U.S. patent application number 16/655188 was filed with the patent office on 2020-02-13 for metal-antibody tagging and plasma-based detection.
The applicant listed for this patent is Purdue Research Foundation. Invention is credited to Euiwon Bae, Valery P. Patsekin, Bartlomiej P. Rajwa, Joseph Paul Robinson.
Application Number | 20200049629 16/655188 |
Document ID | / |
Family ID | 55459652 |
Filed Date | 2020-02-13 |
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United States Patent
Application |
20200049629 |
Kind Code |
A1 |
Robinson; Joseph Paul ; et
al. |
February 13, 2020 |
METAL-ANTIBODY TAGGING AND PLASMA-BASED DETECTION
Abstract
An apparatus and method for characterizing a target, e.g.,
microbial samples or biological toxins, includes labeling the
target with a biomolecular recognition construct and measuring an
atomic-spectra signal of the biomolecular recognition construct.
The method can include heating the labeled target before measuring
the atomic-spectra signal. The atomic-spectra signal can be
measured by performing laser-induced breakdown spectroscopy. The
atomic-spectra signal can be measured by performing spark induced
breakdown spectroscopy. The biomolecular recognition construct can
be prepared by tagging a biological scaffolding with a metal atom
or ion. In an aspect in which the target includes a microbial
sample, the biological scaffolding can include an antibody against
epitopes present on bacterial surface, the antibody linked to a
heavy metal. In an aspect in which the target includes a biological
toxin, the biological scaffolding can include an antibody against
the biological toxin linked to heavy metals.
Inventors: |
Robinson; Joseph Paul; (West
Lafayette, IN) ; Rajwa; Bartlomiej P.; (West
Lafayette, IN) ; Patsekin; Valery P.; (West
Lafayette, IN) ; Bae; Euiwon; (West Lafayette,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Purdue Research Foundation |
West Lafayette |
IN |
US |
|
|
Family ID: |
55459652 |
Appl. No.: |
16/655188 |
Filed: |
October 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15510319 |
Mar 10, 2017 |
10451556 |
|
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PCT/US15/49916 |
Sep 14, 2015 |
|
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16655188 |
|
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|
62049931 |
Sep 12, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/56911 20130101;
G01N 2469/00 20130101; G01N 2469/10 20130101; G01N 21/67 20130101;
G01N 21/25 20130101; G01N 33/58 20130101; G01N 21/718 20130101;
G01N 33/569 20130101 |
International
Class: |
G01N 21/71 20060101
G01N021/71; G01N 33/569 20060101 G01N033/569; G01N 33/58 20060101
G01N033/58; G01N 21/25 20060101 G01N021/25; G01N 21/67 20060101
G01N021/67 |
Goverment Interests
STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
Contract No. 59 1935 2 279 awarded by the United States Department
of Agriculture. The government has certain rights in the invention.
Claims
1. A method for characterizing a target within a sample, the method
comprising: applying to the sample a biomolecular recognition
construct comprising a metal and a molecular recognition scaffold,
wherein the molecular recognition scaffold is configured to bind to
the target; generating a plasma of at least some of the sample; and
detecting electromagnetic radiation emitted by the plasma to
provide an atomic-spectra signal of the sample.
2. The method according to claim 1, wherein the generating
comprises heating at least part of the sample.
3. The method according to claim 1, wherein the generating
comprises irradiating at least part of the sample using a
laser.
4. The method according to claim 1, wherein the generating
comprises applying a spark to at least part of the sample.
5. The method according to claim 1, further comprising: determining
presence of the metal in the sample based at least in part on the
atomic-spectra signal by performing at least one of spectral
unmixing or constrained energy minimization (CEM).
6. The method according to claim 1, further comprising: preparing
the biomolecular recognition construct by bonding the metal to the
molecular recognition scaffold, wherein the molecular recognition
scaffold comprises a biological scaffold and the metal comprises a
metal atom or ion.
7. The method according to claim 1, wherein the target includes a
microbe and the molecular recognition scaffold comprises an
antibody against epitopes present on a surface of the microbe.
8. The method according to claim 1, wherein the target includes a
biological toxin and the molecular recognition scaffold comprises
an antibody against the biological toxin.
