METHODS FOR MANUFACTURING AN ELECTROCHEMICAL SENSOR FOR EFFECTIVE DIAGNOSTIC OLIGONUCLEOTIDE DETECTION

Abstract

The present invention features methods for manufacturing an electrochemical sensor for detecting a diagnostic target oligonucleotide. The methods described herein provide for an electrochemical sensor with a higher level of coverage of the probes on its surface, thus allowing for more sensitive detection of a target oligonucleotide. The methods may feature first mixing disulfide terminated oligonucleotides having a free thiol moiety at the 3′ end with a gold substrate and subsequently introducing to the gold substrate a composition for reducing thiol moieties to cause the oligonucleotides to bind to the surface of the gold substrate. In some embodiments, the method comprises removing excess thiol and oligonucleotides, which may help with non-competitive binding. In some embodiments, the method comprises rinsing the gold substrate with water and drying with nitrogen.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a non-provisional and claims benefit of U.S. Provisional Application No. 63/183,504 filed May 3, 2021, the specification of which is incorporated herein in their entirety by reference.

This application is a non-provisional and claims benefit of U.S. Provisional Application No. 63/171,761 flied Apr. 7, 2021 and U.S. Provisional Application No. 63/240,227 filed Sep. 2, 2021, the specification(s) of which is/are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to methods that allow for detecting diagnostic oligonucleotide target sequences.

BACKGROUND OF THE INVENTION

Diagnostic electrochemical sensors, particularly those that can screen and survey for infection in the population, food supply, animals, or the like, have reached a new level of importance. This is because electrochemical sensors allow for a simple, low-cost, sensitive system to measure target nucleic acid (e.g., DNA or RNA). The methods described herein provide for an electrochemical sensor with increased oligonucleotides (e.g., probes) on its surface. This increase of oligonucleotides on the surface of the electrochemical sensors increases the overall signal and allows for increased sensitivity and lower detection levels.

BRIEF SUMMARY OF THE INVENTION

It is an objective of the present invention to provide devices and methods that allow for the detection of diagnostic oligonucleotide target sequences, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

In some embodiments, the present invention may further feature a method of producing an electrochemical sensor for detecting a target oligonucleotide (e.g., single-stranded target oligonucleotide). In some embodiments, the method comprises obtaining and preparing a gold substrate. In some embodiments, the method comprises adhering disulfide terminated oligonucleotides to the gold substrate. In some embodiments, the method comprises reducing the disulfide, causing a thiol to bind directly to a surface of the gold substrate. In some embodiments, the method comprises removing excess thiol and oligonucleotides (which may help with non-competitive binding). In some embodiments, the method comprises adding a molecule comprising a thiol moiety at a first end to the surface of the gold substrate. In some embodiments, the method comprises rinsing the gold substrate with water and drying with nitrogen.

One of the unique and inventive technical features of the present invention is using a two-step method to attach the oligonucleotide (e.g., a DNA probe) to the electrochemical sensor (e.g., the DNA is put onto the surface of the electrochemical sensor first, and then a reducing agent is added afterward to “glue” the DNA probe to the surface of the electrochemical sensor). Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides for a higher level of coverage of the probes on the electrochemical sensor surface. Specifically, when the DNA probes are put onto the surface of the electrochemical sensor first, the DNA probes can occupy all of the binding sites on the surface, thus preventing any of the reducing agents from binding directly to the surface of the electrochemical sensor. Then, once the DNA probe has occupied all of the binding sites on the surface of the electrochemical sensor, a reducing agent is added to transform the probe-surface bond from a loose adhesion to a solid covalent bond. None of the presently known prior references or work has the unique, inventive technical feature of the present invention.

Furthermore, the prior references teach away from the present invention. For example, prior references teach mixing the DNA probe and the reducing agent together and allowing that mixture to react first before adding it to the electrochemical sensor surface, which causes a reduction in the probe coverage on the surface. Specifically, the reducing agent competes with the DNA probe to bind to the electrochemical surface (e.g., a gold surface). Therefore, when the DNA probe is mixed with the reducing agent and put onto the surface together, there is a competition between the DNA probe and the reducing agent for any available binding sites on the surface of the electrochemical sensor. Thus, instead of getting maximum coverage of the DNA probe on the surface of the electrochemical sensor, a mixed coverage between the DNA probe and the reducing agent is observed.

Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

FIG. 1 shows a method for producing an electrochemical sensor, in accordance with some embodiments described herein.

FIGS. 2A and 2B show methods for mixing disulfide terminated oligonucleotides (e.g., oligonucleotide probes) with a gold substrate. FIG. 2A shows a 40 μL drop of a DNA solution (e.g., oligonucleotide probes and a solvent) placed on top of the electrode. FIG. 2B shows the electrochemical sensor submerged in a DNA solution (e.g., oligonucleotide probes and a solvent). For both methods described in FIGS. 2A and 2B, the electrochemical sensor was allowed to sit with the DNA solution for about 30 minutes before a composition for reducing thiol moieties was added.

FIG. 3 shows, in accordance with some embodiments, an electrochemical sensor as described herein.

FIGS. 4A and 4B show the finished electrochemical sensor. FIG. 4A shows a glass test strip. The center circle shows an electrochemical sensor as described herein; the curved arc on top of the circle is the counter electrode. The small electrode on the right is the reference electrode. The probe is only found on the center circle. FIG. 4B shows a gold disk electrode supported by a PEEK material (polyetheretherketone) substrate. This is the finished product for the working electrode (sensor). Counter and Reference not shown.

FIG. 5 shows, in accordance with some embodiments, how the electrochemical sensor technology works when a target oligonucleotide binds to a probe.

FIGS. 6A and 6B show the setup for using the electrochemical sensor described herein. FIG. BA shows a three-electrode setup; the counter electrode is shown with a black lead, the reference electrode is shown with a blue lead, and the working electrode (i.e., the electrode comprising the electrochemical sensor described herein) is shown with a red lead. All electrodes comprising oligonucleotide probes on the surface were placed in a 10 mM PBS solution pH 7.4.

FIG. 7 shows an atomic force micrograph of the gold laminated glass test strips. The image area was five microns by five microns. The color scale was set to fifty nanometers. Peak to peak surface roughness was approximately 600 pm with exclusions due to contamination which was around 80 nm.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of summarizing the disclosure, certain aspects, advantages, and novel features of the disclosure are described herein. It is to be understood that not necessarily al such advantages may be achieved in accordance with any particular embodiments of the disclosure. Thus, the disclosure may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.

Additionally, although embodiments of the disclosure have been described in detail, certain variations and modifications will be apparent to those skilled in the art, including embodiments that do not provide all the features and benefits described herein. It will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed embodiments to other alternative or additional embodiments and/or uses and obvious modifications and equivalents thereof. Moreover, while a number of variations have been shown and described in varying detail, other modifications, which are within the scope of the present disclosure, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the present disclosure. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the present disclosure. Thus, it is intended that the scope of the present disclosure herein disclosed should not be limited by the particular disclosed embodiments described herein.

As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

Referring now to FIG. 1-7, the present invention features devices, compositions, and methods that allow for a point-of-care diagnostic system that allows for the identification of various pathogens. The methods described herein allow for more oligonucleotides (i.e., probes) to bind to the surface of an electrochemical sensor, which increases the overall signal and allows for increased sensitivity and lower detection levels. Additionally, the methods described herein generate an electrochemical sensor that has a 5×-10× increase in probe performance over traditional methods.

The present invention features a method of producing an electrochemical sensor for detecting a target oligonucleotide. In some embodiments, the present invention features a method of producing an electrochemical sensor for detecting a single-stranded target oligonucleotide. In other embodiments, the present invention features a method of producing an electrochemical sensor for detecting a double-stranded target oligonucleotide. The method comprises mixing disulfide terminated oligonucleotides having a free thiol moiety at a 3′ end with a gold substrate. Subsequently, the method comprises introducing to the gold substrate a composition for reducing thiol moieties of the oligonucleotides, thereby causing the oligonucleotides to bind a surface of the gold substrate.

In some embodiments, the method further comprises removing excess thiol and oligonucleotides. In other embodiments, the method further comprises adding a back-filler additive to the surface of the gold substrate. Finally, the method may further comprise rinsing the gold substrate with water and drying with nitrogen.

