MX2007005099A - Nucleic acid enzyme light-up sensor utilizing invasive dna - Google Patents

Abstract

The present invention provides a colorimetric light-up sensor for determining the presence and optionally the concentration of an analyte in a sample. Methods of utilizing the sensor and kits that include the sensor also are provided. The sensor utilizes invasive DNA to assist the analyte dependent disaggregation of an aggregate that includes nucleic acid enzymes, substrates, and particles.

Description

ENZYME LIGHTING SENSOR OF NUCLEIC ACIDS USING INVASIVE DNA RESEARCH OR FEDERALLY SPONSORED DEVELOPMENT The content of this application has been based in part on the following concessions and research contracts: DOE Concession No. DEFG02-01-ER63179, NSF CTS-0120978, and NSF DMR-0117792. The government of E.U.A. You can have rights in this invention.

BACKGROUND The ability to determine the presence of an analyte in a sample has an important benefit. For example, many metals and metal ions, such as lead, mercury, cadmium, chromium, and arsenic, are important health risks when present in drinking water supplies. To avoid contamination of drinking water and other water supplies, it is common to test industrial waste streams prior to release to the water treatment plant. Biological fluids, such as blood and those that originate from body tissues, can also be tested for a variety of analytes to determine if the body has been exposed to harmful agents or if there is a disease state. For example, there has recently been a need to detect trace amounts of anthrax and other biologically damaging agents in a variety of samples. Colorimetric methods are commonly used for the detection of metals and ions in soil, water, waste streams, biological samples, body fluids and the like. In relation to mass methods in analytical instruments, such as atomic absorption spectroscopy, colorimetric methods tend to be fast and require little in terms of equipment or user sophistication. For example, colorimetric tests are available for aquariums that render shades of pink dark when they were added to aqueous samples containing increasing concentrations of nitrate ion (NO3). In this way, colorimetric tests show that the analyte of interest, such as nitrate, is present in the sample and can also provide an indicator of the amount of analyte in the sample through a specific dye of generated color. While colorimetric tests are extremely useful, they only exist for a limited group of analytes, often very small quantities or traces of the analyte can not be detected, and depending on the nature of the sample, can generate unacceptable levels of false positive or negative results . False positives occur when the colorimetric reagents produce the color associated with the presence of an analyte when the analyte is not present, while false negatives occur when the analyte of interest is present in the sample, but the expected color is not produced. False positives are often the result of constituents in the sample that can not distinguish the colorimetric test from the analyte of interest. False negatives often result from the constituents of the samples that interfere with the chemical reaction that provides color associated with the analyte. As can be seen from the above description, there is a present need for colorimetric tests that can identify trace amounts of a broader range of analytes. In addition, colorimetric tests that have a lower incidence of false positive and / or negative results could also provide important benefit.

SUMMARY In one aspect of the invention, a sensor system is described that includes a nucleic acid enzyme, a substrate for the nucleic acid enzyme, first particles and invasive DNA. The substrate can include first polynucleotides and the first particles can include second polynucleotides that are coupled to the first particles. The invasive DNA may include four polynucleotides. The first polynucleotides may be at least partially complementary to the second and fourth polynucleotides. The sensor system may also include second particles including third polynucleotides that are at least partially complementary to the first polynucleotides. In another aspect of the invention, describes a method for detecting an analyte that includes combining an aggregate, a sample, and invasive DNA to detect a color change that responds to the analysis. The aggregate may include a substrate and first particles. The aggregate may also include second particles and an endonuclease. In another aspect of the invention, an apparatus for detecting an analyte including a first container containing a system for forming aggregates including first polynucleotides and first particles and a second container containing invasive DNA is disclosed. In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided. The term "sample" or "test sample" is defined as a composition that will be subjected to analysis that is suspected to contain the analyte of interest. Normally, a sample for analysis is in liquid form, and preferably the sample is an aqueous mixture. A sample can be from any source, such as an industrial sample from a waste stream or a biological sample, such as blood, urine or saliva. A sample can be a derivative of an industrial or biological sample, such as an extract, a dilution, a filtrate or a reconstituted precipitate. The term "analyte" is defined as one or more substances potentially present in the sample. The analysis process determines the presence, quantity or concentration of the analyte present in the sample. The term "colorimetric" is defined as an analysis process in which the reagent or reagents that make up the sensor system produce a color change in the presence or absence of an analyte. The term "sensitivity" refers to the lower concentration limit at which a sensor system can detect an analyte. Therefore, the more sensitive a sensor system is for an analyte, the better is the system for detecting lower concentrations of the analyte. The term "selectivity" refers to the ability of the sensor system to detect the desired analyte in the presence of other species.

The term "hybridization" refers to the ability of a first polynucleotide to form at least one hydrogen bond with at least one second nucleotide under mild conditions.

BRIEF DESCRIPTION OF THE DRAWINGS The invention can be better understood with reference to the following drawings and description. The components in the figures are not necessarily to scale and are not intended to represent molecules or their interactions with precision, it is emphasized instead of illustrating the principles of the invention. Fig. 1 depicts a colorimetric analytical method for determining the presence and optionally the concentration of an analyte in a sample. Fig. 2A depicts DNA enzyme that depends on Pb (II) as a cofactor to exhibit catalytic activity. Fig. 2B depicts the separation of a DNA-based substrate by a DNA enzyme. Fig. 3A represents the disintegration of an aggregate in the presence of a Pb (II) analyte and invasive DNA. Fig. 3B depicts the tail-to-tail hybridization of a DNA-based substrate with functionalized oligonucleotide particles.

Fig. 3C depicts the hybridization of a DNA-based substrate with functionalized oligonucleotide particles. Fig. 4 is a graph that refers to extinction ratios of the wavelengths of light emitted from a sample by gold nanoparticles added (solid line) or disaggregated (dotted line). Fig. 5A is a graph showing the change in extinction ratios over time for samples containing invasive DNA (Inva) and Pb (II) (o), invasive DNA (Inva) without Pb (II) (A), and a control sample containing Pb (II) without invasive DNA (|). Figure 5B is a graph showing the change in extinction ratios over time for samples containing invasive DNA (Inva-A) and Pb (II) (o), invasive DNA (Inva-A) without Pb (II) ( A), and a control sample containing Pb (II) without invasive DNA (|). Fig. 6A is a graph plotting the change in extinction ratios as a function of time for each of the preferable, shortened invasive DNA strands (relative to the original Inva strand) with and without the Pb analyte (II ). Fig. 6B is a graph plotting the change in extinction ratios as a function of time for each of the shortened alternating invasive DNA strands (relative to the original Inva strand) with and without the Pb (II) analyte . Fig. 7A is a graph describing the extinction ratios at 522 700 nm plotted as a time function for multiple metal caps. Fig. 7B is a graph describing the correlation between the observed extinction ratios for the color change of the sensor system and the concentration of the Pb (II) analyte after five minutes. Fig. 7C is a graph describing the extinction ratios for concentrations of multiple Pb (II) analytes over a 10 minute period with Inva-6. Fig. 8 is a graph describing the NaCl-dependent stability of aggregates of gold nanoparticles. Fig. 9 is a TEM image photograph of aggregates of gold nanoparticles of 13 mm assembled with DNA enzyme. Figs. 10A-10S describes nucleic acid enzymes that utilize specific analytes as co-factors for catalytic separation reactions.