9. The method according to claim 1, wherein the biomolecular
recognition construct is a first biomolecular recognition
construct, the metal is a first metal, the molecular recognition
scaffold is a first molecular recognition scaffold, and the target
is a first target, the method further comprising: applying, to the
sample, a second biomolecular recognition construct comprising a
second metal and a second molecular recognition scaffold, wherein
the second molecular recognition scaffold is configured to bind to
a second target; determining presence of the first metal in the
sample based at least in part on the atomic-spectra signal;
determining absence of the second metal in the sample based at
least in part on the atomic-spectra signal; determining presence of
the first target in the sample based at least in part on the
presence of the first metal in the sample; and determining absence
of the second target in the sample based at least in part on the
absence of the second metal in the sample.
10. The method of claim 9, further comprising: attaching the sample
to a silicon wafer; and washing the second biomolecular recognition
construct from the silicon wafer.
11. An apparatus for characterizing a biological target in a
sample, the apparatus comprising: a first subsystem configured to
apply, to the sample, a biomolecular recognition construct
comprising a metal and a molecular recognition scaffold, wherein
the molecular recognition scaffold is configured to bind to the
target; a second subsystem configured to generate a plasma using at
least some of the sample; and a third subsystem configured to
detect electromagnetic radiation emitted by the plasma to provide
an atomic-spectra signal of the sample.
12. The apparatus according to claim 11, wherein the second
subsystem comprises a laser.
13. The apparatus according to claim 11, wherein the second
subsystem comprises: a first electrode; a second electrode separate
from the first electrode; and a power supply connected to the first
electrode and the second electrode, the power supply being
configured to selectively produce a spark across the two
electrodes.
14. The apparatus according to claim 11, further comprising: at
least one processor; and memory storing instructions that, when
executed by the at least one processor, cause the at least one
processor to perform operations comprising: determining presence of
the metal in the sample based at least in part on the
atomic-spectra signal by performing at least one of spectral
unmixing or constrained energy minimization (CEM).
15. The apparatus according to claim 11, wherein the target
includes a microbe and the molecular recognition scaffold comprises
an antibody against epitopes present on a surface of the
microbe
16. The apparatus according to claim 11, wherein the target
includes a biological toxin and the molecular recognition scaffold
comprises an antibody against the biological toxin.
17. A method, comprising: applying, to a sample, a biomolecular
recognition construct comprising a metal and a molecular
recognition scaffold, wherein the molecular recognition scaffold is
configured to bind to a target; generating a plasma using at least
some of the sample; detecting electromagnetic radiation emitted by
the plasma to provide an atomic-spectra signal of the sample;
determining presence of the metal in the sample based at least in
part on the atomic-spectra signal; and determining presence of the
target in the sample based at least in part on the presence of the
metal in the sample.
18. The method of claim 17, further comprising: preparing the
biomolecular recognition construct by bonding the metal to the
molecular recognition scaffold, wherein the molecular recognition
scaffold comprises a biological scaffold and the metal comprises a
metal atom or ion.
19. The method of claim 17, wherein the biomolecular recognition
construct is a first biomolecular recognition construct, the metal
is a first metal, the molecular recognition scaffold is a first
molecular recognition scaffold, and the target is a first target,
the method further comprising: applying, to the sample, a second
biomolecular recognition construct comprising a second metal and a
second molecular recognition scaffold, wherein the second molecular
recognition scaffold is configured to bind to a second target;
determining absence of the second metal in the sample based at
least in part on the atomic-spectra signal; and determining absence
of the second target in the sample based at least in part on the
absence of the second metal in the sample.
20. The method of claim 19, further comprising: attaching the
sample to a silicon wafer; and washing the second biomolecular
recognition construct from the silicon wafer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of, and claims the benefit
of, commonly assigned, co-pending U.S. patent application Ser. No.
15/510,319, filed Mar. 10, 2017, which is a national stage
application of International Patent Application No. PCT/US15/49916,
filed Sep. 14, 2015, which is related to and claims the priority
benefit of U.S. Provisional Patent Application Ser. No. 62/049,931,
filed Sep. 12, 2014, the contents of which are hereby incorporated
by reference in their entirety into this disclosure.
TECHNICAL FIELD
[0003] The present disclosure generally relates to biological
detection, and in particular to detection of biological pathogens
using antibody tagging.
BACKGROUND
[0004] This section introduces aspects that may help facilitate a
better understanding of the disclosure. Accordingly, these
statements are to be read in this light and are not to be
understood as admissions about what is or is not prior art.
[0005] The fields of microbiology, biosafety and biosurveillance
employ multiple detection technologies paired with various
reporting modalities. The most common approaches use traditional
optical labeling techniques such as fluorescence, phosphorescence
or formation of color chromophores. The optical labels are
typically connected to molecular recognition molecules such as
antibodies.
