of the sample, and analyzes those
signals to determine the presence or absence or concentration of
the selected analytes. Then, based on the measurement results, a
physician may then diagnose the patient's disease.
[0185] In one embodiment of the systems described above, the system
detects analyte by measuring signals from the liquid, the signals
being related to the magnetic field. Specifically, the signals are
sensitive to the distinct magnetic field in the special region
around the nanoparticles. When analyte binds to the corresponding
antibody or other binding agent, the analyte is caused to remain in
the special region, and thus in the distinct magnetic field. The
analyte displaces the liquid from that region, so the liquid no
longer emits magnetic resonance signals characteristic of the
magnetic field in that region. Also, it is important to note that
the analyte does not emit magnetic resonance signals, or at least
does not emit signals which are similar to those of the liquid.
This is because the analyte is held tightly to the solid
nanoparticle, causing the analyte to exhibit the short T2
characteristic of solids. Thus, in one embodiment, the analyte,
while occupying the special region, does not produce signals that
mimic the liquid.
[0186] Agglomeration can cause a change in T2 but not T1, whereas
both T1 and T2 change in response to increased concentration of
nanoparticles. Therefore, a measurement of T1 can be used as a
calibration or an independent measure of nanoparticle
concentration. In one embodiment, the system measures both the T1
and T2 of the sample, applies analysis relating the T1 value to
determine the nanoparticle concentration, and the T2 value to
detect analyte. Alternatively, other methods are available to
measure the iron content, and hence nanoparticle concentration, in
the sample.
[0187] The data processing step performed by the controller
includes fitting the data for parameters related to the presence of
analyte, such as a T2 change in CPMG data. Normally the echo train
in CPMG is fit to a single exponential formula, a three-parameter
fit for amplitude, time constant, and background. A simple but
efficient way to accomplish this is a grid search in which all
three parameters are first estimated from the data, and then a
three-dimensional grid of values is generated by varying all three
parameters above and below the estimated values. Then the best
values are selected as the minimum chi-square, or mean squared
deviation of the data from the formula. Starting from the best
value, a new search grid is again calculated, the deviations
calculated, and the best values again derived. This process is
repeated a number of times (typically 9) to obtain the best global
fit. Optionally, the scale of the grid may be reduced by a factor
(typically 0.95) each time it is used, so that the same values are
not appearing repeatedly.
[0188] The primary subsystems of the magnetic resonance system are
the pulse generator, the signal receiver, and the controller. These
subsystems may reside on separate boards, interconnected by cables.
Alternatively, the subsystems may be integrated as a single circuit
on a single computer board. The advantage of the latter is that
cable interconnects are not needed, and also that a single time
base may be used for all.
[0189] The system can be battery powered. The system uses very
little power during data acquisition, and can be programmed to use
essentially zero power in a sleep mode.
[0190] In one embodiment the system also includes a radiation
detector interfaced to the controller. The purpose of the radiation
detector is to detect radioactive materials in the sample. The
radiation detector may be any radiation sensor, preferably
sensitive to gamma rays, such as semiconductor, scintillator, and
gas-filled counters. The detector may be positioned proximate to
the sample collection means, the sample mixing system, or a holding
chamber placed downstream of the magnetic resonance system.
[0191] Insects such as spiders may obscure the air inlets and
collectors. First barrier to entry for these bugs are filters. For
outside installations, a slow release insecticide, preferably
harmless to humans and pets, can be incorporated. Such insecticides
can be implemented along the shaft of the inlet or near the mouth
of the inlet.
[0192] In one embodiment the systems and methods detect explosives
and chemical weapon materials. The systems and methods can perform
the detection using nanoparticles as disclosed above, wherein
specific binding sites on the nanoparticles bind to the explosive
or chemical weapon molecules. Alternatively, the systems and
methods can detect explosives or chemical weapon materials by
measuring magnetic resonance signals from the sample material
itself, without use of nanoparticles. The system may employ the
Spin Nuclear Overhauser Effect to detect chemical weapons and
explosives. No nanoswitches are required in this case. Another
configuration could be a hybrid system incorporating gas
chromatography, mass spectroscopy, ion mobility spectroscopy, other
analytical techniques, and NMR with or without nanoparticles
[0193] An advantage of the inventive systems and methods is that
confirmation tests may be carried out for certain analytes using
the same apparatus. For example, a confirming test for explosives
comprises measuring the T1 parameter using a magnetic resonance
system, since the T1 for most explosives is extremely long (many
seconds). As another example, a confirming measurement for chemical
weapons such as nerve agents is a magnetic resonance scan for
fluorine or phosphorus based on the characteristic Larmor
frequencies of those elements.
