Patent Application
20230221306
Advanced sensor capabilities for onsite detection of chemical, biological, and radiological/nuclear (CBRN) threats are important to significantly limit the risk of exposure to persons at such sites and allow for the rapid, in-field collection of essential scientific data and critical evidence. However, current scientific and commercial sensing capabilities generally require highly specific and ultra-sensitive methods that are often energy-intensive or require offsite post-analysis for positive detection, leaving onsite personnel vulnerable. Moreover, current in-field sensors are typically limited to detecting only one class of threat at a time.
Current detection techniques for CBRN are generally complex and require highly specific and ultra-sensitive methods. For example, biological threats, such as anthrax or ricin, are routinely analyzed offsite through a laboratory response network, where samples of interest are first sent to a sentinel lab, followed by a reference laboratory, and then, if the biological threats are not ruled out at the first two laboratories, the samples are sent to a national laboratory. This network uses fixed laboratory equipment, such as mass spectroscopy (MS) techniques, which are highly sensitive (ricin reported detection limits of 0.64 ng/mL in the literature). Unfortunately, they require complex data analysis, especially for biological samples, which have numerous MS fragments and potential matrix effects that can interfere with the determination of the analyte.
Accordingly, improved sensor technology for simultaneously detecting multiple CBRN threats, including different types of threats, would be welcomed in the technology. Sensors that are capable of use in-field for detection of multiple threat types would be of great benefit.
Aspects and advantages of the present subject matter will be set forth in part in the following description, or may be obvious from the description, or may be learned through practice of the present subject matter.
According to one embodiment, disclosed is a threat detection sensor that includes a first energy transfer pair and a second energy transfer pair. Each energy transfer pair includes a donor and an acceptor. The first energy transfer pair and the second energy transfer pair share a donor or an acceptor, e.g., a single particle that functions as a donor or as an acceptor for both pairs. The donor and acceptor of the first energy transfer pair are physically and energetically coupled to one another by a first linking agent that includes a first polynucleic acid, the first linking agent containing a specific binding ligand for a first CBRN threat. The donor and acceptor of the second energy transfer pair are also physically and energetically coupled to one another with a second linking agent that includes a second polynucleic acid, the second polynucleic acid containing a specific binding ligand for a second chemical threat.
Also disclosed is a method for detecting a plurality of different CBRN threats by use of a single sensor. The method includes contacting a sensor with a sample. The sensor includes a first energy transfer pair and a second energy transfer pair. Each energy transfer pair includes a donor and an acceptor, with the different energy transfer pairs sharing a donor or an acceptor. The donor and acceptor of each energy transfer pair is physically and energetically coupled to one another with each linking agent including a polynucleic acid that includes a specific binding ligand for a particular chemical threat. Upon binding of a first agent to the first linking agent of the first energy transfer pair, the energetic coupling of the first energy transfer pair is altered causing a first optically detectable signal. Upon binding of a second agent to the second linking agent of the second energy transfer pair, the energetic coupling of the second energy transfer pair is altered causing a second, different optically detectable signal. In the presence of both the first and the second agent, a third optically detectable signal can be obtained.
A full and enabling disclosure of the present subject matter, including the best mode thereof, directed to one of ordinary skill in the art, is set forth in the specification, which makes reference to the appended figures, in which:
Reference now will be made in detail to embodiments of the invention, one or more examples of which are illustrated in the drawings. Each example is provided by way of explanation of the invention, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment can be used with another embodiment to yield still a further embodiment. Thus, it is intended that the present invention covers such modifications and variations as come within the scope of the appended claims and their equivalents.
The present subject matter relates to the simultaneous detection of multiple chemical, biological, and radiological/nuclear (CBRN) threats and, more particularly, to multiplexed FOrster Resonance Energy Transfer based (FRET-based) or Nanometal Surface Energy Transfer based (NSET-based) sensors capable of simultaneous detection of multiple threats, including one or more CBRN threats. Specifically, the present subject matter relates to a multifunctional sensor that can simultaneously detect two or more different CBRN threats of the same or different classifications on a single nano-sized platform using energy transfer-based technology. The disclosed sensors can be used to simultaneously detect CBRN threats with high selectivity, high confidence, and reduced false positives.