In some embodiments, the surface of the electrochemical sensor (e.g. the surface of the gold substrate) may be washed with water. In some embodiments, the water is deionized water. In other embodiments, the water is Type I deionized water. In further embodiments, the water is Type I deionized water with a resistivity of greater than 18 MOhms. In other embodiments, the surface of the electrochemical sensor (e.g., the surface of the gold substrate) may be washed with a polar solvent, including but not limited to acetonitrile.

The present invention may also feature a method of producing an electrochemical sensor for detecting a single-stranded target oligonucleotide. In other embodiments, the present invention features a method of producing an electrochemical sensor for detecting a double-stranded target oligonucleotide. The method comprises mixing disulfide terminated oligonucleotides having a free thiol moiety at a 3′ end with the gold substrate. Subsequently, the method comprises introducing to the gold substrate a composition for reducing the thiol moieties of the oligonucleotides, thereby causing the oligonucleotides to a surface of the gold substrate. The method may also comprise removing excess thiol and oligonucleotides and adding a back-filler additive to the surface of the gold substrate. The method may further comprise rinsing the gold substrate with water and drying with nitrogen.

In preferred embodiments, the disulfide terminated oligonucleotides (e.g., the probes) are mixed with the gold substrate 30 minutes before the probes are chemically bound using a reducing agent (e.g., TCEP). In other embodiments, the disulfide terminated oligonucleotides (e.g., the probes) are mixed with the gold substrate for about 15 to 240 minutes, or about 15 to 210 minutes, or about 15 to 180 minutes, or about 15 to 150 minutes, or about 15 to 120 minutes, or about 15 to 90 minutes, or about 15 to 60 minutes, or about 15 to 30 minutes, or about 30 to 240 minutes, or about 30 to 210 minutes, or about 30 to 180 minutes, or about 30 to 150 minutes, or about 30 to 120 minutes, or about 30 to 90 minutes, or about 30 to 60 minutes, or about 60 to 240 minutes, or about 60 to 210 minutes, or about 60 to 180 minutes, or about 60 to 150 minutes, or about 60 to 120 minutes, or about 60 to 90 minutes, or about 90 to 240 minutes, or about 90 to 210 minutes, or about 90 to 180 minutes, or about 90 to 150 minutes, or about 90 to 120 minutes, or about 120 to 240 minutes, or about 120 to 210 minutes, or about 120 to 180 minutes, or about 120 to 150 minutes, or about 150 to 240 minutes, or about 150 to 210 minutes, or about 150 to 180 minutes, or about 180 to 240 minutes, or about 180 to 210 minutes, or about 210 to 240 minutes, before the probes are chemically bound using a reducing agent (e.g., TCEP). In some embodiments, the disulfide terminated oligonucleotides (e.g., the probes) are mixed with the gold substrate for about 15 minutes, or about 30 minutes, or about 60 minutes, or about 75 minutes, or about 90 minutes, or about 105 minutes, or about 120 minutes, or about 135 minutes, or about 150 minutes, or about 165 minutes, or about 180 minutes, or about 195 minutes, or about 210 minutes, or about 225 minutes, or about 240 minutes before the probes are chemically bound using a reducing agent (e.g., TCEP).

In preferred embodiments, the reducing agent (e.g., TCEP) is added to the gold substrate comprising disulfide terminated oligonucleotides (e.g., probes) for 30 to 60 minutes. In some embodiments, the reducing agent (e.g., TCEP) is added to the gold substrate comprising disulfide terminated oligonucleotides (e.g., probes) for about 15 to 120 minutes, or about 15 to 90 minutes, or about 15 to 60 minutes, or about 15 to 30 minutes, or about 30 to 120 minutes or about 30 to 90 minutes, or about 30 to 60 minutes, or about 60 to 120 minutes, or about 60 to 90 minutes, or about 90 to 120 minutes. In some embodiments, the reducing agent (e.g., TCEP) is added to the gold substrate comprising disulfide terminated oligonucleotides (e.g., probes) overnight (e.g., for about 12 to 16 hours). In other embodiments, the reducing agent (e.g., TCEP) is added to the gold substrate comprising disulfide terminated oligonucleotides (e.g., probes) for a minimum of about 5 minutes. In some embodiments, the reducing agent (e.g., TCEP) is added to the gold substrate comprising disulfide terminated oligonucleotides (e.g., probes) for about 15 minutes, or about 30 minutes, or about 60 minutes, or about 90 minutes, or about 120 minutes.