DETAILED DESCRIPTION In a related application, E.U.A. Not to be. / 144,679, filed on May 10, 2002, entitled "Simple catalytic DNA biosensors for ions based on color changes", describes a colorimetric sensor that in one aspect uses heat to accelerate the disintegration catalyzed by analytes of an aggregate. In this prior sensor system, a sample was added to a DNA / substrate / particle aggregate enzyme. The mixture was then heated to cause disintegration of the aggregate in the sample including the selected analyte. The present invention uses the discovery that the addition of invasive DNA to DNA-RNA enzyme / substrate / particle aggregate can accelerate aggregate disintegration without heating. In this way, a colorimetric illumination sensor is provided that undergoes the desired color change in response to a selected analyte at room temperature, thus overcoming a disadvantage of the sensor system described in the E.U.A. No. 10 / 144,679. Figure 1 depicts a colorimetric analytical method 100 for determining the presence and optionally the concentration of an analyte 105 in a sample 102 (not shown). At 110, the analyte 105 for which the method 100 will determine whether presence / concentration is selected.

In one aspect, the analyte 105 can be any ion that can serve as a cofactor for a separation reaction, as discussed below. Preferred monovalent metal ions having a formal oxidation state +1 (I) include Li (I), TI (I), and Ag (I). Preferred divalent metal ions having a formal oxidation state +2 (II) include Mg (II), Ca (II), Mn (II), Co (II), Ni (II), Zn (II), Cd ( II), Cu (II), Pb (II), Hg (II), Pt (II), Ra (II), Sr (II), Ni (II), and Ba (II). Preferred trivalent and higher metal ions having formal oxidation states +3 (III), +4 (IV), +5 (V), or +6 (VI) include Co (III), Cr (III), Ce ( IV), As (V), Ü (VI), Cr (VI), and lanthanide ions. The most preferred analyte ions include Ag (I), Pb (II), Hg (II), Ü (VI), and Cr (VI) due to the toxicity of these ions to living organisms. At present, and the ion of especially preferred analytes is Pb (II). Once the analyte 105 is selected at 110, at 120, direct evolution 122 can be carried out to isolate nucleic acid enzymes, such as DNA 124 enzyme or RNA 126 enzyme, which will catalyze the separation of substrates in the presence of the analyte. . Direct evolution 122 preferably is a type of in vitro selection method that selects molecules on the basis of their ability to interact with another constituent. Therefore, the procedure of direct evolution 122 can be selected to provide the. DNA-RNA enzyme that demonstrates improved substrate separation in the presence of the selected analyte 105 (thus providing sensor sensitivity). The method can also be selected to exclude DNA-RNA enzymes that demonstrate separation in the presence of selected analytes, but additionally demonstrate separation in the presence of unselected analytes and / or other species present in sample 102 (thus providing sensor selectivity). The direct evolution 122 can be any routine selection that provides nucleic acid enzymes that will catalyze the separation of a substrate in the presence of the desired analyte with the desired sensitivity and selectivity In one aspect, the direct evolution 122 can be initiated with a bank of DNA that includes a large collection of strands (eg, 1016 sequence variants), each having a different base variation.The phosphoramidite chemistry can be used to generate the strands.The DNA bank is then sieved for strands These strands are isolated and amplified as by CPR, and the amplified strands can then be be used to reintroduce variation. These strands are then sieved for strands that bind more effectively to the analyte. By repeating the sequence of selection, amplification and mutation while increasing the amount of binding efficiency required for selection, the strands that bind more effectively to the analyte can be generated, thus providing greater sensitivity. In one aspect, a technique called in vitro selection and evolution can be used to perform direct evolution 122. Details regarding this technique can be found in Breaker, RR, Joyce, GF, "A DNA enzyme with Mg2 + dependent RNA phosphoesterase activity ", Chem. Biol. 1995, 2: 655-660; and in Jing Li, et al., "In Vitro Selection and Characterization of a Highly Efficient Zn (II) -dependent RNA-Cleaving Deoxyribozyme", Nucleic Acids Res. 28, 481-488 (2000). In another aspect, nucleic acid enzymes that have higher selectivity to a specific analyte can be obtained by introducing a negative selection process in the direct evolution 122. After selecting, the strands that have high sensitivity to the analyte, a selection, can be apply a sequence of selection, amplification and mutation, but to be selected, the strand should not be tightly bound to the related analytes. For example, a DNA enzyme that specifically binds to Pb (II) can be selected, while not significantly binding to Mg (II), Ca (II), Co (II), or other competing metal ions. In one aspect, this can be accomplished by isolating DNA enzymes that bind to Pb (II), then removing any DNA enzymes that bind to Mg (II), Ca (II), or Co (II). In another aspect, DNA enzymes that bind to Mg (Il), Ca (II), or Co (II) are first discarded and then those that bind to Pb (II) are isolated. In this way, the selectivity of the DNA enzyme can be increased. Details regarding a method to increase the selectivity of the DNA enzyme can be found in Bruesehoff, PJ, et al., "Imporoving Metal Ion Specificity During Vitro Selection of Catalytic DNA", Combinatorial Chemistry and High Throughput Screening, 5, 327 -355 (2002). The DNA-RNA enzymes 124, 126 are nucleic acid enzymes that have the ability to catalyze chemical reactions, such as hydrolytic separation, in the presence of a co-factor. DNA enzyme 124 includes deoxyribonucleotides, while enzyme RNA 126 includes ribonucleotides. The nucleotides of which the DNA-RNA enzyme 124, 126 is formed can be natural, unnatural, or modified nucleic acids. Peptide nucleic acids (ANP), which includes a base structure of polyamide and nucleoside bases (available from Biosearch, Inc., Bedford, MA, for example), may also be useful. The following table lists specific analytes, in the Figure, in which the sequence of corresponding nucleic acid enzymes can be found uses the analyte as a separation cofactor, and the reference or references where each sequence of nucleic acid enzymes is described . Figs. 10A-10D describes trans-acting nucleic acid enzymes that are specific for metal ions that have formal 2 * oxidation states. Figures 10K-10L describe trans-acting nucleic acid enzymes that can also serve as suitable nucleic acid enzymes. Figs. 10E-10F and 10H-10J describe cis-acting nucleic acid enzymes that are specific for metal ions having formal +2 oxidation states. Figs. 10M-10S describe cis-acting nucleic acid enzymes that also serve as suitable nucleic acid enzymes. Preferably, the cis-acting nucleic acid enzymes can be cut into two strands (truncated), such as by separation of the GAAA loop presented on the right side of the enzymes 10M to 10Q, to provide a catalytic system. Any of these, and other nucleic acid sequences can be adapted for use as the DNA-RNA enzymes 124, 126. The trans-and cis-acting enzymes are further treated with respect to Fig. 2A.