[0006] Other lesser-known methods for pathogen recognition and/or
detection include detection of antibody immobilized bacteria using
surface plasmon resonance (SPR) sensors, interferometric
biosensors, acoustic wave biosensor platforms based on the
thickness shear mode (TSM) resonator, and piezoelectric-excited
millimeter-sized cantilever (PEMC) sensors. There has been also
experimental work reported on detection involving microfluidic
microchips coated with antibodies. The chips have an electric
current passed through them. When the chip surface comes into
contact with bacteria, the system shows changes in potentiometric,
amperometric or impedimetric/conductimetric characteristics
demonstrating bacterial presence.
[0007] Most of the listed techniques do not offer good multiplexing
capability, as they are specifically designed to announce the
presence of a specific type or category of bacteria. They are also
not easily extendable to detect other biological hazards, such as
present of biological toxins. Therefore, improvements are needed in
the field.
SUMMARY
[0008] In one aspect, a method for characterizing a biological
target, is disclosed, the method comprising labeling the target
with a biomolecular recognition construct and measuring an
atomic-spectra signal of the biomolecular recognition construct.
The labeled target may be heated before measuring the
atomic-spectra signal. The atomic-spectra signal may be measured by
performing laser-induced breakdown spectroscopy. The atomic-spectra
signal may also be measured by performing spark induced breakdown
spectroscopy. The biomolecular recognition construct may be formed
by tagging a biological scaffolding with a metal atom or ion. The
target may include a microbial sample and the biological
scaffolding may comprise an antibody against epitopes present on
bacterial surface, the antibody linked to a heavy metal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a diagram showing the components of a system for
detecting a biological target in a sample.
[0010] FIG. 2 is a plot showing exemplary data that were collected
in an experiment that where samples containing bacteria were
labeled with two different metal-tagged antibodies, Sb and Pr
according to one embodiment.
[0011] FIG. 3 is an annotated graphical representation of a
photograph of an exemplary configuration of a silicon wafer to hold
sample(s).
[0012] FIG. 4 is a plot showing spectral measurement of a sample
containing antitoxin antibodies labeled with Lu and Pr in the
400-600 nm range according to one embodiment.
[0013] FIG. 5 is a plot showing spectral measurement of a sample
containing antitoxin antibodies labeled with Lu and Pr in the
320-380 nm range according to one embodiment.
[0014] FIG. 6 is a plot showing spectral measurement of a sample
containing antitoxin antibodies labeled with Gd 156 and a blank
sample in the 340-380 nm range according to one embodiment.
[0015] FIG. 7 is a plot showing spectral measurement of a sample
containing antitoxin antibodies labeled with Pr in the 340-460 nm
range according to one embodiment.
[0016] FIG. 8 is a plot showing spectral measurement of a sample
containing antitoxin antibodies labeled with Dy in the 240-360 nm
range according to one embodiment.
[0017] FIG. 9 is a plot showing initial dose response to two
different agents, Shiga Toxin Stx-2-2 labeled with Pr 141 and Ricin
labeled with Dy 162.
[0018] FIG. 10 is a plot showing spectral measurement of the 240
nm-360 nm window, where there are possible peaks that only exists
on certain regions of the spectra (for Pr, Lu, Gd, and Dy).
[0019] The attached figures are for purposes of illustration and
are not necessarily to scale.
DETAILED DESCRIPTION
[0020] For the purposes of promoting an understanding of the
principles of the present disclosure, reference will now be made to
the embodiments illustrated in the drawings, and specific language
will be used to describe the same. It will nevertheless be
understood that no limitation of the scope of this disclosure is
thereby intended.
[0021] The present disclosure provides a method and apparatus for
metal-antibody tagging and plasma-based detection (MAPD) which
involves the use of metal-labeled recognition macromolecules to tag
infectious agents (such as bacterial cells) or toxic biological
products and substances (e.g. Ricin, mycotoxins, bacterial toxins,
Shiga toxin, Botulinum) for subsequent detection using
laser-induced breakdown spectroscopy (LIBS), spark induced
breakdown spectroscopy (SIBS), laser ablation molecular isotopic
spectrometry (LAMIS) or other detection modalities relying on
atomic spectra evaluation after plasma formation.
[0022] Various herein-described detection techniques use atomic
optical emission spectroscopy. They employ a laser and a focusing
lens (LIBS and LAMIS), or a spark (SIBS) to generate a plasma from
the vaporized tagged sample.
[0023] Some aspects herein are described in terms that can be
implemented as software programs. The equivalent of such software
can also be constructed in hardware, firmware, or micro-code.