As previously stated, the methods described herein may comprise removing excess thiol and oligonucleotides. In some embodiments, the excess thiol and oligonucleotides are physically removed, e.g., by flicking the electrochemical sensor. In other embodiments, the excess thiol and oligonucleotides are removed by wicking the solution off, e.g., by using a tissue to wick the solution off.

The methods described herein may further comprise removing excess back-filler additives. In some embodiments, the back-filler agent is physically removed, e.g., by flicking the electrochemical sensor. In other embodiments, the back-filler agent is removed by wicking the solution off e.g., by using a tissue to wick the solution off.

As used herein, a “back-filler addictive” refers to additional molecules which bind onto the surface of the electrochemical sensor (e.g., gold) to fil any space on the surface of the electrochemical sensor not occupied by the oligonucleotides described herein. Such molecules include, but are not limited to, hydrocarbon chains comprising at least one terminal thiol group. In other embodiments, additives may include molecules comprising internal di-sulfide groups that can be reduced to form a two-terminal thiol group. In some embodiments, the back-filer additive binds to a portion of space on the gold substrate not occupied by the oligonucleotides.

In some embodiments, the back-filler additive is a molecule that already has a sulfur terminated end and that naturally binds to the surface of the electrochemical sensor (e.g., gold) not occupied by the oligonucleotides (e.g., exposed gold). The back-filler additive naturally forms a self-assembled monolayer. In some embodiments, the method comprises adding a back-filler additive to the surface of the gold substrate without any other additions. In other embodiments, the method comprises adding a back-filler additive to the surface of the gold substrate with a reducing agent (e.g., TCEP). Without wishing to limit the present invention to any theory or mechanism, it is believed that adding a reducing agent (e.g., TCEP) along with the back-filler addictive to the surface of the gold substrate increases the strength of the sulfur-gold bond by guaranteeing that all of the sulfur has been fully reduced.

In some embodiments, the back-filer additive is organic. In some embodiments, the back-filler additive comprises a thiol moiety (i.e., a sulfur group) at a first end. In some embodiments, the thiol moiety binds to the surface of the electrochemical sensor (e.g., the surface of a gold substrate).

In some embodiments, the back-filler additive further comprises a carbon chain linked to the thiol moiety at the first end of the back-filler additive. The carbon chain of the back-filler additive may help to physically separate the surface of the electrochemical sensor (e.g., the gold surface) from the solution. In some embodiments, the carbon chain is a hydrocarbon chain. In preferred embodiments, the hydrocarbon chain comprises a six hydrocarbon chain. In some embodiments, the hydrocarbon chain comprises a chain of about four hydrocarbons, or about five hydrocarbons, or about six hydrocarbons, or about seven hydrocarbons, or about eight hydrocarbons. Without wishing to limit the present invention to any theories or mechanisms it is believed that the carbon chain (e.g., the hydrocarbon chain) of the back-filler additive should be long enough to prevent other ions from getting close to the surface of the electrochemical sensor (e.g., the gold surface) which may cause additional electrochemical reactions, while not being too long to prevent the indicator (e.g., Methylene Blue) from reacting. In some embodiments, back-filler additives with shorter carbon chains (e.g., a three hydrocarbon chain) result in additional noise. In other embodiments, back-filler additives with longer carbon chains (e.g., a nine hydrocarbon chain) results in no signal from the desired reaction.

In some embodiments, the back-filer additive comprises a hydrophilic moiety at a second end. The hydrophilic moiety allows for water to make contact with the back-filler additive and not be repelled. In preferred embodiments, the hydrophilic moiety comprises a hydroxyl group. In other embodiments, the hydrophilic moiety comprises a polar group including but not limited to an amino group, a cyano group, or a carboxylic acid group.

In some embodiments, the carbon chain is linear. In other embodiments, the carbon chain is branched. In some embodiments, the back-filler additive comprises a hydrocarbon chain linked to a thiol moiety. In further embodiments, the back-filler additive is nonreactive. In certain embodiments, the back-filler additive is mercaptohexanol.