Disclosed sensors are based upon the energy transfer from an excited donor molecule to an acceptor through nonradiative dipole-dipole interactions. As illustrated in
As illustrated in
The detectable response of a particular sensing pair 12, 14 upon binding of a CBRN threat 18 to a binding ligand 16 of a linking agent 10 can be utilized to identify a particular type of threat. For example, different types of threats can be identified by a different optically detectable response (e.g., increased, quenched, or otherwise altered emission) upon analyte binding to a particular sensing pair of a sensor, leading to a unique indicator of the FRET-based biosensor. Moreover, in the case of binding multiple threats to a single sensor, a different response can be obtained as compared to the responses for each threat individually.
In implementation, a sensor can utilize FRET-based sensing, NSET-based sensing, or a combination thereof. The primary difference between FRET-based sensing and NSET-based sensing is the distance range of the sensing pairs. FRET-based sensing pairs are generally designed for a distance range between the donor and acceptor of from about 1 to about 20 nm, while NSET can extend that range to about 40 nm. In addition, for FRET, both the donor and acceptor are considered as point dipoles in which an excited donor (e.g., a molecule, molecular complex, ion, or nanoparticle) transfers the excitation energy non-radiatively to an acceptor, and the process is driven by dipole-dipole interactions between the transition dipole moment of donor emission and acceptor absorption in energetic resonance. As such, it is necessary to have a spectral overlap between the donor emission and the acceptor absorption. In NSET systems, the acceptor is considered a nanometric surface modeled as a collection of multiple point dipoles. The FRET point dipole-point dipole interaction results in the rate of energy transfer between the two components being dependent on the inverse of the distance to the sixth power, whereas the NSET point dipole-surface dipole interaction results in the rate of energy transfer being dependent on the inverse of the distance to the fourth power. Either system or a combination of both systems can be utilized in disclosed sensors to provide for real-time detection of multiple chemical threats.
A sensor can include a single instance of any one of the multiple coupled pairs or multiple instances. For instance, a sensor can include from 1 to about 500 instances of a single type of a coupled pair of a sensor (e.g., from 1 to about 500 instances of coupled pair 22, coupled pair 24, and coupled pair 26 of the sensor of
Each of the different coupled donor/acceptor coupled pairs 22, 24, 26 can share either a donor or an acceptor. For instance, in the illustrated embodiment, each of the coupled donor/acceptor pairs 22, 24, 26 can include the same acceptor 14, but a different donor 21, 23, 25, respectively. The component that differs in each of the different coupled pairs can be selected to ensure that the detectable signals of each coupled pair 22, 24, 26 will likewise differ. As such, a detectable signal of a coupled pair 22 that includes a donor 21 and an acceptor 14 will be detectably different as compared to a detectable signal between a coupled pair 24 that includes a donor 23 and the acceptor 14, and so on for each type of coupled pair of a sensor.
Donor/acceptor pairs as may be incorporated in a sensor are not particularly limited, provided each different donor acceptor pair of a single sensor can be identifiably detected from the others upon analyte binding. For instance, any optically active material that can emit light in the range from about 100 nm to about 100 microns (e.g., from ultraviolet to infrared) can be utilized as a donor, such as from about 380 nm to about 25 microns (e.g., from visible light to mid-infrared light), or from about 380 nm to about 10 microns (e.g., from visible light to near-infrared) can be used as a donor in disclosed sensors. In one embodiment, a donor can emit light in the visible spectrum, which may provide for ease of use in an in-field sensing application.
