Patent Application
20220136973
Typical affinity agents used in analytical techniques are anchored onto a substrate and are not freely movable in the test solution containing the analyte. Such affinity agents are also usually deigned to be highly specific to the target analyte.
Applicant has found that by using linear polymers with suitable functional groups, the polymers may be allowed to freely move in the solution and to bind to the analyte by hydrogen bonding. A relatively high concentration of polymer may be present in the solution, allowing for the capture of a greater amount and/or larger number of analytes. The polymer-analyte complex may then be applied to a sensing substrate for spectral analysis, e.g., by surface-enhanced Raman scattering (SERS). The analytes may be any suitable small molecules that are capable of hydrogen bonding with the functional groups of the polymer. Two or more analytes may be sensed at the same time.
The system and method of the present disclosure involve the use of a polymer affinity agent, a sensing substrate, and SERS. The system and method of the present disclosure are suitable for detection of multiple analytes at the same time. The system and method of the present disclosure are suitable for detection of analyte molecules that are capable of hydrogen bonding. The system and method of the present disclosure are particularly suitable for detection of contaminants in foods and beverages.
Detection of mycotoxins, for example two or more mycotoxins simultaneously, is of particular interest. The number of known mycotoxins is very high, and various mycotoxins may be present in the same food or beverage product. However, due to their varying molecular structures, analysis of multiple mycotoxins at the same time is challenging, and often different mycotoxins need to be analyzed separately. For example, while various analytical techniques have been used to individually detect deoxynivalenol (DON) or ochratoxin A (OTA) at low limits of detection, direct multiplex detection of these toxins together is new. Collecting data in other types of multiplex sensing has often involved post-measurement chemometric analyses to distinguish between different toxins. Various aspects of the present disclosure relate to a linear polymer affinity agent used to directly detect both DON and OTA simultaneously via surface-enhanced Raman scattering without the need for chemometric analysis. Paired with density functional theory (DFT), the vibrational stretches of each small molecule are predicted, which provides additional insight on association of the analyte and polymers at the molecular level. This multiplex sensing scheme is simple, relatively inexpensive, and requires minimal analysis, displaying the overall potential a linear polymer affinity agent has for detecting various classes of small molecules.
In one aspect, the present disclosure relates to a method of using a sensor. The method includes mixing a linear polymer affinity agent in a sample solution. The method also includes subjecting a metal substrate to the sample solution to attach the linear polymer affinity agent to the metal substrate. The method may also include generating, via Raman Spectroscopy, spectral data representing the at least one linear polymer affinity agent attached to the metal substrate. The method also includes determining whether two or more analytes, such as toxins, are present in the solution at respective minimum threshold concentrations based on the spectral data.
In another aspect, the present disclosure relates to a sensor. The sensor includes a metal substrate including a plasmonic metal. The sensor also includes at least one linear polymer affinity agent. In one exemplary embodiment, the linear polymer affinity agent is synthesized via polymerization of N-(2-aminoethyl) methacrylamide hydrochloride (polymerization of AEMA, or pAEMA). The linear polymer affinity agent may bind to one or more analytes by hydrogen bonding. The linear polymer affinity agent may be attached to the metal substrate.
In another aspect, the present disclosure relates to a method of calibrating a sensor. The method includes subjecting a metal substrate to a calibrating solution including at least one linear polymer affinity agent and at least two analytes at respective known concentrations. The method also includes generating, via surface-enhanced Raman spectroscopy, spectral data representing the at least one linear polymer affinity agent being bound to the at least two toxins and being attached to the metal substrate. The method also includes generating calibration data based on the spectral data to detect the at least two analytes at respective minimum threshold concentrations. The calibration data includes identification of different peaks associated with each analyte.