While both DNA enzymes and RNA enzymes can double with a DNA-based substrate, such as substrate 134 described below, the RNA / substrate enzyme duplex can be less stable than the DNA / substrate enzyme duplex. Additionally, DNA enzymes are easier to synthesize and more robust than their RNA enzyme counterparts. The deoxyribonucleotides of the DNA enzyme 124 and the strand of the complementary substrate 134 can be replaced with their corresponding ribonucleotides, thus providing the RNA 126 enzyme. For example, one or more ribocitocins can be substituted for the cytosines, one or more can be substituted ribo-guanines by guanines, one or more ribo-adenosines can be substituted by adenosines and one or more uracils can be replaced by thymines. In this manner, nucleic acid enzymes including DNA bases, RNA bases, or both, can be independently hybridized with strands of complementary substrates including DNA bases, RNA bases, or both. After selecting an enzyme of appropriate nucleic acids or enzymes at 120, an aggregate 132 can be formed at 130. Aggregate 132 includes nucleic acid enzymes; the substrate 134; and particles functionalized with oligonucleotides 136. Considering the physical size of their components, aggregate 132 may be very large. In fact, studies of transmission electron microscopy (TEM) suggest that individual aggregates can vary from 100 nm to 1 miera, and can agglomerate to form larger structures. The substrate 134 can be any oligonucleotide that can be hybridized with, and separated by, the enzyme from nucleic acids in the presence of analyte 105. The oligonucleotide can be modified with a sort of separation, which allows the separation of the substrate into two fragments by the enzyme of nucleic acids. In one aspect, the substrate 134 is a strand complementary to the nucleic acid enzyme and can be extended to form a 12 mer pendant on each end to hybridize with the functionalized particles of oligonucleotides 136. For example, if a functionalized oligonucleotide particle a sequence of bases of 5 '-CACGAGTTGACA, a hanging sequence appropriate for the substrate could be of 3'-GTGCTCAACTG. Because the particles 136 demonstrate optical properties that depend on distance, the particles have a color when they are held closely in the aggregate 132 and undergoes a color change as the distance between the particles increases. For example, when the particles 136 are gold nanoparticles, the aggregate 132 shows a blue color in aqueous solution that changes to red as the disintegration proceeds. Disintegration occurs when the substrate 134 which holds the functionalized particles 136 together is removed, thereby allowing the aggregate particles 132 to separate. Therefore, as they extend away from the aggregate 132, the solution changes from blue to red. The particles 136 can be any species that demonstrates optical properties that are distance dependent and compatible with the operation of the sensor system. Suitable particles can include metals, such as gold, silver, copper and platinum; semiconductors such as CdSe, CdS, and CdS or CdSe coated with ZnS; and magnetic colloidal materials, such as those described in Josephson, Lee, et al., Ange andte Chemie, International Edition (2001), 40 (17), 3204-3206. Useful specific particles can include ZnS, ZnO, TiO2, Agi, AgBr, Hgl2, PbS, PbSe, ZnTe, CdTe, In2S3, Cd3P2, Cd3As2, InAs, and GaAs. In a preferred aspect, the particles are gold nanoparticles (Au) and have an average diameter of 5 to 70 nanometers (nm) or 10 to 50 nm. In a particularly preferred aspect at present, gold nanoparticles having an average diameter of 10 to 15 nm are functionalized with the oligonucleotides. For a more detailed treatment of how to prepare gold functionalized oligonucleotides, see Patent of E.ü.A. No. 6,361,944; Mirkin, et al., Nature (London) 1996, 382, 607-609; Storhoff, et al., J. Am. Chem. Soc. 1998, 20, 1959-1064; and Storhoff, et al., Chem. Rev. (Washington, D.C.) 1999, 99, 1849-1862. While gold particles are currently preferred, other fluorophores, such as dyes, inorganic crystals, quantum dots, and the like which undergo a color change depending on the distance, can also be linked to oligonucleotides and used. In 140, aggregate 132 of 130 can be combined with sample 102 and invasive DNA 144. In 150 sample 102 is monitored for a color change. If there is no color change, then the analyte 105 is monitored for a color change. If a color change occurs at 160, the analyte 105 is present in the sample 102. Therefore, the analytical method 100 provides an "illumination" sensor system because a color change occurs in the presence of the analyte 105 The color change means that analyte 105 is an appropriate co-factor to catalyze the separation of substrate 134, which hybridizes with particles functionalized with oligonucleotide 136. This separation is thought to cause substrate 134 to be divided into two fragments , thus allowing the particles 136 to expand away from the aggregate 132 and into the solution of the sample 102. While it is thought that this separation of the substrate 134 proceeds at room temperature, it is thought that a significant portion of the 9 base pairs forming each separate portion of the substrate remains hybridized with the nucleic acid enzyme. Therefore, for disintegration to occur, it is preferred that it interrupts this hybridization. It is thought that the invasive DNA 144"invades" the aggregate 132 and helps to release the separated substrate fragments. While not wishing to be bound by any particular theory, it is thought that equilibrium forces cause competition for the sites in the separate portions of the substrate 134 between the nucleic acid enzyme and the invasive DNA 144. Because this equilibrium favors the hybridization of the substrate 134 with the invasive DNA 144, the bound particles 136 expand away from the aggregate 132 and provide the desired color change. Although the term "invasive DNA" is used throughout this specification and appended claims for consistency, if the substrate 134 includes ribonucleotides, the invasive DNA 144 may also include ribonucleotides. While the invasive DNA 144 can be any oligonucleotide that is at least partially complementary to the separated fragments of the substrate 134, preferably, the invasive DNA 144 includes relatively small pieces of DNA. In one aspect, the invasive DNA 144 includes at least two types of DNA strands, each being partially complementary to one of the two separate substrate fragments. In another aspect, at least one base terminates from each of the separated substrate fragments is complementary to at least one base terminates from each of the strands of invasive DNA. In yet another aspect, the invasive DNA 144 includes at least two types of DNA strands, each being completely complementary to one of the two separate substrate fragments. In yet another aspect, the invasive DNA 144 has from 2 to 10 or from 4 to 8, which includes 2, 4, 6, or 8 bases less capable of hybridizing with the corresponding separate substrate fragment. At present, especially preferred invasive DNA strands have 6 fewer complementary bases than the fragment of the separated substrate.