An acceptor for use with any particular donor should be selected so as to provide a detectable change in the emission upon the expected variation in conformation between the two upon analyte binding. One criterion that can be considered in determining suitable pairings is the relative emission/fluorescence spectrum of the donor compared to that of the acceptor. The emission spectrum of a donor and the absorbance spectrum of the acceptor should be in suitable range to one another (e.g., overlapping) such that the emission spectrum of the donor is at a wavelength that is able to interact with the absorbance spectrum of the acceptor at a first proximity and orientation relative to the acceptor and that upon change in that proximity and orientation due to analyte binding, the emission of the coupled pair is altered and thereby provide for an optically detectable signal signifying the analyte binding.
By way of example, when the donor is excited, for instance by visible light, a portion of the energy emitted by the donor can be transferred to the acceptor when the acceptor is spatially close enough to the donor (i.e., the distance between them is approximate to or less than the FOrster radius). Once the acceptor absorbs the energy, it in turn fluoresces in its characteristic emission wavelength, resulting in a recognizable emission of the pair. Upon analyte binding, the distance between the two can increase causing a change in the emission of the pair (e.g., quenching of the acceptor fluorescence) and an optically detectable change in the emission.
In some embodiments, upon binding a target analyte, the detectable signal can be the quenching of a signal as compared to no analyte binding. For instance, a signal of a donor/acceptor pair can be quenched by at least 1%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including all percentages in between each percentage listed.
In some embodiments, a sensor can include a nanoparticle of a plasmonic metal as a donor or an acceptor of a system, e.g., a plasmonic metal nanoparticle of gold, silver, copper, platinum, nickel, or palladium as an acceptor or a donor of a sensor. A metallic component is not limited to these materials, however, and other metals as may be incorporated in a system can include but are not limited to lithium, sodium, copper, aluminum, magnesium, barium, potassium, rubidium, and cesium. A particle may comprise mixtures or combinations of metal atoms. A nanoparticle component can generally have a largest cross-sectional dimension (e.g., a diameter) of about 1 nm or greater, such as from about 1 nm to about 100 nm, such as from about 1 nm to about 80 nm, such as from about 1 nm to about 50 nm, such as from about 5 nm to about 100 nm, such as from about 5 nm to about 50 nm, such as from about 10 nm to about 50 nm, such as from about 10 nm to about 30 nm.
In some embodiments, a metal nanoparticle component can be the donor or acceptor component that is shared among the multiple coupled donor/acceptor pairs of a sensor. For instance, a gold nanoparticle can be a common acceptor for all of the coupled pairs of a system.
Another class of energy transfer components as may be incorporated in a sensor can include semiconductors formed on a nanometer scale, generally referred to as quantum dots. Exemplary quantum dot materials can include, without limitation, binary materials such as CdTe, CdSe, CdS, ZnSe, PbS, PbSe, ternary materials such as ZnCdSe, CdSeS, CdPbTe, quaternary materials such as ZnCdSSe, as well as doped materials such as Mn doped ZnSe or Mn doped ZnS. In embodiments, a quantum dot of a system can include a core/shell quantum dot, e.g., a quantum dot including a CdSe core and a ZnS shell.
Quantum dots may be employed in a system as donors, acceptors, or both. For instance, a quantum dot of a first type may be utilized as a common donor or acceptor for a system and this common component can be coupled to a plurality of different quantum dots or other types of materials (or a combination thereof) to form a sensor.
The emission of quantum dot components incorporated in a system as a donor can be controlled through size of the particle or, in the case of a core/shell quantum dot, size of the core and/or thickness (i.e., number of atomic layers) of the shell.
Energy transfer components can also include organic molecular components such as fluorescent dyes, proteins, or peptides. Non-limiting examples of a fluorescent organic dyes include fluorescein and its derivatives, rhodamine and its derivatives, Texas Red, Cy5, acridine orange, 2,7-dichlorofluorescein, eosin, rose bengal, 1,2-dihydroxyanthraquinone, 1,4-dihydroxyanthraquinone, 1,8-dihydroxyanthraquinone, 1,3,8-trihydroxy-6-ethylanthraquinone, 1,2,5,8-tetrahydroxyanthraquinone, 1-aminonaphthalene, and 2-aminonaphthalene. Fluorescent derivatives of any of the foregoing are further examples of suitable fluorophores. The foregoing are merely examples and are not intended to be exhaustive.
