Abstract:
Triazole-based molecules, methods of making and using the same are provided. Triazole-based molecules may be used as reading molecules and incorporated into or operatively-linked with electrodes, for example, and used in recognition tunneling systems to identify individual and/or sequences of molecules (e.g., DNA bases, carbohydrates, proteins, peptides, and/or amino-acids).

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority to U.S. provisional application No. 61/945,659 titled “TRIAZOLE-BASED READER MOLECULES AND METHODS FOR SYNTHESIZING AND USE THEREOF”, filed Feb. 27, 2014, the entire disclosure of which is incorporated herein by reference. 
     
    
     STATEMENT OF FEDERALLY SPONSORED RESEARCH 
       [0002]    The invention(s) disclosed herein were made with government support under U54 CA143862 awarded by the National Institute of Health. The United States government has certain rights in the invention(s). 
     
    
     FIELD OF THE DISCLOSURE 
       [0003]    This disclosure relates to the identification of DNA bases, sugars, proteins/peptides/amino acids, and/or pharmaceuticals (drug molecules) by means of electronic detection using recognition tunneling. Reading molecules, methods of preparing such reading molecules and methods of using the same are provided. Reading molecules, according to at least some of the embodiments of the present disclosure, may be attached to palladium surfaces/electrodes, for example, and used in the detection of single molecules and/or sequences thereof (e.g., nucleic acids, proteins, carbohydrates, and/or the like). 
       BACKGROUND 
       [0004]    The human proteome, which is encoded by just some 25,000 genes, consists of millions of proteins variants, due to single nucleotide polymorphisms (SNP), somatic DNA rearrangements, RNA splicing, and post translational modifications (PTM). 
         [0005]    In recent years, a need for a parallel method has emerged, which requires the ability to read human proteomes with high throughput and low cost. In contrast to the human genome in which DNA exists as diploid, the proteome has a wide dynamic range, for example the abundance of proteins in human plasma spans more than 10 orders of magnitude. Some proteins are expressed in a low quantity. Proteomics has no tool equivalent to the polymerase chain reaction (PCR) for protein sample amplification. There is no cost effective way to faithfully reproduce a protein population from a source. Thus, protein analysis must be carried out by extracting materials from samples removed from humans in some quantity. 
         [0006]    There have been remarkable advances in sample preparation, and sequencing techniques, most notably based on mass spectroscopy as a proteomic tool. However, mass-spectrometers are large, costly machines. Their size is dictated by the need for very high mass resolution to obtain accurate identification of the amino acid components (and even then, readout is complicated by isobaric amino acids). Accordingly, there is a need for an alternative method for identifying amino acids, particularly in small quantities, and ideally at the single-molecule level. “Recognition Tunneling” (RT) has emerged as such a method, which is purely physical and does not rely on the reactions of a DNA polymerase or ligase, but also is able to recognize any chemical residue provided that it generates a distinctive tunneling current signal. 
         [0007]    More specifically, the mechanism of recognition tunneling for reading nucleic acids, sugars, and amino acid sequences is based on the trapping of an analyte (i.e., a molecule of a nucleic acid, a sugar, an amino acid) by “reading molecules”, which are chemically tethered to two closely spaced electrodes, which generate a distinct tunneling signal upon a potential being applied across the electrodes. Specifically, the reading molecules are chemically bonded to the metal electrodes through a short linker while non-covalently interacting with the target molecule(s) at the other end. As target molecules of nucleic acids, sugar, amino acids, drug molecules pass through the tunnel, and a potential is applied between the electrodes, interaction of each such molecule with the reading molecules temporarily traps the analytes and produces tunneling signals, which comprises a particular current. The tunneling signal can be used to identify the analytes. Prior to the present disclosure, reading molecules for RT included imidazole-based reading molecules, namely, 4(5)-(2-mercaptoethyl)-1H-imidazole-2-carboxamide (ICA) (Liang, F.; Li, S.; Lindsay, S.; Zhang, P.  Chem. Eur. J.  2012, 18, 5998-6007) and 5(6)-mercapto-1H-benzo[d]imidazole-2-carboxamide (see, U.S. provisional patent application No. 61/829,229). 