The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
In general, the present disclosure relates to multiplex surface-enhanced Raman scattering detection of one or more analytes using a linear polymer affinity agent and a sensing substrate. The present disclosure further relates to polymer affinity agents including one or more functional groups capable of hydrogen bonding, and to use of the affinity agents to detect analytes that are capable of hydrogen bonding with the functional groups of the polymer. The system and method of the present disclosure are suitable for detection of multiple analytes at the same time. The system and method of the present disclosure are particularly suitable for detection of contaminants in foods and beverages. Exemplary analytes include mycotoxins, cyanogenic glycosides, and other small molecule toxins.
As an example, the system and method of the present disclosure can be used to detect deoxynivalenol (DON) and ochratoxin A (OTA) with a linear polymer affinity agent. While various analytical techniques have been used to individually detect DON and OTA at low limits of detection, direct multiplex detection of these toxins together is new. Collecting data in other types of multiplex sensing has often involved post-measurement chemometric analyses to distinguish between different toxins. Various aspects of the present disclosure relate to a linear polymer affinity agent used to directly detect both DON and OTA simultaneously via surface-enhanced Raman scattering without the need for chemometric analysis. Paired with density functional theory (DFT), the vibrational stretches of each small molecule are predicted, which provides additional insight on association of the analyte and polymers at the molecular level. This multiplex sensing scheme is simple, relatively inexpensive, and requires minimal analysis, displaying the overall potential a linear polymer affinity agent has for detecting various classes of small molecules.
This disclosure describes the use of basic molecular hypotheses to design a linear polymer affinity agent that can bind multiple targets. According to an embodiment, the linear polymer affinity agent includes repeating monomer units, which include one or more functional groups capable of hydrogen bonding with an analyte. For example, the linear polymer affinity agent may include repeating monomer units that have an amine group, a hydroxyl group, or another functional group capable of hydrogen bonding. Further according to an embodiment, the linear polymer affinity agent has or may be reacted with a functional end group that is capable of bonding with the sensing substrate. For example, the linear polymer affinity agent may have or may be reacted with a functional end group that contains sulfur and is capable of bonding with the sensing substrate. Any suitable polymer backbone may be used. For example, the repeating monomer units may include (meth)acrylate, (meth)acrylamide, or any variation of acrylates and acrylamides. The size of the polymer may be selected such that the enhancement field of the SERS can be utilized. In some embodiments, the polymer has 10 or more, 15 or more, or 20 or more monomer repeating units. The polymer may have 40 or less, 35 or less, or 30 or less monomer repeating units. For example, the polymer may have from 10 to 35 or from 15 to 30 monomer repeating units.
One example of a suitable linear polymer affinity agent is pAEMA, which is a simple and inexpensive linear polymer. pAEMA may be synthesized to have from 10 to 35 or from 15 to 30 (e.g., 29) methacrylate units. pAEMA can be used to complex multiple analytes and anchor onto a sensing substrate through, for example, trithiocarbonate and gold interactions. For example, pAEMA can be used to complex at least two small molecule mycotoxins, DON and OTA, and anchor onto a sensing substrate through trithiocarbonate and gold interactions. This disclosure describes detection of both toxins individually using surface-enhanced Raman scattering (SERS), with no additional sensing probe molecule. Both toxins may be sensed simultaneously and visibly distinguished without any chemometric analysis of the data. The OTA Raman spectrum has been computationally modeled for the first time and added to the overall vibrational labeling of the DON Raman spectrum. Moreover, DFT can assist not only in the labeling of strong vibrational modes, but the ability to monitor the stretches in real-time facilitates clear conclusions about the fundamental interactions occurring between target and affinity agent during sensing. Hydrogen bonding may associate the two mycotoxins to the polymer through the amine groups. Additionally, hydrogen bonding may occur at multiple sites on the small molecules based on the location of the vibrational shifts. The polymer affinity agent may be selected such that multiple analytes (e.g., multiple toxins) are able to interact and complex to the polymer at concentrations relevant to their level of interest, such as possible regulation limits. For example, in the case of mycotoxins, both DON and OTA are able to complex to pAEMA at concentrations relevant to their regulation limits: 1 part per million (ppm) and 5 parts per billion (ppb) for DON and OTA, respectively. This further shows that linear polymer affinity agents can serve as unique capture agents due to their easily modifiable pendant groups, inexpensive nature, and non-specificity towards solely one target.