[0194] In one embodiment the system detects toxins and biological
weapons in mail envelopes, by testing particulate matter collected
from mail. In this application, the system would preferably include
means for extracting particulate matter from envelopes, such as
shaking, vibrating, blowing air through the mail piece or
compressing the envelopes. The system may include means for cutting
envelopes to retrieve powder, preferably only after other sensors
had directed suspicion at a particular mail piece.
[0195] A preferred embodiment for applications sampling air
includes an air inlet, a collector, concentrator and an automated
fluidic system. The air inlet includes a filter to exclude dirt and
insects, and a cyclone to separate sample particles from air.
Inlets may use "impactor" or "pre-separator", or "fractionator" and
serves the role of preventing large (e.g., particles with sizes
greater than about 10 micrometers aerodynamic diameter) from
entering the detector or identifier. The large-particle
fractionator is an integral component in the ambient sampler--it is
the combination of the internal nozzle and the plate that is normal
to the nozzle. For the HVAC unit or the occupied environment
sampler, there could be an optional pre-separator cartridge that is
placed downstream of the inlet. In addition, for the ambient
sampler, there could be a bug screen that is placed just upstream
of the exhaust port. The collector includes concentrator means
including a virtual impactor to insert the sample particles into a
liquid medium. The fluidic system then mixes the sample with
nanoparticles.
[0196] In one embodiment the systems and methods are adapted to
inspect shipping containers, for example to detect hazardous
materials or drugs or microbes among items in a shipping container.
The embodiment includes means for drawing air from the interior
space of the shipping container, means for collecting or
concentrating any material suspended or entrained in that air,
means for mixing the material with nanoparticles, and means for
presenting that mixture to the magnetic resonance system for
testing. The inspection may be carried out by opening a door of the
shipping container. Alternatively, the interior air may be drawn
through a port or reclosable opening on the shipping container.
Further details are provided in Provisional Application Ser. No.
60/669,019, filed Apr. 7, 2005, titled SHIPPING CONTAINER
INSPECTION DEVICE.
[0197] Those of skill will further appreciate that the various
illustrative logical blocks, modules, circuits, and algorithm steps
described in connection with the embodiments disclosed herein can
often be implemented as electronic hardware, computer software, or
combinations of both. To clearly illustrate this interchangeability
of hardware and software, various illustrative components, blocks,
modules, circuits, and steps have been described above generally in
terms of their functionality. Whether such functionality is
implemented as hardware or software depends upon the particular
application and design constraints imposed on the overall system.
Skilled persons can implement the described functionality in
varying ways for each particular application, but such
implementation decisions should not be interpreted as causing a
departure from the scope of the invention. In addition, the
grouping of functions within a module, block, circuit or step is
for ease of description. Specific functions or steps can be moved
from one module, block or circuit without departing from the
invention.
[0198] The various illustrative logical blocks, modules, and
circuits described in connection with the embodiments disclosed
herein can be implemented or performed with a general purpose
processor, a digital signal processor (DSP), an application
specific integrated circuit (ASIC), a field programmable gate array
(FPGA) or other programmable logic device, discrete gate or
transistor logic, discrete hardware components, or any combination
thereof designed to perform the functions described herein. A
general-purpose processor can be a microprocessor, but in the
alternative, the processor can be any processor, controller,
microcontroller, or state machine. A processor can also be
implemented as a combination of computing devices, for example, a
combination of a DSP and a microprocessor, a plurality of
microprocessors, one or more microprocessors in conjunction with a
DSP core, or any other such configuration.
[0199] The steps of a method or algorithm described in connection
with the embodiments disclosed herein can be embodied directly in
hardware, in a software module executed by a processor, or in a
combination of the two. A software module can reside in RAM memory,
flash memory, ROM memory, EPROM memory, EEPROM memory, registers,
hard disk, a removable disk, a CD-ROM, or any other form of storage
medium. An exemplary storage medium can be coupled to the processor
such the processor can read information from, and write information
to, the storage medium. In the alternative, the storage medium can
be integral to the processor. The processor and the storage medium
can reside in an ASIC.
[0200] The above description of the disclosed embodiments is
provided to enable any person skilled in the art to make or use the
invention. Various modifications to these embodiments will be
readily apparent to those skilled in the art, and the generic
principles defined herein can be applied to other embodiments
without departing from the spirit or scope of the invention. Thus,
the invention is not intended to be limited to the embodiments
shown herein but is to be accorded the widest scope consistent with
the principles and novel features disclosed herein.
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