       SUMMARY 
       [0008]    In some embodiments, triazole-based compounds may be used as universal reader molecules for functionalization onto electrodes/substrates of RT systems. For example, triazole-based compound 5-(2-mercaptoethyl)-4H-1,2,4-triazole-3-carboxamide (TCA), the chemical structure of which can be found in  FIG. 1 , is a reading molecule which can be used in a RT system for the identification of an analyte containing, for example, DNA nucleotides. In some embodiments, TCA can be used to distinguish, for example, between DNA bases (including methylated cytosine), sugars, and proteins/peptides/amino acids. TCA can exist in tautomers ( FIG. 2 ). 
         [0009]    In some embodiments, the thickness of the triazole compound on an electrode/substrate (self assembled monolayer, or SAM) in an RT system may be between approximately 1-15 Å, and in some embodiments, between approximately 5-10 Å, and in some embodiments, between approximately about 8 and 10Å, including all values and subranges in between. For example, a calculated thickness of the triazole compound on an electrode/substrate in a RT system was found to be about 9.90 Å (using ChemDraw3D), and according to one experiment/example, was found to be about 8.41 Å, +/−0.24 Å (thickness determined using ellipsometry). Other characterization of the triazole reader&#39;s SAM can be found with references to  FIGS. 3-5 . 
         [0010]    In some embodiments, simulation of random thermal motion of analytes in tunnel gap is provided. In a strongly binding environment, analytes can experience higher fluctuations compared to an environment where analytes are weakly bonded. As a consequence of the exponential dependence of tunnel current with distance, weakly bonded analytes may give a wider range of turning current amplitude. 
         [0011]    In some embodiments, a universal reader molecule for functionalization onto an electrode/substrate of a recognition tunneling molecule identification system are provided, which may include a triazole compound. In some embodiments, the triazole compound includes TCA. 
         [0012]    In some embodiments, a compound of formula (I) is provided: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0013]    In some embodiments, a method of using a triazole compound (e.g., TCA) as a reader molecule in a recognition tunneling molecule identification system for identifying and/or sequencing one or more individual DNA bases and/or a DNA sequence is provided. 
         [0014]    In some embodiments, a method of using a triazole compound (e.g., TCA) as a reader molecule in a recognition tunneling molecule identification system for identifying and/or sequencing one or more individual sugars and/or a chain of sugars is provided. 
         [0015]    In some embodiments, a method of using a triazole compound (e.g., TCA) as a reader molecule in a recognition tunneling molecule identification system for identifying and/or sequencing one or more individual amino acids and/or protein/peptide is provided. 
         [0016]    In some embodiments, a method for preparing a triazole compound is provided which includes contacting benzyl mercaptan with 3-bromopropanenitrile in the presence of a base and a first solvent to obtain 3-(benzylthio)propanenitrile, contacting 3-(benzylthio)propanenitrile with hydrochloric gas in the presence of a second solvent to obtain benzyl 3-(benzylthio)propanimidothioate, contacting 3-(benzylthio)propanimidothioate with oxamic acid hydrazide in the presence of a third solvent to obtain 5-(2-(benzylthio)ethyl)-4H-1,2,4-triazole-3-carboxamide, and contacting 5-(2-(benzylthio)ethyl)-4H-1,2,4-triazole-3-carboxamide with sodium metal in liquid ammonia to obtain 5-(2-mercaptoethyl)-4H-1,2,4-triazole-3-carboxamide. 
         [0017]    In some embodiments:
       the first solvent may be dimethylformamide;   the base may be sodium hydride;   the second solvent may be diethyl ether; and/or   the third solvent may be pyridine.       
 
         [0022]    In some embodiments a method for preparing 3-(benzylthio)propanenitrile is provided which includes contacting benzyl mercaptan with 3-bromopropanenitrile in the presence of a base. In some embodiments, the solvent is dimethylformamide. In some embodiments, the base is sodium hydride. 
         [0023]    In some embodiments a method of preparing benzyl 3-(benzylthio)propanimidothioate is provided which includes contacting 3-(benzylthio)propanenitrile with hydrochloric gas in the presence of a solvent. In certain embodiments of this method the solvent is diethyl ether. In some embodiments a method of preparing 5-(2-(benzylthio)ethyl)-4H-1,2,4-triazole-3-carboxamide is provided which may comprise contacting 3-(benzylthio)propanimidothioate with oxamic acid hydrazide in the presence of a solvent. In certain embodiments of this method the solvent is pyridine. 
         [0024]    In some embodiments a method of preparing 5-(2-mercaptoethyl)-4H-1,2,4-triazole-3-carboxamide is provided which includes contacting 5-(2-(benzylthio)ethyl)-4H-1,2,4-triazole-3-carboxamide with sodium metal in ammonia. 