Various aspects of the present disclosure relate to polymers used as affinity agents to capture and detect a wide variety of analytes. Synthetic affinity agents are relatively inexpensive, robust, and are readily synthesized to tune and exploit certain interactions. Whether in conjunction with other affinity agents for increased specificity or synthesized specifically to bind to a target, these agents can be used to identify and detect biological toxins, food contaminants, and other small molecules.
Molecular imprinting polymers (immobilizing a target as a template in a polymer matrix) has long served as the synthetic mechanism to generate polymer-based affinity agents for sensors. However, these systems may be difficult to characterize and reproduce, due to their insoluble nature, and may be challenging to use as sensors in food samples with significant background signal due to the amount of other small molecules, proteins, sugars, etc. masking sensing identification, i.e. complex matrices. In addition, because they are cast to bind a specific target, these polymer templates cannot detect multiple targets at once without adding additional synthetic steps. These relatively thick polymer templates make it difficult to use surface analytical techniques for target detection, for example, due to the majority of the sensing volume being occupied by the polymer matrix rather than the matrix with captured analytes.
On the other hand, linear polymer affinity agents facilitate synthetic control of the chain length of the polymer and multivalent display of functional groups, where each monomer repeat unit serves as a potential binding site for the target analyte. In one example, a single-point-attachment polymer affinity agent with pendant saccharide moieties on the repeat unit structure was designed to specifically bind to a pocket of a protein used as a bioterror agent. Leveraging simple chemistry with an attractive analytical technique like SERS, one can monitor binding of the target to the polymer in both purified and complex matrices. Expanding on this concept, if one allows for similar bonding interactions, the choice of the polymer repeat unit can facilitate multiplex capture of an entire class of molecules.
In light of multiplex detection, SERS may be used as a signal transduction mechanism because of its low limit of detection, its ability to provide a “fingerprint” unique to the analyte of interest, and its compatibility with aqueous samples. When an analyte is immobilized near a plasmonic metal surface, one can observe an enhanced intensity of the analyte's signature “fingerprint” due to the vibrational modes of the analyte inelastically scattering light. These surfaces supporting localized plasmons are usually characterized by nanoscale roughness to generate a small-volume, but intense, electromagnetic (EM) field. This EM field extends only a few nanometers from the plasmonic surface. Both affinity agent and target analyte may be contained within the enhancement field to fully take advantage of SERS capabilities. Polymer chain length of the affinity agent may be a factor when using polymer affinity agents for SERS detection. With a long chain length (e.g., greater than 40), the analyte signature may not be seen. With a short chain length (e.g., fewer than 10), insufficient repeat unit binding sites for target-analyte interaction due to dense packing of the short polymer chains at the substrate surface may occur.
Various aspects of this disclosure relate to the use of SERS and linear polymers as affinity agents for multiplex analyte detection. In some embodiments, the optimization of linear polymer affinity agents may include the order of polymer and target attachment to the sensing substrate to reach relevant levels of detection. While traditional affinity agents are often anchored to the sensing substrate first, polymer affinity agents may exhibit heightened flexibility in solution, which may enable optimal polymer-target binding that is generally not achievable when pre-anchored to the substrate. This increases the amount of analyte that can readily associate with the polymer. The potential of linear polymer affinity agents may be demonstrated, for example, by multiplex detection of two different small molecule targets of interest, such as deoxynivalenol (DON) and ochratoxin A (OTA). These molecules are mycotoxins, small molecule toxins that are naturally produced from fungi that contaminate various types of crops and feedstocks. Mycotoxins are an interesting class of small molecules to detect, for example, due to their toxicity at very low exposure levels and ability to biomagnify in the environment. Both DON and OTA toxins are relevant targets for detection due to their prevalence in food, dangerous effects on livestock and humans, and their toxicity at very low concentrations. Multiplex detection of the two, simultaneously, is also important because they both contaminate grains and grain products.