The degree of color changes in response to analyte 105 can be quantified by colorimetric quantization methods known to those of ordinary skill in the art at 170. Various color comparison wheels, such as those available from Hach Co. , Loveland, CO or LaMotte Co., Chestertown, MD may be adapted for use with the present invention. Normal samples containing known quantities of the selected analyte can be further analyzed in the test sample to increase the accuracy of the comparison. If superior precision is desired, different types of spectrophotometers can be used to plot a Beer curve on the desired concentration scale. The color of the test sample can then be compared with the curve and the concentration of the analyte present in the determined test sample. Suitable spectrophotometers include Hewlett-Packard 8453 and Baushch & Lomg Spec-20. In yet another aspect, method 100 can be modified to determine the sensitivity and selectivity of an endonuclease, such as a nucleic acid enzyme, to detect analyte 105. In this aspect, an aggregate of substrate 134 and particles 136 are formed, but without DNA-RNA enzymes 124, 126 at 130. This aggregate is combined with the analyte of interest and the invasive DNA at 140. Then the endonuclease is added, such as one created by direct evolution 122. If the endonuclease can separating the substrate 134 with the desired sensitivity and selectivity in the presence of the analyte 105 in a colorimetric sensor system. In this regard, the endonuclease or nucleic acid enzyme can also be considered an analyte. In this way, multiple endonucleases generated from direct evolution 122 can be tested for use in a colorimetric sensor system. Fig. 2A describes a DNA enzyme 224 that depends on Pb (II) as a co-factor to exhibit catalytic activity. As can be seen from the base pairs, the DNA enzyme 224 can hybridize to a strand of complementary substrate 234 that includes a sort of separation, such as ribo-adenosine 235. Unlike the riboadenosine separation species 235, it is forms the strand of complementary substrate 234 described from deoxyribonucleosides. While a base sequence for the DNA enzyme and the complementary substrate strand is shown, the bases can be changed over both strands to maintain the pairs. For example, any of C in any strand can be changed to T, while the base is changed in pair from G to A. The base pair regions of the DNA enzyme 224 and the complementary substrate strand 234 can be extended or truncated, while there are sufficient bases to maintain the desired separation of the substrate. While various modifications to the enzyme and substrate are possible, modifications made to the catalytic core region of the enzyme may have important effects on the catalytic efficiency or specificity of analytes of the enzyme. A more detailed discussion of such modifications are the resulting effects on the catalytic activity that could be found in Bro n, A., et al., "A Lead-dependent DNAzyme with a Two-Step Mechanism", Biochemistry, 42, 7152-7661 ( 2003). The ribo-adenosine (rA) 235 provides a cleavage site 237, where it is thought that the DNA enzyme 224 hydrolytically cleaves the substrate 234 in the presence of the cofactor, as described in Fig. 2B. This separation reaction results in the substrate 234 being separated into its 3 'and 5' fragments as described in Fig. 2B. In addition to the ribo-adenosine 235, the separation species used with a DNA enzyme, such as DNA 224 enzyme, may also include ribo-cytosine (rC), ribo-guanine (RG), and Oracil (U). Similarly, if the nucleic acid enzyme were an RNA enzyme (not shown) the appropriate separation species may also include rA, rC, rG, and ü. DNA Enzyme 224 and complementary substrate strand 234 can be separate strands, as described in Fig. 2A, or the DNA enzyme and the substrate can be part of the same strand of nucleic acids. When the AD enzyme and the complementary substrate are different strands of nucleic acids, the DNA enzyme can be referred to as a "trans action enzyme". The trans-acting enzymes have the advantage of being able to separate multiple complementary substrates. If the DNA enzyme and the complementary substrate are part of the same strand of nucleic acids, as described for example in FIG. 10E, the DNA enzyme can be referred to as a "cis-acting enzyme". Fig. 3A describes the disintegration of an aggregate 332 in the presence of an analyte of Pb (II) 305 and invasive DNA 344. Aggregate 332 was formed of a DNA enzyme 324 and a strand of substrate 334, which hybridizes to particles functionalized thiol-olgonucleotides 3 'and 5' 336 and 337, respectively. The strand of substrate 334 was extended over the 3 'and 5' ends for the 12 bases, allowing hybridization with the functionalized DNA particles 12-mer 336, 337. The catalytic core of the DNA enzyme 324 includes the motif of the DNA enzyme "8-17", which exhibits high activity in the presence of the Pb (II) cation. Invasive DNA 344 includes a 3 '387 strand and a 5' 386 strand. In the presence of analyte 305 and invasive strands 386 and 387, blue aggregate 332 begins to disintegrate from partial aggregate 390. This partial disaggregation adds a red color to the blue solution as the particles extend away from the aggregate 332, thus giving a purple solution. If enough analyte 305 is present in the sample, the reaction will continue until the aggregate 332 is completely disintegrated to give 395. This results in a red solution due to the larger distance between the nanoparticles. The alignment of the particles (tail to tail or head to tail) with respect to each of them can influence the way in which the portions that form the aggregate are hermetically joined. Figs. 3A and 3B describe that aggregate 332 can be formed from DNA enzyme 324 and substrate strand 344 where functionalized particles, such as 337, are hybridized in a tail-to-tail array (Fig. 3B) with the strand of Substrate 344. Hybridization of tail glue or head fits (Fig. 3C) can be selected by inverting the end of the oligonucleotide to which the particle binds. Therefore, head-to-tail alignment can be selected by the use of a single strand of thiol-modified DNA, such as 337, while tail-to-tail alignment of both threads of modified ADN with 3 'and 5' thiol they can attach to the particles. Currently, the tail-hybridization arrangement of FIG. 3A and 3B is preferred because the head-to-tail hybridization arrangement of FIG. 3C can produce aggregates that aesthetically hide the catalytic activity of the DNA enzyme. . However, this steric hydration can be reduced through a reduction in the average diameter of the particles or through the use of a longer substrate, for example. The ionic strength of the sample can influence the way in which the portions that make up the aggregate are tightly bound. Higher salt concentrations favor aggregation, thus decreasing the sensor response, while lower salt concentrations may lack the ionic strength necessary to maintain the aggregates. In one aspect, the sample may include or be modified with a reagent to include a concentration of monovalent metal ions of 30 mM and greater. The ionic strength of the sample can be modified with Na + ions, for example. In a preferred aspect, the monovalent metal ion concentration of the sample, which contains the aggregate, is from 28 to 40 mM. At present, a concentration of especially preferred monovalent metal ions is about 30 mM. The pH can also influence the union of the aggregate, can be attributed possibly to the protonation of base pairs of polynucleotides at lower pH. In one aspect, an approximately neutral pH is preferred.