A fluorescent protein may be, for example, a green fluorescent protein (e.g., TurboGFP, Azami Green, ZsGreen, Emerald), a blue fluorescent protein (e.g., EBFP2, mTagBFP2), a yellow fluorescent protein (mYFP, mVenus, mCitrine, TurboYFP), a red fluorescent protein (TurboRFP), mRuby2, mStrawberry, FusionRed). Fluorescent peptides are also embraced herein.
Each of the donor and acceptor can include suitable derivatization so as to be covalently or otherwise bonded to a coupling agent. For instance, a nanoparticle (a metal nanoparticle or a quantum dot) may include a coating on a surface that includes a polymer that can provide a hydrophobic, hydrophilic, or otherwise charged surface, or providing functional groups or groups that can subsequently be modified to provide functional groups that can directly or indirectly bind the desired coupling agent. A molecular donor or acceptor can be derivatized to include a functional group or a group that can subsequently be modified to include a functional group that can directly or indirectly bind the desired coupling agent. For instance, a donor and acceptor of a system can be functionalized to include a carboxyl, amine, or sulfhydryl group that can covalently bind an amine, carboxyl, or disulfide of a polynucleotide coupling agent under suitable reactive conditions, e.g., by reduction of a disulfide and subsequent binding to a sulfhydryl of an energy transfer component. Other suitable ligand exchange and coupling chemistries are known in the art as may be utilized.
Referring again to
A linking agent can include a double stranded polynucleic acid as in
The length of a polynucleotide linking agent can allow energy transfer to occur at least in the non-analyte bound conformation. For instance, a polynucleotide linking agent 10 having a length of about 100 bases or less, e.g., from about 5 to about 100 bases, from about 10 to about 90 bases, or from about 15 to about 85 bases, such as 5, 10, 15, 20, 25, 30, 40, 60, 80, or any number of bases therebetween in length can allow for energy transfer to occur and provide stability to a sensor.
When considering a single stranded linking agent 10 as in
As used herein, the terms “complementary” is used in reference to polynucleotides related by the base-pairing rules. For example, the sequence “5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.” Complementarity may be “partial,” in which only some bases of a polymer are matched according to the base pairing rules.
As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermodynamics of the formed hybrid. “Hybridization” methods involve the non-covalent bonding of one nucleic acid to another, complementary nucleic acid.
The linking agent 10 of each acceptor/donor pair of a system can also include one or more binding ligands 16. The binding ligand of each pair is specific for a particular analyte that represents a CBRN threat. For instance, an analyte can be the threat itself, e.g., a radiological threat such as strontium, cesium, etc. as well as a metabolite or adduct of a threat, e.g., a sarin metabolite, an anthrax metabolite, etc. In some embodiments, two or more binding ligands for each analyte of interest can be added to a sensor for secondary positive identification. Such an approach can decrease the risk of false positives for more reliable detection.
In various embodiments, the selective binding of the binding ligand 16 to a particular CBRN threat can be an innate component of the linking agent 10. For instance, a polynucleotide linking agent 10 can include a binding ligand 16 as an innate portion of the polynucleotide structure, e.g., a portion of the linking agent sequence, e.g., less than about 10 bases of the complete linking agent 10, that is a specific binding agent for the analyte. For example, as illustrated in
In some embodiments, the selective binding of a binding ligand 16 to a particular CBRN threat can be provided through addition of a binding functionality to a pre-formed linking agent 10, for instance through modification of a single base or a series of bases of a linking agent to include one or more inserted sequences that provides a specific binding ligand 16.