         [0025]    In some embodiments, a recognition tunneling system is provided which includes at least a pair of electrodes, where at least one of the electrodes includes a one or more triazole-based molecules functionalized thereto. In some embodiments, the triazole molecule includes TCA. 
         [0026]    Such system embodiments may be used to identify and/or sequence: one or more individual DNA bases and/or DNA sequences, one or more individual carbohydrates and/or chains of sugars, and/or one or more individual amino acids and/or proteins/peptides. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0027]      FIG. 1  is an illustration of a triazole based reading molecule 5-(2-mercaptoethyl)-4H 1,2,4-triazole-3-carboxamide (TCA), according to some embodiments of the disclosure. 
           [0028]      FIG. 2  is an illustration of three tautomers of the triazole-carboxamide reader, according to some embodiments of the present disclosure. 
           [0029]      FIG. 3  is an illustration of a table showing an analysis of atomic ratio using X-ray photoelectron spectroscopy (XPS) of a SAM of TCA on a palladium substrate. 
           [0030]      FIG. 4  is a graph of Fourier Transform Infrared Spectroscopy (FTIR) data of a SAM of TCA on a palladium substrate and TCA powder. 
           [0031]      FIG. 5  is an illustration of measured contact angles of a SAM of TCA on a palladium substrate and a bared palladium substrate. 
           [0032]      FIGS. 6-7  are charts illustrating the percent accuracy between DNA nucleotides (using support vector machine (SVM) analysis) vs. parameters for analytes for triazole ( FIG. 6 ) and imidazole ( FIG. 7 ). 
           [0033]      FIG. 8A  is a diagram of a Recognition Tunneling measurement setup according to some embodiments of the disclosure 
           [0034]      FIGS. 8B-8C  illustrate the simulated result of random thermal fluctuations of analytes in strongly ( FIG. 8B ) and weakly ( FIG. 8C ) binding in a tunnel gap. 1    
           [0035]      FIG. 9  is a graph showing example tunneling spectra (i.e., current spikes) obtained from a control experiment of an RT system using TCA as a molecular reader in 1 mM PB buffer 
           [0036]      FIGS. 10A and 10B  are a series of graphs is a graph of example tunneling spectra of a DNA nucleoside monophosphate dGMP obtained from an RT system using TCA as a molecular reader: 10A shows data obtained from 0-60 sec., and  10 B shows data from 19.6 to 20.6 sec. 
           [0037]      FIGS. 11A and 11B  are a series of graphs of example tunneling spectra of a DNA nucleoside monophosphate dTMP obtained from an RT system using TCA as a molecular reader:  11 A shows data obtained from 0-60 sec., and  11 B shows data from 26.4 to 27.4 sec. 
           [0038]      FIGS. 12A and 12B  are a series of graphs of example tunneling spectra of a DNA nucleoside monophosphate dAMP obtained from an RT system using TCA as a molecular reader:  12 A shows data obtained from 0-60 sec., and  12 B shows data from 11.3 to 12.3 sec. 
           [0039]      FIGS. 13A and 13B  are a series of graphs of example tunneling spectra of a DNA nucleoside monophosphate dCMP obtained from an RT system using TCA as a molecular reader:  13 A shows data obtained from 0-60 sec., and  13 B shows data from 14.3 to 15.3 sec. 
           [0040]      FIGS. 14A and 14B  are a series of graphs of example tunneling spectra of a DNA nucleoside monophosphate 5-methyl-dCMP obtained from an RT system using TCA as a molecular reader:  14 A shows data obtained from 0-60 sec., and  14 B shows data from 33.18 to 34.18 sec. 
           [0041]      FIGS. 15A and 15B  are a series of graphs of example tunneling spectra of a sugar molecule galactose obtained from an RT system using TCA as a molecular reader:  15 A shows data obtained from 0-60 sec., and  15 B shows data from 45.64 to 47.99 sec. 
           [0042]      FIG. 16  is an illustration of a reaction scheme, according to some embodiments, for the synthesis/preparation of TCA. 