Deoxynivalenol (DON), also known as vomitoxin, is a toxin naturally produced by Fusarium bacteria species (F. graminearum and F. culmorum). It is one of the most common mycotoxin contaminants in grains such as wheat and corn. When ingested, DON is an immunotoxin and can cause severe dehydration due to vomiting and diarrhea. Some jurisdictions regulate the DON in the range of 1 ppm for humans.
Ochratoxin A (OTA), is a toxin naturally produced by various Aspergillus and Penicillium bacteria species (A. ochraceus, P. verrucosum, A. carbonarius, and A. nigier). OTA tends to contaminate various grains, pork, and alcoholic beverages such as beer and wine. OTA is a nephrotoxin, a teratogen, a potential carcinogen, and has been linked to neurodegenerative diseases. Some jurisdictions regulate OTA at a much lower concentration compared to DON, such as 5 ppb, due to its extremely adverse effects.
In some embodiments, the metal substrate may include at least one of gold, copper, aluminum, or silver. In a preferred embodiment, the metal substrate includes gold. The metal substrate may include one of a film of gold over a support substrate. The support substrate may include a silica nanosphere matrix, a colloidal gold substrate, or both.
According to an embodiment, the linear polymer affinity agent may be configured to bind to at least two analytes by hydrogen bonding. In some embodiments, at least one linear polymer affinity agent may be configured to bind to at least two mycotoxins. At least one linear polymer affinity agent may be configured to bind deoxynivalenol, ochratoxin A, or both.
The sensor may be used in any suitable manner. In one example, a method may include mixing a linear polymer affinity agent in a sample solution. The method may include hydrogen bonding of an analyte (e.g., two or more different analytes) to the linear polymer affinity agent. The method may also include subjecting a metal substrate to the sample solution to attach the linear polymer affinity agent to the metal substrate. The method may also include generating, via Raman Spectroscopy, spectral data representing the at least one linear polymer affinity agent attached to the metal substrate. The method may further include determining whether two or more toxins are present in the solution at respective minimum threshold concentrations based on the spectral data.
The linear polymer affinity agent may have at least 10 or at least 15 and up to 40 or up to 35 monomer repeating units. The linear polymer affinity agent may include one or more functional groups that are capable of hydrogen bonding. The linear polymer affinity agent may include methacrylamide. The linear polymer affinity agent may include amine groups, hydroxyl groups, or other functional groups capable of hydrogen bonding. In some embodiments, the linear polymer affinity agent may be synthesized via polymerization of N-(2-aminoethyl) methacrylamide hydrochloride.
In some embodiments, determining whether two or more analytes (e.g. toxins) are present may include determining whether two or more mycotoxins are present. Determining whether two or more analytes (e.g. toxins) are present may include determining whether deoxynivalenol, ochratoxin A, or both are present. Determining whether two or more analytes (e.g. toxins) are present may include detecting one or more hydrogen bonds between the linear polymer affinity agent and at least one of the analytes (e.g. toxins).
In some embodiments, determining whether two or more analytes (e.g. toxins) are present may include identifying a peak in the spectral data not associated with the linear polymer affinity agent or the two or more analytes (e.g. toxins). Determining whether two or more analytes (e.g. toxins) are present may include identifying bonds with a different functional group of each analyte (e.g. toxin) bonding with the linear polymer affinity agent.
Determining whether two or more toxins are present may include identifying a peak in a range from 300 to 1700 cm−1 as corresponding to ochratoxin A. Determining whether two or more toxins are present may include identifying a peak in a range from 300 to 1800 cm−1 as corresponding to deoxynivalenol.
The sensor may be calibrated using any suitable method. In one example, a method may include subjecting a metal substrate to a calibrating solution including at least one linear polymer affinity agent and at least two analytes at respective known concentrations. The method may also include generating, via Raman Spectroscopy, spectral data representing the at least one linear polymer affinity agent being bound to the at least two analytes and being attached to the metal substrate. The method may further include generating calibration data based on the spectral data to detect the at least two analytes at respective minimum threshold concentrations. The calibration data may include identification of different peaks associated with each analyte.