Therefore, the performance of the sensor can be improved by adjusting the ionic strength and pH of the sample before combining it with the aggregate. Depending on the sample, it may be convenient to add the sample or analyte "to a solution containing the aggregate (where the ionic resistance and pH can be controlled) or vice versa." The sensor system, including the substrate, functionalized oligonucleotide particles and DNA. In one aspect, the kit includes the desired endonuclease or nucleic acid specific for analytes that is at least partially complementary to the substrate.In yet another aspect, the kit excludes the endonuclease / enzyme. of nucleic acid, which is provided by the user or is provided separately In this regard, the equipment can also be used to determine the specificity and / or selectivity of several endo-nucleases for a selected analyte. use to select an appropriate endonuclease in addition to detecting the analyte In yet another aspect, the equipment includes an outer package that describes a DNA enzyme, a complementary substrate, functionalized particles of oligonucleotides and invasive DNA. One or more of these components can be separated into individual containers, or they can be provided in their aggregate state. If it is separated, the aggregate can be formed before introducing the sample. The invasive DNA can be kept in a separate container so that it can be added to the sample before it is combined with the aggregate. Additional regulators and / or pH modifiers can be provided in the equipment to adjust the ionic strength and / or pH of the sample. The containers may be in the form of bottles, tubes, sacks, envelopes, pipes, ampoules and the like, which may be formed in whole or in part from plastic, glass, paper, aluminum foil, MYLAR®, wax and the like. The containers can be equipped with completely or partially separable covers which can initially be part of the containers or can be fixed to the containers by mechanical, adhesive or other means. The containers can also be equipped with stops, allowing access to the contents by syringe needle. In one aspect, the outer package can be made of paper or plastic, while the containers are glass ampules. The outer package may include instructions regarding the use of the components. Comparators of color; normal analyte solutions, such as a 10 μ solution? of analyte solution; and display aids, such as thin layer chromatography (TLC) plates, test tubes, and cuvettes, may also be included. The outer package may also include filters and dilution reagents that allow the preparation of the sample for analysis. In another aspect, in addition to the sensor system of the present invention, the equipment may also include multiple sensor systems to further increase the reliability of analyte determination and reduce the probability of user error. In one aspect, multiple "light" sensor systems may be included in accordance with the present invention In another aspect, a "down light" sensor system may be included with the lighting sensor system of the present invention. Currently claimed sensor system can be considered a sensor because a color change occurs (blue or red) in the presence of the analyte.Reversely, in a downlight sensor system, a color change is not observed in the presence of the Thus, a lighting system can give a false result by illuminating upwardly when the analyte is absent, while a downlight sensor system may not undergo color change when the analyte is present Combining a sensor system using chemistry Downlighting with the lighting sensor claimed herein may reduce the likelihood of an analyte determination or imprecise.

Descending lighting sensors suitable for inclusion in the equipment claimed herein may be based on DNA enzyme / substrate / aggregates in particles that are not formed in the presence of the selected analyte. Therefore, for these sensors, a color change of aggregate formation is observed when the selected analyte is not present in the sample. In one aspect, these down lighting sensors may be based on an array of tail-to-tail particles coupled with nanoparticles having average diameters of approximately 43 nm to provide aggregation at room temperature in the absence of the analyte. A more detailed description of downstream lighting sensor systems suitable for inclusion in the currently claimed equipment can be found, for example, in the patent application of E.Ü.A. 10 / 756,825, filed January 13, 2004, entitled "Biosensors Based on Directed Assembly of Particles", which is incorporated herein by reference. The above description is not intended to limit the scope of the invention to the preferred embodiments described, but instead allow a person with ordinary skill in the art to make and use the invention. Similarly, the following examples should not be construed as limiting the scope of the appended claims or their equivalents and are provided for illustration only. It should be understood that numerous variations may be made to the following procedures, which are within the scope of the appended claims and their equivalents.

EXAMPLES All DNA samples were purchased from Integrated DNA Technology Inc., Coralville, IA. Enzymes and enzyme portions of the DNA enzyme were purified by HPLC before use. Gold nanoparticles having an average diameter of 13 nm were prepared and functionalized with 12-mer thiol-modified DNA following literature procedures, such as those described in Storhoff, J., et al., "One-pot colorimetric differentiation of polynucleotides with single base imperfections using gold partióle probes ", JACS 120: 1959-1964 (1998), for example. The average diameter of the gold nanoparticles was verified by transmission electron microscopy (JEOL 2010).

Example 1: Blue aggregate formation The enzyme (17E, 400 nM), substrate (35Sub¾u, 100 nM), ADNAu 3 '(6 nM), and ADN¾U 5' (6 nM) were mixed with a buffer solution of 25 mM Tris acetate, pH 8.2, 300 mM NaCl. The mixture (usually in volume of 1 ml) was heated at 65 ° C for 3 minutes and allowed to cool slowly to room temperature for about 4 hours. Aggregates of blue nanoparticles were formed and precipitated. Optionally, Iso aggregates were further precipitated with a centrifuge and the supernatant was removed. The precipitated aggregates were washed three times with a buffer containing 100 m NaCl and 25 mM tris acetate (pH 8.2) and redispersed in 200 μ? 25 mM fresh tris acetate solution, but with 100 mM NaCl. The concentration of the aggregates in this undiluted mixture was standardized by adding 10 μ? of the aggregate containing mixture for 80 μ? of deionized water to disperse the aggregates. Extinction of this 9x diluted mixture was then measured at 522 nm. From this measurement, the amount of pH buffer required to provide an extinction value of 1 to 522 nm was calculated for the undiluted mixture. The appropriate amount of pH buffer containing 100 mM NaCl was then added to the undiluted mixture. In this way, the aggregate concentration in the pH buffer was adjusted so that when it broke up, the mixture could provide an ~ 9 or ~ 1 extinction after 9 dilutions at 522 nm. The sequences of the DNA enzyme 17E (SEQ ID NO: 1) and the substrate 17DS (SEQ ID NO: 2; r denote a single ribonucleotide) extended on each end with 12 bases SubSu (SEQ ID NO: 3: r denotes a only ribonucleotide) to hybridize to the 5 'DNA¾U (SEQ ID NO: 4) and gold nanoparticles functionalized with 3' ADNAu oligonucleotides (SEQ ID NO: 5) are given in the following Table 1.

Table 1 Example 2: Addition of Analyte and Invasive DNA A solution of 80. μ? including 21 mM NaCl, 25 mM tris acetate (pH 8.2), 2.25 μ? of invasive DNA, and a concentration of Pb (OAc) 2 12.5% higher than that desired in the test sample was combined with 10 μ? of the 100 mM NaCl solution containing the aggregates of Example 1. The resulting test sample had concentrations of 30 mM NaCl, 2 μ? Invasive DNA, and the desired concentration of Pb (II). The color change of the solution was determined after 5 minutes at ~ 22 ° C.