       
    
    
     DETAILED DESCRIPTION 
       [0043]    Some embodiments include triazole-containing reading molecules which improve the ability for reading DNA bases over, for example, imidazole-based reading molecules in an RT apparatus. Such improvements can be attributed to, for example, a lesser number of parameters to reach a level of (for example) 95.5% accuracy to distinguish different nucleoside monophosphates. This can be seen, for example, with reference to  FIGS. 6-7 , charts which illustrate the percent accuracy between DNA nucleosides (using support vector machine (SVM) analysis) vs. parameters for analytes for both TCA ( FIG. 6 ) and ICA ( FIG. 7 ). As one of ordinary skill in the art will appreciate, the triazole reader molecules provide similar accuracy with a lesser number of parameters. 
         [0044]    The triazole-based reading molecules may also interact with carbohydratees (e.g. galactose), as evident with their interaction with DNA bases, as well as with amino acids. 
         [0045]      FIG. 8A  illustrates a substrate and tunneling measurement (STM) instrument used for the recognition tunneling measurements, which includes of a sample stage/plate with a teflon liquid cell, scanner and microscope head. Prior to an STM measurement, the teflon cell was cleaned with freshly prepared piranha solution (a 3:1 mixture of sulfuric acid and hydrogen peroxide), followed by sonication in nanopure water three (3) times and in ethanol three (3) times, then blow-dry with nitrogen. The functionalized palladium tip and palladium substrate were rinsed thoroughly with ethanol, and blow-dried with nitrogen. A new tip and substrate set was used for each experiment for different DNA monophosphates. The same procedure was followed for immobilization of triazole reader as was done for imidazole reader immobilization. 
         [0046]    Initially, for an STM measurement, the tip was inserted into the scanner and connected to the microscope head. The voltage was adjusted to zero by using the adjustment screw on the top of the scanner while checking the oscilloscope, to measure the variation voltage or other electrical signal as a function of time. Voltage was also adjusted to zero (e.g., via picoview software). Labview software was used for tracking, recording and further analysis of current signal as a function of time. Before starting the measurement, the oscilloscope, the picoview and the Labview software should all be reading 0 V applied bias (voltage) and 0 pA current. A sodium phosphate buffer (10 mM, pH 7.0, 120 uL) was added to the teflon cell. The value of leakage current with an applied bias of (−0.5 V) was checked, keeping the tip far enough from the surface of the palladium substrate (i.e., not in tunneling regime). The tip was discarded if the value was more than 1 pA. All measurements were done with tips having a leakage current below 1 pA. After the leakage check, the tip was made to approach the bottom electrode until the tunneling current reached 2 pA (also termed as “set point”) under an applied bias of (−0.5V) and definite set of values for I gain and P gain (1=1.5, P=1.5) in picoview. 
         [0047]    After the tip engaged, a few STM images were taken to verify that the tip was not over-coated. After a well defined image of the palladium substrate was obtained, the tip was withdrawn for 20 microns and the system was stabilized for 2 hours. Then the tip was arranged adjacent to the surface (2 pA set point, −0.5 V bias, 1=1.5, P=1.5). Both I and P values were changed to 0.1, and then control data was recorded at two different values of set point (2 pA &amp; 4 pA). Thereafter, the tip was withdrawn and the phosphate buffer was discarded carefully, followed by the addition of a DNA monophosphate solution (100 μM) to the liquid cell. The tip was then arranged in the same manner and withdrawn for 20 microns prior to system stabilization of 2 hours. After 2 hours, the tip was arranged again and the tunneling data of the DNA monophosphates was recorded at two different values of set point (2 pA &amp; 4 pA). The data were then analyzed using support vector machine (SVM) analysis. 
         [0048]      FIGS. 10A-14B  are graphs illustrating example tunneling current spikes for different DNA nucleotides.  FIGS. 15A-B  are graphs illustrating example tunneling current spikes of a sugar molecule (galactose). 