In some embodiments, at least two analytes may include two mycotoxins. At least two toxins may include deoxynivalenol, ochratoxin A, or both.
A linear, methacrylamide polymer affinity agent was explored to capture two mycotoxins, deoxynivalenol (DON) and ochratoxin A (OTA), for multiplex surface-enhanced Raman scattering (SERS) detection. These mycotoxins are naturally occurring small molecules from fungi that can be dangerous at low concentrations. SERS detection was completed for each polymer-toxin complex at concentrations relevant to current safety regulation by the FDA: 1 ppm for DON and 5 ppb for OTA. Visibly distinguishable vibrational modes were observed in the multiplex spectra that were attributed to each mycotoxin individually, thus, not requiring any additional chemometric analysis. Density functional theory (DFT) was used to model DON and OTA to accurately label the vibrational modes in the experimental spectra as well as provide insight on the binding between both targets and the affinity agent. Fully modeled vibrations of these toxins are novel contributions due to OTA never being modeled and only a few published vibrational modes of DON. DFT guides empirical observations regarding hydrogen bonding at multiple sites of each mycotoxin target molecule through the amine groups on the polymer, confirming the capabilities of a single polymer affinity agent to facilitate multiplex detection of a class of molecules through less-specific interactions than traditional affinity agents.
N-(2-aminoethyl) methacrylamide hydrochloride (AEMA.HCl) was purchased from Polysciences, Inc, (Warrington, Pa.) and was purified by recrystallization in ethanol.35 Deoxynivalenol from Fusarium graminearum and Fusarium culmorum cultures and ochratoxin A from Petromyces albertensis (OTA, ≥98%) were purchased from Sigma-Aldrich. The polymerization initiator, 4,4′-azobis(4-cyanovaleric acid) (V501, ≥98.0%), was purchased from Sigma-Aldrich as well. The chain transfer agent (CTA), 4-cyano-4-(propylsulfanylthiocarbonyl)-sulfanylpentanoic acid (CPP), was synthesized as previously reported in literature.36 Silica nanospheres, with a 590 nm diameter (10% solids), were purchased from Bangs Laboratories, Inc (Fishers, Ind.). Pure gold (99.999%) was purchased from Kurt J. Lesker, (Clairton, Pa.). Purchased reagents did not undergo any further purification unless noted.
Synthesis of this 29 repeat unit polymer (pAEMA29) is fully described in a previous paper. (Szlag, V. M. et al. Isothermal Titration calorimetry for the Screening of Aflatoxin B1 Surface-Enhanced Raman Scattering Sensor Affinity Agents, Anal. Chem. 2018, 90 (22), 13409-13418.) Briefly, AEMA.HCl was dissolved in 90% 1 M acetate buffer, alongside 10% ethanol, the initiator, 4,4′-azobis(4-cyanovaleric acid) (V501), and the CTA 4-cyano-4-(propylsulfanylthiocarbonyl) sulfanylpentanoic acid (CPP). The reaction mixture was degassed via 3 cycles of freeze-pump-thaw and polymerized at 70-80° C. overnight (˜18 h). The polymerization was stopped by exposing the mixture to ambient air. The mixture was dialyzed in a 100-500 Da bag against 3 L of Milli-Q water for 24 h. This was then lyophilized resulting in a dry, light yellow solid with a yield of 78%. The polymer molecular weight was characterized via aqueous mobile phase (0.1 M Na2SO4 in 1.0 v % acidic acid) size exclusion chromatography (SEC). The instrument is an Agilent 1260 Infinity Quaternary LC System with Eprogen columns [CATSEC1000 (7 μm, 50×4.6), CATSEC100 (5 μm, 250×4.6), CATSEC300 (5 μm, 250×4.6), and CATSEC1000 (7 μm, 250×4.6)]. The system was equipped with a Wyatt HELEOS II light scattering detector (λ=662 nm) and an Optilab rEX refractometer (λ=658 nm).