Because data-manipulation algorithms and systems are well known,
the present description is directed in particular to algorithms and
systems forming part of, or cooperating more directly with, systems
and methods described herein. Other aspects of such algorithms and
systems, and hardware or software for producing and otherwise
processing signals or data involved therewith, not specifically
shown or described herein, are selected from such systems,
algorithms, components, and elements known in the art. Given the
systems and methods as described herein, software not specifically
shown, suggested, or described herein that is useful for
implementation of any aspect is conventional and within the
ordinary skill in such arts.
[0024] FIG. 1 is a diagram showing the components of an exemplary
recognition system 101 for analyzing sample data and performing
other analyses described herein, and related components. The system
101 includes a processor 186, a peripheral system 120, a user
interface system 130, and a data storage system 140. The peripheral
system 120, the user interface system 130 and the data storage
system 140 are communicatively connected to the processor 186.
Processor 186 can be communicatively connected to network 150
(shown in phantom), e.g., the Internet or a leased line, as
discussed below. Lasers, sample addition devices, substrate
handlers, and other devices herein can each include one or more
processor(s) 186 or one or more of systems 120, 130, 140, and can
each connect to one or more network(s) 150. Processor 186, and
other processing devices described herein, can each include one or
more microprocessors, microcontrollers, field-programmable gate
arrays (FPGAs), application-specific integrated circuits (ASICs),
programmable logic devices (PLDs), programmable logic arrays
(PLAs), programmable array logic devices (PALs), or digital signal
processors (DSPs).
[0025] Processor 186 can implement processes of various aspects
described herein. Processor 186 and related components can, e.g.,
carry out processes for performing assays using recognition
macromolecules as described in Paper 1.
[0026] Processor 186 can be or include one or more device(s) for
automatically operating on data, e.g., a central processing unit
(CPU), microcontroller (MCU), desktop computer, laptop computer,
mainframe computer, personal digital assistant, digital camera,
cellular phone, smartphone, or any other device for processing
data, managing data, or handling data, whether implemented with
electrical, magnetic, optical, biological components, or
otherwise.
[0027] The phrase "communicatively connected" includes any type of
connection, wired or wireless, for communicating data between
devices or processors. These devices or processors can be located
in physical proximity or not. For example, subsystems such as
peripheral system 120, user interface system 130, and data storage
system 140 are shown separately from the processor 186 but can be
embodied or integrated completely or partially within the processor
186. In an example, processor 186 includes an ASIC including a
central processing unit connected via an on-chip bus to one or more
core(s) implementing function(s) of systems 120, 130, or 140.
[0028] The peripheral system 120 can include or be communicatively
connected with one or more devices configured or otherwise adapted
to provide digital content records to the processor 186 or to take
action in response to signals or other instructions received from
processor 186. For example, the peripheral system 120 can include
digital still cameras, digital video cameras, spectroscopic
detector 196, or other data processors. The processor 186, upon
receipt of digital content records from a device in the peripheral
system 120, can store such digital content records in the data
storage system 140.
[0029] Processor 186 can, via peripheral system 120, control
subsystems 190, 192, 194, and spectroscopic detector 196.
Biological sample 198 is carried on substrate 199, which can be,
e.g., a silicon (Si) wafer. Target 197 is shown in sample 198 for
illustration. Sample 198 can include liquid, gas, powder, bulk
solid, or any combination or mixture thereof. Substrate 199 can be
manipulated by a wafer-handling or other motion subsystem (not
shown). Subsystem 190 (graphically represented as an eyedropper) is
configured or otherwise adapted to add a biomolecular recognition
construct to the sample 198, e.g., a dispenser or sample-deposition
device such as those used in automatic dry- or wet-slide bioassays
or in flow cytometry. Subsystem 192 is configured to wash unbound
recognition construct out of the sample 198. Subsystem 194 is
configured to heat the sample-construct mixture so that metals in
the biomolecular recognition construct in the washed sample emit
photons at characteristic wavelengths. This subsystem 194 can
include a laser, e.g., of a type used in laser-induced breakdown
spectroscopy (LIES). Subsystem 194 can also include a spark induced
breakdown spectroscopy (SIBS) spark generator, e.g., a
closely-spaced electrode pair connected to a high-voltage power
supply so that a high voltage can be introduced across the
electrodes to produce a spark. Spectroscopic detector 196 (depicted
as a camera; dashed-line connector used for clarity only) is
configured to detect light emitted by the metals, e.g., by metal
atoms or ions in the recognition macromolecules. In this example,
apparatus for detecting a target 197 in a sample 198 includes
subsystem 190 for adding a biomolecular recognition construct to
the sample, subsystem 192 for washing unbound recognition construct
out of the sample, subsystem 194 for ionizing the sample-construct
mixture into a plasma. The plasma signal emitted by atomic and
ionic species of the metals used to tag the antibodies attached to
the sample can be collected by a spectrometer. Sample emits photons
at characteristic wavelengths, and spectroscopic detector 196 is
used for detecting photons emitted by the metal ions. The plasma
generation subsystem 194 can include a laser, or can include at
least two electrodes and a high-voltage power supply connected to
the at least two electrodes and configured to selectively produce a
spark across the at least two electrodes.