Example 3: Sensor performance monitoring The color change of the sample of Example 2 was monitored by UV-vis extinction spectroscopy. Fig. 4 is a graph that refers to the extinction ratios provided at specific wavelengths of a sample during disintegration. The dotted line in Fig. 4 shows the strong extinction peak at 522 nm exhibited by separate 13 nm nanoparticles, which provides a deep red color. As can be seen from the solid line in Fig. 4, when added, the 522 nm peak decreases in intensity and changes to a longer wavelength, while the extinction in the 700 nm region increases, resulting in a transition from red to blue. Therefore, a higher extinction ratio of 522 to 700 nm is associated with the red color of separate nanoparticles, while a low extinction ratio is associated with the blue color of aggregated nanoparticles. This extinction ratio was used to monitor the aggregation status of nanoparticles. Fig. 5A is a graph describing the change in extinction ratios over time for samples containing invasive DNA (Inva) and Pb (II) (o), invasive DNA (Inva) without Pb (II) (A), and a control sample containing Pb (II) without Invasive DNA (|). Fig. 5B is a similar graph that uses the Inva-A threads instead of the Inva threads. The extinction ratio increased rapidly over time for the invasive DNA / Pb (II) samples, indicating a rapid color change from blue to red and the presence of the Pb (II) analyte. For samples that only have invasive DNA, a color change from blue to red occurs, however, at a slower rate indicated by the slower increase in the extinction ratio. This test established that an undesirable color change could be generated only by the Invasive DNA, Inva or Inva-A strands. Therefore, the very "invasive" of a DNA can cause the aggregate to disintegrate without the analyte (co-factor), which can result in a false positive or an undesirable background level of color change. The control, which grows from invasive DNA and Pb (II), showed a very slow increase in the extinction ratio, indicating little color change. These experiments demonstrated the utility of the sensor system for detecting an analyte, but suggested that selection of the appropriate invasiveness of the invasive DNA could provide a sensor with diminished background color change and a decreased propensity to give false positives.

Example 4: Refining DNA Invasivity Invasive To find a less invasive DNA, a series of strands of invasive DNA having a reduced number of base pairs with the separated fragments of the DNA substrate were tested in the following manner. A UV-vis quantum spectrophotometer cell (Hellma, Germany) was prepared as a target by combining 60.3 μ? of 25 mM tris acetate (pH 8.2), 17 μ? of 10 mM NaCl-25 mM tris acetate (pH 8.2), 1.8 μ? of 0.1 mM of invasive DNA, and 1 μ? of 1 mM Pb (OAc) 2 · After measuring the base line, 10 μ? of the mixture of aggregates of Example 1 were added to the cell. This addition gave a final NaCl concentration of 30 mM and a final concentration of invasive DNA of 2 μ? for each strand of DNA. The final concentration of Pb (II) was 10 μ ?. Samples without the Pb (II) analyte were prepared in a similar manner, except that 61.3 μ were added? instead of 60.3 μ? of 25 mM tris acetate pH buffer (pH 8.2) to form the sample volume. Preferred reduced base pair pairs of DNA that were prepared as described above are listed as Inva-2 (SEQ ID NO: 8 and SEQ ID NO: 9, from left to right, respectively), Inva-4 (SEC ID NO: 10 and SEQ ID NO: 11), Inva-6 (SEQ ID NO: 12 and SEQ ID NO: 13), and Inva-8 (SEQ ID NO: 14 and SEQ ID NO: 15) in the following Table 2. Inva refers to the 22-mer invasive DNA strands (SEQ ID NO: 6 and SEQ ID NO: 7) used to generate the data for FIG. 5A. The initial Inva strands that underline the preferred sequences are completely complementary to the separated fragments of the substrate.

Table 2 The invasive strands of additional reduced base pairs that were tested are listed as Inva-2A (SEQ ID NO: 18 and SEQ ID NO: 19, from left to right, respectively), Inva-4A (SEQ ID NO: 20 AND SEC ID NO: 21), Inva-6A (SEQ ID NO: 22 and SEQ ID NO: 23) and Inva-8A (SEQ ID NO: 24 and SEQ ID NO: 25) in the following Table 3. Inva-A refers to to the 23 and 21-mer invasive DNA strands (SEQ ID NO: 16 and SEQ ID NO: 17) used in Example 3 and to generate the data for Fig. 5B.The initial Inva-A strands underscoring the Additional sequences are partially complementary to the separated fragments of the substrate, each strand being "deviated" by a base. Therefore, the invasive 23-mer Inva-A strand includes an "extra" base, while the 21-mer invasive Inva-A strand includes one less mase than the separated substrate fragments. In this way, an "inequality" is formed between the Inva-A invasive strands and the separated substrate fragments.

Table 3 Fig. 6A and 6B are graphs plotting the change in extinction ratios as a function of time for each of the shortened invasive DNA strands (relative to the original Inva or Inva-A strands) with and without the analyte of Pb (II). As the strands shorten and the number of base pairs with the separated portions of the substrate decreases, the rate of color change in the absence of the Pb (II) analyte decreases. The color change regime was always faster in the presence of the Pb (II) analyte with the same invasive DNA, thus establishing the ability of the sensor to detect the analyte. The preferred Inva · DNA sequences are completely complementary to each of the two fragments of the separated substrate, while the Inva-A DNA sequences are partially complementary, unpaired by a base. While the reduction in the number of base pairs for each of the completely complementary Inva or Inva-A strands is uneven, the overall invasiveness of the DNA decreases and provided a convenient reduction in the level of color change of the phono without the analyte. Inva-6 strands of reduced base maintained a rapid rate of disintegration in the presence of the analyte. Therefore, the strands of Inva-6, which have 6 bases less than the separate portions of the substrate, were chosen as the best compromise between the rate of color change in response to the analyte and the level of background color change that it can be attributed solely to the disintegration of invasive DNA. For these reasons, the Inva-6 strands were used to test the sensitivity and selectivity of the sensor. While not wishing to be bound by any particular theory, it is thought that the number of complementary base pairs has a greater effect on invasiveness (thermodynamic control), while the disintegration regime depends more strongly on the capacity of the fragment ends. of the separated substrate to hybridize initially with the strands of invasive DNA (kinetic control). By altering these parameters of invasive DNA, the background levels and color change regime can be optimized for a specific DNA-RNA and / or analyte enzyme. In addition to reducing complementarity by reducing the number of bases of strands of invasive DNA relative to the separated substrate fragments, other methods can also be used to reduce complementarity. For example, the invasive DNA strands may include bases that do not hybridize effectively with the bases of the separated substrate fragments. In another aspect, the bases from which the substrate and the invasive DNA are assembled can be selected to hybridize more weakly relative to other base pairs. Other methods can also be used to reduce the strength of hybridization between the separated substrate fragments and invasive DNA strands known to those of ordinary skill in the art.