Isothermal titration calorimetry was carried out using an ITC-200 microcalorimeter. ITC measurements were performed using a MicroCal PEAQ-ITC Automated (Malvern Instruments, Westborough, Mass.) at 25° C. as previously discussed in previous literature. (Szlag, V. M. et al., Anal. Chem. 2018; Szlag, V. M. et al. Optimizing Linear Polymer Affinity Agent Properties for Surface-Enhanced Raman Scattering Detection of Aflatoxin B1, Mol. Syst. Des. Eng. 2019.)
The sample cell and injection syringe were cleaned with 20% Contrad 70 detergent, water, and methanol. The instrument syringe was flushed with 10% bleach twice after each experiment. A 22 vol % dimethylsulfoxide, 16 vol % methanol, 62 vol % acetate buffer (pH 5) mixture was used to make a 4.0 mM polymer repeat unit solution and 0.26 mM OTA and DON samples. The instrument automatedly transferred polymer into the mycotoxin samples (or into blank solvent for the background titration). The titration used a 1.5 μL injection volume and 150 s injection intervals. Raw ITC profiles, as shown in
FONs were fabricated as previously reported in literature. (Szlag, V. M. et al. Mol. Syst. Des. Eng. 2019; Kim, D. et al. Microfluidic-SERS Devices for One Shot Limit-of-Detection, Analyst 2014, 139 (13), 3227-3234; Le Ru, E. C. et al. Surface Enhanced Raman Scattering Enhancement Factors: A Comprehensive Study, J. Phys. Chem. C 2007, 111 (37), 13794-13803) 590-nm-diameter silica nanospheres were dropcast on 1 cm×1 cm silicon wafers to form a nanosphere mask. A 95.3 nm pure gold film was deposited under vacuum, measured by a quartz crystal microbalance (Denton Vacuum, Moorestown, N.J.). FONs with a localized surface plasmon resonance (LSPR) λmax between 750 and 850 nm, measured using a fiber optic probe (Ocean Optics, Dunedin, Fla.) with a flat gold film as the reflective standard, were used.
A 1 mM polymer solution (40:60 MeOH/water) was mixed with 50% by volume solutions of varying concentrations of DON and OTA and left to interact for 6 h. FON substrates were then incubated in 200 μL of the complexed mixture in a 24-wellplate for 18 h. Substrates were then washed with 1-2 mLs of Milli-Q water and air dried. Measurements were performed using a Snowy Range Instruments SnRI ORS System with a 785 nm laser, 9 mW incident power, and a 10 s integration time. Each condition was measured on three substrates, and five spots on each substrate was measured for a total of 15 averaged spectra. The FON average spectrum was baselined in OriginLab's Origin 9.1 (using eleven anchor points created by the first and second derivative with a Savitsky-Golay smoothing and connected by B-spline interpolation, with the same number of points as the input spectrum) and normalized by the incident power and integration time.
Non-resonant Raman spectra of DON and OTA were calculated using density functional calculations in GaussView 6.0.16 with a basis set of B3LYP/6-311G++ (d, p) following previous literature that calculated a small number of DON vibrational bands. (Yuan, J. et al. Rapid Raman Detection of Deoxynivalenol in Agricultural Products, Food Chem 2017, 221, 797-802.) Rotational and vibrational bands were labeled for each molecule and can be found in Table 1.
Computational modeling of the molecule was completed here to fully assign all possible peaks in the experimental spectrum for DON. To determine vibrational band assignments for OTA itself, DFT calculations were performed for both mycotoxins, and their vibrational band assignments were labeled accordingly. The Raman spectrum of DON was computed following the same basis set as previously reported and agreed well with published calculations. As can be seen in
SERS Detection of Deoxynivalenol with pAEMA29.
In contrast to traditional affinity agents, which have been anchored to the sensing substrate and then flowing or attaching the target, DON and pAEMAa29 were complexed in solution, through what is hypothesized to be hydrogen bonding, and then this complex was anchored to the FON substrate. Comparing the anchored complex at various concentrations of DON to DON-free polymer and the 0 ppm condition (just the solution conditions the toxin is diluted in) in
SERS Detection of Ochratoxin A with pAEMA29.