In some embodiments, in the absence of an analyte, a binding ligand 16 can hybridize with a complementary segment of a linking agent 10. In general, however, in the absence of the analyte a binding ligand 16 can be present in the linking agent 10 as a non-hybridized portion, as illustrated in
In one embodiment, a binding ligand 16 can be a component of an aptamer designed or identified to exhibit specific binding with an analyte of interest. A large number of aptamers have been developed and are known to those of skill in the art and as such are not enumerated herein. For example, aptamers as have been developed to exhibit specific binding to sarin and other nerve agents, anthrax (e.g., PA63, anthrax protective antigen, etc.), organophosphates (e.g., 2(diisopropylamino)ethyl] methylphosphonothiolate), botulism, metals (e.g., lead, uranium, etc.), any of which could be incorporated in a sensor as disclosed herein.
The configuration of a sensing system is not particularly limited. One embodiment of a liquid-based sensing system is illustrated in
A lateral flow system can include one or more zones through which a sample can flow, e.g., an application zone 54, a conjugate zone 55, a detection zone 56 and a calibration zone 57. Optionally, a system can include multiple areas within each zone, e.g., multiple conjugation areas that together form a conjugate zone 55, multiple calibration areas of a calibration zone 57, etc. Moreover, in some embodiments, a system need not include all of the zones as are illustrated in the embodiment of
To initiate the detection of one or more threat agents 50 within the test sample, a user may directly apply the test sample 43 to a portion of the substrate 52, e.g., to an application zone 54, through which it can then travel to reach one or more detection zones 56 and calibration zones 57. Alternatively, the test sample may first be applied to a sampling pad that is in fluid communication with the substrate 52.
In the illustrated embodiment, the test sample 43 travels from the application zone 54 to a conjugate zone 55 (as shown by the directional arrow 45 in
The detection zone 56 may contain an immobilized capture reagent 59. Although not required, it may be desired that the capture reagent 59 be formed from the same class or category of materials (e.g., aptamers) as the specific binding ligands 16 used to form the sensors 50. These capture reagents 59 serve as stationary binding sites for the sensor 50/threat agent 58 complexes. In some instances, the threat agents 58 can be capable of binding multiple specific binding agents (either the same or different from one another). Upon reaching the detection zone 56, one of these binding sites is occupied by the specific binding ligand of the sensor 50/threat agent 58 complex. However, the free binding site of the threat agent 58 can bind to the immobilized capture reagent 59. Upon being bound to the immobilized capture reagent 59, the newly formed ternary complex that includes the sensor 50, the threat agent 58, and the capture reagent 59 signals the presence of the threat agent 58.
As illustrated, a lateral flow-type sensing system can include a calibration zone 57. In this embodiment, the calibration zone 57 is formed on the porous membrane of the substrate 52 and is positioned downstream from the detection zone 56. The calibration zone 57 is provided with a binding agent 47 that is capable of binding to any remaining sensors 50 that pass through the length of the membrane substrate 52. In particular, upon being contacted with the test sample 43, any sensors 50 that do not bind any threat agent 58 migrate through the detection zone 56. In the detection zone 56, as set forth above, the conjugated sensors 50 bind to capture reagents 59 and remain immobilized. However, the unbound sensors 50 and in some embodiments also a portion of the sensor 50/threat agent 58 conjugates continue to migrate through the detection zone 56 and enter the calibration zone 57 of the substrate 52. At the calibration zone 57 these unbound sensors and/or conjugates thereof then bind to the binders 47. When immobilized in the calibration zone 57, any sensors can be detected, and a user can compare the signal intensity in the detection zone 56 to the signal intensity in the calibration zone 57.
The calibration zone 57 may generally provide any number of distinct calibration regions so that a user can better determine the concentration of a particular threat agent 58 within a test sample. In most embodiments, for example, the calibration zone 57 includes two or more calibration distinct calibration regions (e.g., lines, dots, etc.). A comparison may be made between the intensity levels of the calibration regions of a calibration zone 57 and that of the detection zone 56 to calculate the amount and type of threat agents present in the test sample.
A sensing system can carry the individual sensor components according to any known approach. For instance, and as illustrated in
Advanced and portable sensor capabilities for the simultaneous onsite detection of specific CBRN threats as possible with disclosed sensors can significantly limit the risk of exposure to personnel and allow for the rapid, in field collection of essential scientific data and critical evidence. Disclosed sensors can provide effective identification of multiple threats with a much faster, real-time response and a simpler signal readout as compared to previously known CBRN sensors. In this regard, disclosed sensors can be utilized for in field detection due to the low detection limits, small size, and low power requirements.