Step 5: Selectivity Confirmation and Sensor Sensitivity In a ÜV-vis quartz spectrophotometer cell (Hellma, Germany), 60.3 μ were combined? of 25 mM tris acetate (pH 8.2), 17 μ? of 100 mM NaCl-25 mM tris acetate (pH 8.2), 1.8 μ? of 0.1 mM of invasive DNA Inva-6, and 1 μ? of a 0.5 mM solution containing a metal salt. The samples were prepared to include the following metal salts: Pb (OAc) 2 / - COCl2, ZnCl2, CdCl2, NCICI2, CUCI2, MgCl2 and CaCl2. After a measurement of the base line, 10 μ? of the aggregate mixture of Example 1 was added to each cell. This addition gave a final NaCl concentration of 30 mM, a final Inva-ß invasive DNA concentration of 3 μ? of each strand of DNA and a final metal ion concentration of 5 μ? for each tested metal. After completing the dispersion, the extinction at 522 nm was ~ 1. The dispersion kinetics for each metal ion was monitored as a function of time using a Hewlett-Packard 8453 spectrophotometer. Fig. 7A is a graph describing the extinction ratios at 522 and 700 nm plotted as a function of time. As can be seen in the traces, only Pb (II) gave a significant increase in the extinction ratio as a function of time, while the other metal ions Zn (II), Co (II), Cd (II), g (II), Cu (II), NI (II) and Ca (II), provided a color change that matches the background. Therefore, the high selectivity of the sensor was confirmed. Fig. 7B is a graph describing the correlation between the observed extinction ratios for the color change of the sensor system and the concentration of the Pb (II) analyte after five minutes of aggregation. The exceptional linearity of the sensor system was evident from approximately 0.1 to around 2 μ ?. Fig. 7C is a graph describing the extinction ratios for multiple Pb (II) analyte concentrations over a 10 minute period with Inva-6. The graph demonstrates the ability of the sensor system to effectively differentiate between different analyte concentrations within a few minutes. Therefore, the ability of the sensor system to provide accurate quantitative information was described. In addition to the instrumental method of Fig. 7B, the color developed by the sensor was conveniently observed by running the sensor solution on an alumina TLC plate. A color progression from blue to red was observed as the concentration of Pb (II) was increased from 0 to 10 μ ?. The other metal ions gave a color similar to that of the background.

Example 6: Determination of the Preferred Ion Resistance Environment for the Sensor To facilitate the rapid dispersion of the aggregates of Example 1, the aggregates were suspended in a pH-regulating solution containing NaCl to determine the concentration of lower NaCl capable of stabilizing the aggregates. Fig. 8 is a graph describing the stability that depends on NaCl of the aggregates. The data were acquired in a Hewlett-Packard 8453 spectrophotometer. The buffer solution was 25 mM Tris acetate, pH 7.6, having NaCl concentrations of 20, 25, 30 and 40 mM. Because the sample container was a UB-vis quartz cell, instead of a 96-well plate, the extinction ratio is different from the values obtained in the previous Examples. Within half an hour, the aggregates were stable when the NaCl concentration was about 30 mM and higher. Therefore, a solution of 30 mM NaCl was chosen having an appropriate ionic strength to stabilize the aggregates while not having a substantial adverse effect on the response time of the sensor.

Example 7: Characterization of the Aggregate Fig. 9 is a transmission electron microscopy (MET) image of aggregates of 13 mm gold nanoparticles assembled with DNA enzyme. The scale bar corresponds to 200 nm. It is clear from the image that the aggregates contain substantial numbers of gold nanoparticles. As any person with ordinary experience in the art will recognize from the description, figures and examples provided, modifications and changes can be made without departing from the scope of the invention defined by the following claims and their equivalents.

Claims (50)