         [0049]    Synthesis of a Triazole-Based Universal Reading Molecule 
         [0050]    In some embodiments, methods for synthesizing triazole-based compounds are provided. The synthesized triazole-based compounds may be used as a universal reader molecule in a recognition tunneling molecule identification system (see above). For example, in some embodiments, such a method for synthesizing the triazole compound 5-(2-mercaptoethyl)-4H-1,2,4-triazole-3-carboxamide (TCA) is provided. Such an example method is outlined in  FIG. 16 , with reference to the following compounds:
       (1) 3-(benzylthio)propanenitrile   (2) 3-(benzylthio)propanimidothioate (Lunt, E.; Newton, C., G.; Smith, C.; Stevens, G., P.; Stevens, M., F., G.; Straw, C., G.; Walsh, R., J., A.; Warren, P., J.; Fizames, C.; Lavelle, F.; Langdon, S., P.; Vickerss, L., M.  J. Med. Chem.  1987, 30, 357-366)   (3) 5-(2-(benzylthio)ethyl)-4H-1,2,4-triazole-3-carboxamide (Chudinov, M., V.; Konstantinova, I., I).; Ryzhova, O., I.; Esipov, R., S.; Yurkevich, A., M.; Shvets, V., I.; A., I.  Pharmaceutical Chemistry Journal,  2005, 39, 212-215)   (4) 5-(2-mercaptoethyl)-4H-1,2,4-triazole-3-carboxamide (Liang, F.; Li, S.; Lindsay, S.; Zhang, P.  Chem. Eur. J.  2012, 18, 5998-6007)       
 
         [0055]    In some embodiments, (1) is synthesized and then used to synthesize (2), which is then in turn synthesized to synthesize (3), which is then used to synthesize (4). 
       EXAMPLE 
     Synthesis of 3-(benzylthio)propanenitrile (Product 1) 
       [0056]    Benzyl mercaptan A (1.05 g, 19.0 mmol) was added into a stirred solution of sodium hydride (60% in mineral oil, 1.16 g, 24.0 mmol) in anhydrous DMF (50 mL) at 0° C. under nitrogen. After the addition was complete, the reaction mixture was stirred for another 30 min, followed by the slow addition of 3-bromopropanenitrile B (2.68 g, 20.0 mmol). The resulting mixture was allowed to warm at room temperature, stirred for 12 hours until benzyl mercaptan was consumed. The solvent was removed by rotary evaporation, followed by addition of a saturated NH 4 Cl aqueous solution (20 mL), and extracted with chloroform (3×20 mL). The combined organic extracts were washed with brine (30 mL), and dried over magnesium sulfate. The solution was then filtered and concentrated by rotary evaporator. The crude product was purified by silica gel flash column chromatography. 3-(Benzylthio)propanenitrile (Product 1) was obtained as pale yellow liquid (2.25 g, 65%).  1 H NMR (400 MHz, CDCl 3 ): δ=7.24-7.33 (5H, m, Ar H ), 3.78 (2H, s, PhC H   2 ), 2.64 (2H, t, J=8.0 Hz, C H   2 ), 2.47 ppm (2H, t, J=8.0 Hz, C H   2 );  13 C NMR (100 MHz, CDCl 3 ): δ=137.2, 128.9, 127.2, 118.3, 36.0, 26.2, 18.3 ppm; HRMS (APCI+): m/z calculated for C 10 H 11 NS+H: 178.0690; found: 178.0688. 
       EXAMPLE 
     Synthesis of benzyl 3-(benzylthio)propanimidothioate from 3-(benzylthio)propanenitrile (Product 2) 
       [0057]    Product 1 (2.0 g, 11.3 mmol) and benzyl mercaptan (2.0 mL, 16.93 mmol) were added subsequently into anhydrous ethyl ether (120 mL) under nitrogen. The resulting solution was cooled in an ice bath, and HCl (gas, anhydrous) was bubbled into it for 2 h. It was stirred for 24 h at room temperature. Then it was left unstirred for another 2 h. The product was crystallized in the reaction mixture, and filtered through a Buchner funnel. The crystals were washed with three portions (each 20 mL) of cold ethyl ether and dried in vacuum. Product 2 was obtained as white crystals (3.7 g, 97%).  1 H NMR (400 MHz, CDCl 3 ): δ=7.21-7.38 (10H, m, Ar H ), 4.78 (2H, s, CSC H   2 Ph), 3.86 (2H, s, PhC H   2 S-CH 2 ), 3.20 (2H, t, J=7.2 Hz, SC H   2 -), 2.88 (2H, t, J=7.2 Hz, —C H   2 -), 1.61 ppm (1H, s, broad, NH);  13 C NMR (100 MHz, CDCl 3 ): δ=193.3, 137.8, 131.3, 129.8, 129.6, 129.4, 129.3, 129.0, 127.6, 39.3, 37.5, 36.3, 29.7 ppm; HRMS (APCI+): m/z calculated for C 17 H 19 NS 2 +H: 302.1037; found: 302.1036. 