Following the same protocol as with DON, FONs were incubated in an OTA and pAEMA29 mixture at varying concentrations, and the captured spectra can be seen in
Multiplex SERS Detection of Deoxynivalenol and Ochratoxin a with pAEMA29
In an effort to detect both toxins simultaneously, both DON and OTA were placed in solution with pAEMA29 to give both the opportunity to complex with the polymer affinity agent via the hypothesized hydrogen bonding/association. The multiplexed spectra, seen in
The association between mycotoxin and our polymer system was hypothesized to be through hydrogen bonding interactions, while other mycotoxin and affinity agent sensing systems have relied on much more specific interactions. Previous computational modeling to screen various monomers to bind to two different mycotoxins for chromatography applications has revealed a strong binding energy (−41.94 kcal/mol) between OTA and a monomer structure with amine groups. (Piletska, E. et al., Development of the Custom Polymeric Materials Specific for Aflatoxin B1 and Ochratoxin A for Application with the ToxiQuant T1 Sensor Tool, J Chromatogr A 2010, 1217 (16), 2543-2547.) The monomer modeled in that work was not easily amenable to controlled polymerizations. In this example, the same interactions were exploited with a sensing system of the present disclosure due to the amine groups on the polymer and the moieties on both OTA and DON that readily interact with amines through hydrogen bonding. By monitoring the stand-alone, unique peaks to each mycotoxin in the multiplex spectra, the types of interactions occurring were confirmed while sensing the two small molecules simultaneously.
The carboxyl group on the phenylalanine moiety of OTA can form hydrogen bonds between amino groups on a monomer and the carboxyl group of the phenylalanine moiety and electrostatically interact with the amino group on the primary amine monomers. (Piletska, E. et al., J. Chromatogr A 2010.) In the multiplex spectra, the 1535 cm−1 shift, unique to OTA, was referenced to the 1527 cm−1 shift in the computational spectrum. When monitoring this computational mode in real time, the stretch was attributed to strong vibrations on the benzyl ring of the phenylalanine moiety of OTA. This may indicate hydrogen bonding at the carboxyl of the OTA phenylalanine. The second stand-alone peak in the multiplex spectra, at the 916 cm′ shift, was referenced to the 917 cm−1 shift in the computational spectrum due to strong asymmetric stretching of the tertiary carbons attached to the carboxyl group previously mentioned. This further confirmed hydrogen bonding between OTA and the linear polymer affinity agent. An enlarged image of the multiplex spectra and hypothesized binding can be seen in
Following the same hypotheses for OTA and pAEMA29, observing the unique DON peaks in the multiplex spectra, molecular details about DON interactions with the polymer affinity agent were examined. The peak at 1455 cm−1 shift in the multiplex spectra was referenced to 1450 cm−1 shift from the computational spectrum, strong asymmetric rocking of the hydrogens on a tertiary carbon bound to a hydroxyl group, indicating hydrogen bonding at the hydroxyl. At 1234 cm−1 shift, the polymer and blank spectra have a broadened peak that shifts and sharpens into a peak at 1241 cm−1 shift that were referenced to the 1245 cm−1 shift in the computational spectrum of DON. This vibrational mode was attributed to both strong stretching of the hydrogens on the bicyclic ring near the hydroxyl (O3) and symmetric stretching of the hydrogens in the out of plane hydroxyl group (O5) as labeled in
Thus, various embodiments of linear polymer affinity agent substrate for surface-enhanced Raman spectroscopy are disclosed. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern.
References explicitly incorporated herein include, but are not limited to, the following:
This application claims the benefit of U.S. Provisional Application Ser. No. 63/107,117, filed Oct. 29, 2020, which is incorporated by reference herein.
This invention was made with government support under DMR-1420013 awarded by the National Science Foundation and under OD017982 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63107117 | Oct 2020 | US |