1. CLAIMS 1. - A sensor system for detecting an analyte, comprising: a nucleic acid enzyme; a substrate for the nucleic acid enzyme, comprising first polynucleotides; first particles comprising second polynucleotides, the second polynucleotides coupled with the first particles, wherein the first polynucleotides are at least partially complementary to the second polynucleotides; and the invasive DNA, comprising polynucleotide quarters, the polynucleotide quarters are at least partially complementary to the first polynucleotides.
2. 2. The sensor of claim 1, further comprising second particles comprising third polynucleotides, the third polynucleotides coupled with the particles at the 5 'end, wherein the second polynucleotides are coupled to the first particles at the 3' end and the first polynucleotides are at least partially complementary to the third polynucleotides. 3. - The sensor of any of the preceding claims, wherein the nucleic acid enzyme comprises DNA. 4. - The sensor of any of the preceding claims, wherein the first group of particles comprises a material selected from the group consisting of metals, semiconductors, magnetizable materials and combinations thereof. 5. - The sensor of any of the preceding claims, wherein the first group of particles and the second group of particles comprises gold. 6. - The sensor of any of the preceding claims, the first group of particles having an average diameter of 5 nm to 70 nm. 7. - The sensor of any of the preceding claims, the first group of particles having an average diameter of 10 nm to 15 nm. 8. - The sensor of any of the preceding claims, wherein the analyte activates or deactivates the nucleic acid enzyme. 9. - The sensor of any of the preceding claims, wherein the analyte is selected from the group consisting of Ag (I), Pb (II), Hg (II), As (III), Fe (III), Zn ( II), Cd (II), Cu (II), Sr (II), Ba (II), Ni (II), Co (II), As (V), U (VI), and Cr (VI). 10. - The sensor of any of the preceding claims, wherein the analyte comprises a metal ion having a formal oxidation state +2. 11. The sensor of any of the preceding claims, wherein the analyte comprises Pb (II). 12. The sensor of any of the preceding claims, wherein the nucleic acid enzyme comprises a polynucleotide having a sequence selected from the group consisting of SEQ ID NOs: 26-44 and conservatively modified variants thereof. 13. - The sensor of any of the preceding claims, wherein the nucleic acid enzyme comprises a polynucleotide having a sequence of SEQ ID NO: 1 and conservatively modified variants thereof and the first polynucleotides comprise a polynucleotide having a sequence of SEQ ID NO: 3 and conservatively modified variants thereof. The sensor of any one of the preceding claims, wherein the polynucleotide rooms comprise at least two different strands, each having at least one terminate base that is complementary to at least one terminal base of a separate substrate strand. , when the substrate is separated by the nucleic acid enzyme. 15. - The sensor of any of the preceding claims, wherein the fourth polynucleotides comprise at least two different strands, each having 2 to 10 fewer bases capable of hybridizing with a separate substrate strand than a completely complementary strand, when the substrate it is separated by the enzyme from nucleic acids. 16. - The sensor of any of the preceding claims, wherein the fourth polynucleotides comprise at least two different strands, each having 6 fewer bases capable of hybridizing with a separate substrate strand than a completely complementary strand, when the substrate is separated by the nucleic acid enzyme. 17. The sensor of any one of the preceding claims, wherein the fourth polynucleotides comprise a polynucleotide selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEC ID NO: .22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and conservatively modified variants thereof. 18. The sensor of any of the previous claims, wherein the rooms 0 polynucleotides comprise polynucleotides having a sequence of SEQ ID NO: 12 and conservatively modified variants thereof and SEQ ID NO: 13 and conservatively modified variants thereof. 19. - A method to detect an analyte, which comprises: combining an aggregate, a sample and invasive DNA; and detecting a color change responsive to the analyte, the aggregate comprising: a substrate, comprising first polynucleotides, and first particles comprising second polynucleotides, the second polynucleotides coupled with the first particles, wherein the first polynucleotides are at least partially complementary to the second polynucleotides. 20. - The method of any of the preceding claims, which further comprises adjusting the ionic strength of the sample. 21. - The method of any of the preceding claims, wherein the sample and the invasive DNA are added to the aggregate. 22. - The method of any of the preceding claims, wherein the aggregate is added to the sample and the invasive DNA. 23. - The method of any of the preceding claims, wherein the aggregate further comprises: second particles comprising third polynucleotides, the third polynucleotides coupled to the particles at the 5 'terminus, wherein the second polynucleotides, are coupled to the first particles at the 3 'termination and the first polynucleotides are at least partially complementary to the third polynucleotides. 24. - The method of any of the preceding claims, wherein the particles comprise gold. 25. - The method of any of the preceding claims, wherein the aggregate further comprises an endonuclease comprising a binding site for the analyte, wherein the endonuclease is at least partially complementary to the substrate. 26. The method of any of the preceding claims, wherein the endonuclease comprises a nucleic acid enzyme. 27. The method of any of the preceding claims, wherein the nucleic acid enzyme comprises a polynucleotide having a sequence selected from the group consisting of SEQ ID NOS: 26-44 and conservatively modified variants thereof. 28. The method of any of the preceding claims, wherein the nucleic acid enzyme comprises a polynucleotide having a sequence of SEQ ID NO: 1 and conservatively modified variants thereof and the first polynucleotides comprise a polynucleotide having a sequence of SEQ ID NO: 3 and conservatively modified variants thereof. 29. The method of any of the preceding claims, wherein the invasive DNA competes with the nucleic acid enzyme to hybridize to the substrate. 30. - The method of any of the preceding claims, wherein the color change is completed by at least 95% 5"minutes after combining the aggregate, the sample and the invasive DNA 31. - The method of either of the preceding claims, wherein the combination occurs from 20 to 30 ° C. 32. - The method of any of the preceding claims, wherein the aggregate disintegrates in response to the analyte. 33. - The method of any of the preceding claims, wherein the response is proportional to the amount of the analyte in the sample. 34. - The method of any of the preceding claims, wherein the analyte activates or deactivates the nucleic acid enzyme. 35. The method of any of the preceding claims, wherein the analyte is selected from the group consisting of Ag (I), Pb (II), Hg (II), As (III), Fe (III), Zn ( II), Cd (II), Cu (II), Sr (II), Ba (II), Ni (II), Co (II), As (V), U (VI), and Cr (VI). 36. - The method of any of the preceding claims, wherein the analyte comprises Pb (II). 37. The method of any of the preceding claims, wherein the fourth polynucleotides comprise at least one terminal base that is complementary to at least one base terminates from a separate substrate strand. 38. The method of any of the preceding claims, wherein the fourth polynucleotides comprise at least two different strands, each having 2 to 10 fewer bases capable of hybridizing with a separate substrate strand than a completely complementary strand. 39. The method of any of the preceding claims, wherein the fourth polynucleotides comprise at least two different strands, each having 6 fewer bases capable of hybridizing with a separate substrate strand than a completely complementary strand. The method of any of the preceding claims, wherein the fourth polynucleotides comprise a polynucleotide selected from the group consisting of comprising a polynucleotide selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO. : 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 , SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and conservatively modified variants thereof. 41. The method of any of the preceding claims, wherein the fourth polynucleotides comprise polynucleotides having a sequence of SEQ ID NO: 12 and conservatively modified variants thereof and SEQ ID NO: 13 and conservatively modified variants thereof. 42. - The method of any of the preceding claims, wherein the sample originates from a biological source. 43. - The method of any of the preceding claims, wherein the sample originates from an industrial waste stream. 44. The method of any of the preceding claims, wherein the sample originates from a water supply from which water is extracted for human consumption. 45. - The method of any of the preceding claims, which further comprises quantifying the color change. 46. - A device for the detection of an analyte, comprising: a system for forming aggregates, comprising: comprising first polynucleotides, first particles comprising second polynucleotides, the second polynucleotides coupled with the first particles, wherein the first polynucleotides by at least they are partially complementary to the second polynucleotides; at least one first container containing the aggregate formation system; Invasive DNA, comprising fourth polynucleotides, the fourth polynucleotides are at least partially complementary to the first polynucleotides: at least one first container containing the aggregate formation system; Invasive DNA, comprising fourth polynucleotides, the fourth polynucleotides at least partially complementary to the first polynucleotides; at least one second container containing the invasive DNA, wherein a sample can be added to a container selected from the group comprising the first container, the second container, and a third container. 47. The kit of any of the preceding claims wherein the system further comprises third polynucleotides, the third polynucleotides coupled with the particles at the 5 'terminus, wherein the second polynucleotides are coupled to the first particles at the 3' end and The first polynucleotides are at least partially complementary to the third polynucleotides. 48. The equipment of any of the preceding claims, further comprising a reagent for modifying the ionic strength of the sample. 49. The equipment of any of the preceding claims, further comprising a reagent for modifying the pH of the sample, the reagent selected from the group consisting of acids and bases. 50. - The equipment of any of the preceding claims, which further comprises instructions for forming the aggregate. 51. The equipment of any of the preceding claims, further comprising instructions for modifying the ionic strength of the sample. 52. - The team of. any of the preceding claims, further comprising an endonuclease comprising a binding site for the analyte, wherein the endonuclease is at least partially complementary to the substrate. 53. The kit of any of the preceding claims, wherein the endonuclease comprises a nucleic acid enzyme. 54. The kit of any of the preceding claims, wherein the nucleic acid enzyme comprises a polynucleotide having a sequence selected from the group consisting of SEQ ID NOS: 26-44 and conservatively modified variants thereof. 55. The equipment of any of the preceding claims, wherein the fourth polynucleotides comprise at least two different strands, each having at least one terminal base that is complementary to at least two different strands, each having at least minus a terminal base that is complementary to at least one terminal base of a separate substrate strand, when the substrate is separated by the nucleic acid enzyme. The equipment of any of the preceding claims, wherein the polynucleotide quarters comprises at least two different strands, each having 2 to 10 fewer bases capable of hybridizing with a separate substrate strand than a completely complementary strand, when the substrate is separated by the enzyme from nucleic acids. 57. The kit of any of the preceding claims, wherein the fourth polynucleotides comprise at least two different strands, each having 6 bases less capable of hybridizing with a separate substrate strand than a completely complementary strand, when the substrate is separated by the enzyme nucleic acids. 58. The kit of any of the preceding claims, wherein the fourth polynucleotides comprise a polynucleotide selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and conservatively modified variants thereof. 59. The kit of any of the preceding claims, wherein the fourth polynucleotides comprise polynucleotides having a sequence of SEQ ID NO: 12 and conservatively modified variants thereof and SEQ ID NO: 13 and conservatively modified variants thereof. 60. - The equipment of any of the preceding claims, which further comprises a device for quantifying a responsible color change to the disintegration of the aggregate. 61. - The equipment of any of the preceding claims, wherein the device is selected from the group consisting of spectrophotometers and color comparators. 62. - The equipment of any of the preceding claims, further comprising a downlight sensor system that responds to the analyte.