       EXAMPLE 
     Synthesis of 5-(2-(benzylthio)ethyl)-4H-1,2,4-triazole-3-carboxamide from 3-(benzylthio)propanimidothioate (Product 3) 
       [0058]    Oxamic acid hydrazide C (0.34 g, 3.32 mmol) was added into a solution of product 2 (1.0 g, 3.32 mmol) in anhydrous pyridine (10 mL) at room temperature. The resulting solution was refluxed at 110° C. for 3 h. Pyridine was removed by co-evaporating with toluene (5 mL×2) using a rotary evaporator to obtain yellow gummy liquid. DMSO (15 mL) was added to just dissolve the crude and sufficient water (50 mL) was added to get white precipitate, which was filtered through a Buchner funnel and washed thoroughly with cold water (40 mL), followed by cold ethyl ether (40 mL). The solid was dried in vacuum to obtain 0.53 g of a crude product, which was then recrystallized from boiling ethanol (25 mL), filtered, and dried in vacuum at 40° C. to furnish product 3 as white crystals (0.31 g, 40%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=7.81 (1H, s, broad, —N H   2 ), 7.58 (1H, s, broad, —N H   2 ), 7.18-7.28 (5H, m, Ar H ), 3.70 (2H, s, PhC H   2 -), 2.93 (2H, t, J=7.2 Hz, —C H   2 ), 2.73 ppm (2H, t, J=7.2 Hz, —CH 2 );  13 C NMR (100 MHz, DMSO-d 6 ): δ=160.4, 138.8, 129.2, 128.8, 127.3, 35.3, 29.1, 27.2 ppm; HRMS (APCI+): m/z calculated for C 12 H 14 N 4 OS+H: 263.0967; found: 263.0972. 
       EXAMPLE 
     Synthesis of 5-(2-mercaptoethyl)-4H-1,2,4-triazole-3-carboxamide from 5-(2-(benzylthio)ethyl)-4H-1,2,4-triazole-3-carboxamide (Product 4) 
       [0059]    Product 3 (150 mg, 0.572 mmol) was added into liquid ammonia (2 mL) at −78° C. and stirred for 15 min. Small pieces of freshly cut sodium were added into the solution until a blue color remained unchanged for about 3 min. Then NH 4 Cl was added to quench the reaction until the blue color disappeared. Ammonia was allowed to evaporate under nitrogen flow at room temperature. For separation, the residue was dissolved in methanol, followed by the addition of silica gel. The solvent was removed by rotary evaporation. The crude product was purified by silica gel flash column chromatography while the pure product was eluted out with a gradient of methanol (0 to 10% in 2 h) in dichloromethane. Product 4 was obtained as white solid (98 mg, 31%).  1 H NMR (400 MHz, DMSO-d 6 ): δ=6.79 (1H, s, broad, —N H   2 ), 6.71 (1H, s, broad, —N H   2 ), 2.57 (2H, t, J=6.8 Hz, —C H   2 ); 2.43 (2H, t, J=6.8 Hz, —C H   2 ), 2.08 (1H, t, J=2.0 Hz, —S H ); HRMS (APCI+): m/z calculated for C 5 H 8 N 4 OS+H: 173.0497; found: 173.0493. 
         [0060]    Any and all references to publications or other documents, including but not limited to, patents, patent applications, articles, webpages, books, etc., presented in the present application, are herein incorporated by reference in their entirety. 
         [0061]    Although a few variations have been described in detail above, other modifications are possible. For example, any logic flow depicted in any figure and/or described herein does not require the particular order shown, or sequential order, to achieve desirable results. Other implementations may be within the scope of at least some of the following exemplary claims. 
         [0062]    Example embodiments of formulations and methods have been described herein. As noted elsewhere, these embodiments have been described for illustrative purposes only and are not limiting. Other embodiments are possible and are covered by the disclosure, which will be apparent from the teachings contained herein. Thus, the breadth and scope of the disclosure should not be limited by any of the above-described embodiments but should be defined only in accordance with claims supported by the present disclosure and their equivalents. Moreover, embodiments of the subject disclosure may include formulations, methods, systems and devices which may further include any and all elements from any other disclosed methods, systems, and devices, including any and all elements corresponding to triazole reader molecules and RT systems incorporating such reader molecules. In other words, elements from one or another disclosed embodiments may be interchangeable with elements from other disclosed embodiments. In addition, one or more features/elements of disclosed embodiments may be removed and still result in patentable subject matter (and thus, resulting in yet more embodiments of the subject disclosure).