[0030] The user interface system 130 can convey information in
either direction, or in both directions, between a user 138 and the
processor 186 or other components of system 101. The user interface
system 130 can include a mouse, a keyboard, another computer
(connected, e.g., via a network or a null-modem cable), or any
device or combination of devices from which data is input to the
processor 186. The user interface system 130 also can include a
display device, a processor-accessible memory, or any device or
combination of devices to which data is output by the processor
186. The user interface system 130 and the data storage system 140
can share a processor-accessible memory.
[0031] In various aspects, processor 186 includes or is connected
to communication interface 115 that is coupled via network link 116
(shown in phantom) to network 150. For example, communication
interface 115 can include an integrated services digital network
(ISDN) terminal adapter or a modem to communicate data via a
telephone line; a network interface to communicate data via a
local-area network (LAN), e.g., an Ethernet LAN, or wide-area
network (WAN); or a radio to communicate data via a wireless link,
e.g., WIFI or GSM (Global System for Mobile Communications).
Communication interface 115 can send and receives electrical,
electromagnetic or optical signals that carry digital or analog
data streams representing various types of information across
network link 116 to network 150. Network link 116 can be connected
to network 150 via a switch, gateway, hub, router, or other
networking device.
[0032] In various aspects, system 101 can communicate, e.g., via
network 150, with other data processing system(s) (not shown),
which can include the same types of components as system 101 but is
not required to be identical thereto. System 101 and other systems
not shown communicatively connected via the network 150. System 101
and other systems not shown can execute computer program
instructions to measure constituents of samples or exchange spectra
or other data, e.g., as described herein.
[0033] Processor 186 can send messages and receive data, including
program code, through network 150, network link 116 and
communication interface 115. For example, a server can store
requested code for an application program (e.g., a JAVA applet) on
a tangible non-volatile computer-readable storage medium to which
it is connected. The server can retrieve the code from the medium
and transmit it through network 150 to communication interface 115.
The received code can be executed by processor 186 as it is
received, or stored in data storage system 140 for later
execution.
[0034] Data storage system 140 can include or be communicatively
connected with one or more processor-accessible memories configured
or otherwise adapted to store information. The memories can be,
e.g., within a chassis or as parts of a distributed system. The
phrase "processor-accessible memory" is intended to include any
data storage device to or from which processor 186 can transfer
data (e.g., using components of peripheral system 120). A
processor-accessible memory can include one or more data storage
device(s) that are volatile or nonvolatile, that are removable or
fixed, or that are electronic, magnetic, optical, chemical,
mechanical, or otherwise. Exemplary processor-accessible memories
include but are not limited to: registers, floppy disks, hard
disks, tapes, bar codes, Compact Discs, DVDs, read-only memories
(ROM), erasable programmable read-only memories (EPROM, EEPROM, or
Flash), and random-access memories (RAMs). One of the
processor-accessible memories in the data storage system 140 can be
a tangible non-transitory computer-readable storage medium, i.e., a
non-transitory device or article of manufacture that participates
in storing instructions that can be provided to processor 186 for
execution.
[0035] In an example, data storage system 140 includes code memory
141, e.g., a RAM, and disk 143, e.g., a tangible computer-readable
rotational storage device or medium such as a hard drive. In this
example, computer program instructions are read into code memory
141 from disk 143. Processor 186 then executes one or more
sequences of the computer program instructions loaded into code
memory 141, as a result performing process steps described herein.
In this way, processor 186 carries out a computer implemented
process. For example, steps of methods described herein, blocks of
block diagrams herein, and combinations of those, can be
implemented by computer program instructions. Code memory 141 can
also store data.
[0036] Various aspects described herein may be embodied as systems
or methods. Accordingly, various aspects herein may take the form
of an entirely hardware aspect, an entirely software aspect
(including firmware, resident software, micro-code, etc.), or an
aspect combining software and hardware aspects These aspects can
all generally be referred to herein as a "service," "circuit,"
"circuitry," "module," or "system."