The present invention may be better understood by reference to the examples set out below.
The degree of energy transfer between an energy transfer pair depends upon the distance between the donor and acceptor, as well as on the size and composition of the energy acceptor, which may influence detection limits for detection schemes. To investigate the differences in energy transfer with different metal acceptors, a Python script was created to calculate the theoretical FRET/NSET efficiencies for metal nanoparticles of different sizes and optical properties. FRET/NSET calculations were carried out for 3 different metal nanoparticles (Cu, Ag, Au) between 2-50 nm in diameter.
Gold nanoparticles (AuNPs) were functionalized through ligand exchange with thiol functionalized aptamers.
A first DNA sequence examined was a hairpin sequence such as may be utilized in forming a sensor as schematically illustrated in
A second DNA sequence examined was a relatively short strand including a single TTTAGT (SEQ ID NO: 2) binding site for the sarin metabolite methylphosphonic acid (MePA). The full sequence was as follows:
As shown in
To verify DNA analyte binding with the second conjugate, the melting temperature of AuNP-ssDNA (SEQ ID NO: 3) was measured in the presence and absence of MePA. The melting temperature (Tm) decreased from Tm=45° C. to Tm=33 ° C. in the presence of MePA. This indicates interaction and probable binding for the analyte with the DNA strand. This interaction would thus change the distance between the donor and acceptor in a ds-DNA sensor, leading to a detectible change in FRET response.
The AuNP-DNA conjugates that were created with the MePA-targeting aptamers were calculated to have an average of about 70 DNA per AuNP. This high density of targeting ligands was a result of the large nanoparticle surface area, which can lead to more sensitive detection.
Black Hole Quencher®-2 dye was linked to the surface of a CdSe/ZnS quantum dots (qdot) through dsDNA hybridization of complementary ssDNA conjugated to each component to study the FRET between the qdot donor and the dye quencher acceptor. As shown in
Qdots have the ability to be both FRET donors and acceptors because of their broad absorption spectra and narrow emission peaks. As shown in
One challenge to qdot-qdot FRET pairs is spectral cross talk, which results from the broad qdot absorption profiles. Three-dimensional fluorescence excitation emission matrix (EEM) spectroscopy was measured for a qdot pair without spectral overlap and thus an absence of FRET. The results are shown in
CdSe/ZnS quantum dots (qdots) were functionalized with a DNA aptamer specific for the sarin metabolite, methylphosphonic acid (MePA). The aptamer sequence included multiple copies of the TTTAGT (SEQ ID NO: 2) binding sequence specific for the MePA. The aptamer sequence was as follows:
Qdots with different sizes, absorbance and emission wavelengths as illustrated in
An anthrax detection sensor as described by
Known aptamers could be utilized. Two examples of literature-based aptamers as may be utilized include:
A system with the donor and acceptor of the FRET pairs unbound showed little quenching.
A FRET pair was also designed to target nuclear materials using an aptamer for Strontium (Sr) that was isolated using a cell sorter technique. The aptamer was also selected against common interferents (Ca, Mg, Na) for more sensitive detection. Specifically, SELEX library DNA were bound on magnetic beads and incubated with red (Alexa Fluor® 647/APC filter) and green (3′ FAM/FITC filter) fluorescent probes for 10 minutes before they were incubated with 10 nM Sr. Results are shown in
This written description uses examples to disclose the technology, including the best mode, and also to enable any person skilled in the art to practice the technology, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the technology is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they include structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.
This application claims filing benefit of U.S. Provisional Patent Application Ser. No. 63/297,361, having a filing date of Jan. 7, 2022, which is incorporated herein by reference for all purposes.
This invention was made with government support under Contract No. 893033210EM000080, awarded by the U.S. Department of Energy. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63297361 | Jan 2022 | US |