[0037] Furthermore, various aspects herein may be embodied as
computer program products including computer readable program code
("program code") stored on a computer readable medium, e.g., a
tangible non-transitory computer storage medium or a communication
medium. A computer storage medium can include tangible storage
units such as volatile memory, nonvolatile memory, or other
persistent or auxiliary computer storage media, removable and
non-removable computer storage media implemented in any method or
technology for storage of information such as computer-readable
instructions, data structures, program modules, or other data. A
computer storage medium can be manufactured as is conventional for
such articles, e.g., by pressing a CD-ROM or electronically writing
data into a Flash memory. In contrast to computer storage media,
communication media may embody computer-readable instructions, data
structures, program modules, or other data in a modulated data
signal, such as a carrier wave or other transmission mechanism. As
defined herein, "computer storage media" do not include
communication media. That is, computer storage media do not include
communications media consisting solely of a modulated data signal,
a carrier wave, or a propagated signal, per se.
[0038] The program code can include computer program instructions
that can be loaded into processor 186 (and possibly also other
processors), and that, when loaded into processor 486, cause
functions, acts, or operational steps of various aspects herein to
be performed by processor 186 (or other processor). The program
code for carrying out operations for various aspects described
herein may be written in any combination of one or more programming
language(s), and can be loaded from disk 143 into code memory 141
for execution. The program code may execute, e.g., entirely on
processor 186, partly on processor 186 and partly on a remote
computer connected to network 150, or entirely on the remote
computer.
[0039] In various aspects, a method for characterizing a target,
e.g., microbial samples or biological toxins, includes labeling the
target with a biomolecular recognition construct and measuring an
atomic-spectra signal of the biomolecular recognition construct.
The method can include heating the labeled target before measuring
the atomic-spectra signal. The atomic-spectra signal can be
measured by performing laser-induced breakdown spectroscopy (LIBS).
The atomic-spectra signal can be measured by performing spark
induced breakdown spectroscopy (SIBS). Data of the atomic-spectra
signal can be classified using a computer-based classifier and a
classification score can be assigned to the analyzed sample (e.g.,
spectral unmixing or spectral fingerprint classification).
[0040] Using the system 101, the biomolecular recognition construct
can be prepared by tagging a biological scaffolding with a metal
atom or ion. The biological scaffolding may comprise adNectins,
iMabs, anticalins, microbodies, peptide aptamers, designed ankyrin
repeat proteins (DARPins), affilins, tentranectins, avimers or
other scaffolds. In an aspect in which the target includes a
microbial sample, the biological scaffolding can include an
antibody against epitopes present on bacterial surface, said
antibody linked to a heavy metal. In an aspect in which the target
includes a biological toxin, the biological scaffolding can include
an antibody against the biological toxin linked to heavy
metals.
[0041] The scaffold for the molecular recognition system may be
tagged using various metallic elements such as Al, Ca, Cr, Cu, Fe,
Mg, Mn, Pb, Si, Ti, V and Zn. However, in order to minimize the
background it is advisable to use lanthanide metals (rare earth
elements) which are typically not present in biological material
such as La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and
Lu. The probes can be prepared by coupling the scaffolding for
molecular recognition to polymers equipped with metal-binding
ligands. These polymers contain a functional group enabling them to
be covalently attached to biological macromolecules such as
antibodies, while simultaneously binding to one or more metals,
e.g., metal atoms or ions. We have preformed preliminary studies
and propose to extend the use of lanthanide metals (rare earth
elements), which are typically not present in biological material
(La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu).
However, MEPS can be also employed with chelated heavy metal ions,
assuming that food contamination by heavy metals is not the target
of the specific test. The MEPS probes are prepared by coupling the
scaffolding for molecular recognition to polymers equipped with
metal-binding ligands. These specially designed polymers contain a
functional group enabling them to be covalently attached to
biological macromolecules, while simultaneously binding multiple
ions of metals. Specifically we have tested antibodies tagged via a
reaction involving selective reduction of disulfide bonds in their
hinge region, followed by thiol addition to a maleimide group at
one end of the MCP. Owing to unique and distinguishable atomic
spectral signals from many other metals, MEPS can also take
advantage of alternative non-MCP labeling strategies, for example
using HgS nanoparticles, silver nanoparticles, organic mercury
compounds, or ruthenium compounds. Additionally ions derived from
cadmium, mercury, cobalt, arsenic, copper, chromium and selenium
can also be identified.
[0042] The prepared recognition biomolecular recognition constructs
(macromolecules) are subsequently used to perform the assay. There
are many possible ways for the recognition biomolecular recognition
construct to be used. In one example, a biological specimen
containing pathogens or toxins can be attached to an inert surface
(e.g. such as a silicon wafer). A metal-tagged antibody or an
alternative molecular recognition scaffold is applied over the
surface binding to the exposed antigens. The excess antibody or
other recognition macromolecule is removed by washing the
substrate
[0043] In another example, in an indirect setting following the
attachment of the bacteria cells or toxin macromolecules to the
surface of the inert sample holder a primary antibody or other
recognition macromolecule is added, binding specifically to the
antigens of interest. This primary molecular recognition system is
not tagged, in contrast to the reagents described above.
Subsequently a secondary (metal-tagged) macromolecule is added
binding to the primary macromolecule.
[0044] In a further example, an inert surface is functionalized and
covered with recognition macromolecules. The biological specimen is
added and the antigens of interest are captured by the
surface-bound recognition macromolecules. In the last step, the
metal-tagged recognition macromolecules are added binding to the
immobilized antigen. The excess unbound macromolecules are removed
by a wash.
[0045] Following the tagging step in these and other aspects, the
specimen containing the sample of interest labeled by metal-tagged
recognition molecules is analyzed using system 101 by employing one
of the atomic spectroscopy techniques mentioned above. In an aspect
using LIBS spectroscopy, subsystem 194 focuses a laser beam onto
the inert surface (e.g., the silicon wafer) where the sample 198 is
deposited. Owing to the large power density of the laser the tagged
material starts to evaporate leading to the generation of plasma.
The chemical constituents of the biological material are excited by
the laser beam and emit radiation which is element specific, upon
which the radiation is analyzed by subsystem 196.
[0046] In the described settings, simultaneous (multiplexed)
analysis of many targets 197 within the sample 198 is possible by
utilizing a cocktail of recognition macromolecules (e.g., a mixture
of antibodies), each class of recognition macromolecules labeled
with a different metal. Owing to distinguishability and specificity
of atomic spectra produced by different metals, this tagging
arrangement permits effective multiplexing, i.e., simultaneous
detection of multiple targets (for instance, different bacterial
pathogens or toxins).
[0047] The plasma signal emitted by atomic and ionic species of the
metals used to tag the antibodies attached to the sample can be
collected using a spectrometer, such as subsystem 196. The
naturally occurring chemical constituents of the biological sample
198 can also contribute to the atomic spectra signal. In fact, it
has been disclosed and demonstrated that the LIBS signal from
bacteria alone may lead to recognition of some bacterial species.
However, owing to a high similarity in biochemical composition of
bacterial species, the classification ability of the label-free
methods is relatively low. The spectra are used to determine the
elemental constituents of the sample 198, and such constituents are
similar for many bacteria or other targets. In various aspects,
since the metals used to label the antibodies are either not
naturally present in the tested sample 198 of interest or present
only in very small quantities, the detection of the spectra of
those metals is a direct indicator of a sample type and origin.
[0048] Various aspects include digitization of the recorded
spectra, followed by spectral unmixing (allowing for the
determination of the individual spectral constituents) or spectral
fingerprint classification (involving matching the obtained
spectrum to other spectra present in the database).
[0049] The disclosed system 101 therefore offers faster and more
sensitive detection with reduced sample processing and preparation
compared to prior art schemes. The presently disclosed detection
format allows for multiplexing, e.g. simultaneous detection of
multiple bacterial species, biological toxins, or other
targets.
[0050] In one example, shown in FIG. 2, samples containing bacteria
were labelled with two different types of antibodies. The Sb-tagged
antibodies (indicated by Sb) attached to E. coli can be readily
distinguished from Pr-tagged antibodies (indicated by Pr).
[0051] FIG. 3 shows an example of a Silicon wafer with spotted
samples (numbered 1-6) on the surface. Each spot is analyzed using
the techniques described above. Results described herein were based
on measurements made in this manner.
[0052] FIG. 4 is a plot showing spectral measurement of a sample
containing antitoxin antibodies labeled with Lu and Pr in the
400-600 nm range. FIG. 5 is a plot showing spectral measurement of
a sample containing antitoxin antibodies labeled with Lu and Pr in
the 320-380 nm range. FIG. 6 is a plot showing spectral measurement
of a sample containing antitoxin antibodies labeled with Gd 156 and
a blank sample in the 340-380 nm range. FIG. 7 is a plot showing
spectral measurement of a sample containing antitoxin antibodies
labeled with Pr in the 340-460 nm range. FIG. 8 is a plot showing
spectral measurement of a sample containing antitoxin antibodies
labeled with Dy in the 240-360 nm range. FIG. 9 is a plot showing
initial dose response to two different agents, Shiga Toxin Stx-2-2
labeled with Pr 141 and Ricin labeled with Dy 162. FIG. 10 is a
plot showing spectral measurement of the 240 nm-360 nm window,
where there peaks can be identified on regions of the spectra
representing Pr, Lu, Gd, and Dy simultaneously as shown.
[0053] Steps of various methods described herein can be performed
in any order except when otherwise specified, or when data from an
earlier step is used in a later step. Exemplary method(s) described
herein are not limited to being carried out by components
particularly identified in discussions of those methods.
[0054] In view of the foregoing, various aspects relate to a system
for characterizing a biological target within a sample, the system
comprising: a. a high energy source; b. a sample container for
containment of the sample; c. a recognition scaffold; d. a tag
containing a metal element; e. a system for collection of atomic
spectra; and f a system for extracting features from spectra and
identifying tags. Various aspects relate to such a system, the
biological target comprising microbial samples or biological
toxins.
[0055] In view of the foregoing, various aspects provide
measurement of constituents of a sample. A technical effect of
various aspects is to ablate a small quantity of the sample to form
a plasma and to measure the constituents of the plasma
spectroscopically. A technical effect of various aspects is to
provide a metal-labeled target. A further technical effect of
various aspects is to present a visual representation of the
detected spectra or corresponding abundances of selected
biomolecules on an electronic display. This can permit medical or
scientific personnel to more readily determine whether a sample
contains a target of interest, e.g., at a selected concentration or
quantity.
[0056] In various embodiments of the method according to the
invention can optionally also be made of one and/or other of the
following provisions: [0057] According to one aspect, a method for
characterizing a biological target within a sample, the method
comprising: [0058] labeling the target with a biomolecular
recognition construct; and [0059] measuring an atomic-spectra
signal of the biomolecular recognition construct; [0060] According
to another aspect, heating the labeled target before measuring the
atomic-spectra signal. [0061] According to another aspect, the
atomic-spectra signal is measured by performing laser-induced
breakdown spectroscopy. [0062] According to another aspect, the
atomic-spectra signal is measured by performing spark induced
breakdown spectroscopy. [0063] According to another aspect, data of
the atomic-spectra signal is classified using a computer-based
classifier and assigning a classification score to the analyzed
sample. [0064] According to another aspect, the biomolecular
recognition construct is prepared by tagging a biological
scaffolding with a metal atom or ion. [0065] According to another
aspect, the target includes a microbial sample and the biological
scaffolding comprises an antibody against epitopes present on
bacterial surface, said antibody linked to a heavy metal. [0066]
According to another aspect, the target includes a biological toxin
and the biological scaffolding comprises an antibody against the
biological toxin linked to heavy metals. [0067] In various
embodiments of the apparatus according to the invention can
optionally also be made of one and/or other of the following
provisions: [0068] According to one aspect, an apparatus for
detecting a biological target in a sample, the apparatus
comprising: [0069] a. a construct subsystem configured to add a
biomolecular recognition construct to the sample; [0070] b. a wash
subsystem configured to wash unbound recognition construct out of
the sample; [0071] c. a heating subsystem configured to heat the
sample-construct mixture so that metals in the biomolecular
recognition construct in the washed sample emit photons at
characteristic wavelengths; and [0072] d. a spectroscopic detector
configured to detect light emitted by the metals. [0073] According
to one aspect, the heating subsystem includes a laser. [0074]
According to one aspect, the heating subsystem includes at least
two electrodes and a high-voltage power supply connected to the at
least two electrodes and configured to selectively produce a spark
across the at least two electrodes.
[0075] The invention is inclusive of combinations of the aspects
described herein. References to "a particular aspect" (or
"embodiment" or "version") and the like refer to features that are
present in at least one aspect of the invention. Separate
references to "an aspect" (or "embodiment") or "particular aspects"
or the like do not necessarily refer to the same aspect or aspects;
however, such aspects are not mutually exclusive, unless otherwise
explicitly noted. The use of singular or plural in referring to
"method" or "methods" and the like is not limiting. The word "or"
is used in this disclosure in a non-exclusive sense, unless
otherwise explicitly noted.
[0076] invention has been described in detail with particular
reference to certain preferred aspects thereof, but it will be
understood that variations, combinations, and modifications can be
effected within the spirit and scope of the invention.
* * * * *