In some embodiments, the disulfide terminated oligonucleotides are oligonucleotide probes. As used herein, an oligonucleotide probe may refer to an oligonucleotide with an indicator attached to a 5′ end of said oligonucleotide and may be referred to herein as a probe composition. In some embodiments, the oligonucleotide probes are a reverse complement of the target oligonucleotide. In some embodiments, the oligonucleotide probes are single-stranded oligonucleotide probes. In some embodiments, the single-stranded oligonucleotide probes are a reverse complement of the single-stranded target oligonucleotide. In other embodiments, the single-stranded oligonucleotide probes are complementary to a single strand of a double-stranded target oligonucleotide. In some embodiments, the probes (i.e., the oligonucleotide probes) comprise an indicator attached to a 5′ end of the probe.

As used herein, an ‘indicator’ may refer to an electrochemically active molecule that can be covalently attached to the probe. The indicator comprises methylene blue, methylene violet, Ruthenium hexamine, or ferrocene. Other indicators may be used in accordance with compositions and methods as described herein.

In some embodiments, methods of producing an electrochemical sensor described herein further comprise preparing the substrate (e.g., the gold substrate). In some embodiments, preparing the gold substrate comprises electrochemically cleaning a surface of the gold substrate. In some embodiments, electrochemically cleaning the surface of the gold substrate increases the amount of disulfide terminated oligonucleotides bound to the surface of the gold compared to an unclean surface. In other embodiments, electrochemically cleaning the surface of the gold substrate increases the strength of the gold-sulfur bond (i.e., Au—S bond), by reducing any AuO₂ on the gold surface to Au, which better binds to the sulfur. In some embodiments, removing excess thiol and oligonucleotides helps reduce competitive binding.

In some embodiments, methods of producing an electrochemical sensor described herein further comprise clean-up steps to break interactions between the adjacent oligonucleotides and stabilize the electrochemical sensor. In other embodiments, the methods of producing an electrochemical sensor described herein further comprise clean-up steps, said clean-up steps comprising adding an additive to the gold substrate to break interactions between the adjacent oligonucleotides and stabilize the electrochemical sensor.

In some embodiments, the clean-up steps comprise adding an additive to disrupt the interactions between adjacent probes. In some embodiments, the additive is a buffer, a salt solution, or a combination thereof.

As used herein, an “additive” refers to additional molecules which minimize probe to probe interaction on the surface of the electrochemical sensor. The additives described herein separate the probe-to-probe distance and stabilize the probes, thus reducing their electrostatic interactions. In some embodiments, the additives described herein interact (e.g., via ionic interactions) with the negatively charged backbone of the oligonucleotide probe (e.g., a DNA probe). These interactions (i.e., the interactions between the additive and the probe) stabilize the probe making it less likely to interact with adjacent probes. In some embodiments, the additive is inorganic.

In some embodiments, the buffer comprises a phosphate buffer, a carbonate buffer, a MOPS buffer, a MOPSO buffer, a BES buffer, a TES buffer, a HEPES buffer, a DIPSO buffer, a MOBS buffer, a PBS buffer, or a TRIS buffer. In some embodiments, the salt solution comprises sodium (Na⁺), sodium chloride (NaCl), potassium (K⁺), potassium chloride (KCl), lithium (Li⁺), lithium chloride (LiCl), magnesium (Mg⁺²), magnesium chloride (MgCl₂), calcium (Ca⁺²), chloride (Cl⁻), phosphate (PO₄⁻³), nitrate (NO⁻), acetate (C₂H₃O₂⁻), carbonate (CO₃⁻²), bicarbonate (HCO₃⁻), or a combination thereof.

In certain embodiments, the additive (e.g., the buffer) has a pH of 7 to 7.4. In some embodiments, the additive (e.g., the buffer) has a pH of about 6.0 to 9.0, or about 6.0 to 8.5, or about 6.0 to 8.0, or about 6.0 to 7.5, or about 6.0 to 7.0, or about 6.0 to 6.5, or about 6.5 to 9.0, or about 6.5 to 8.5, or about 6.5 to 8.0, or about 6.5 to 7.5, or about 6.5 to 7.0, or about 7.0 to 9.0, or about 7.0 to 8.5, or about 7.0 to 8.0, or about 7.0 to 7.5, or about 7.5 to 9.0, or about 7.5 to 8.5, or about 7.5 to 8.0, or about 8.0 to 9.0 or about 8.0 to 8.5, or about 8.5 to 9.5. In other embodiments, the additives described herein have a pH of about 6.0, or about 6.5, or about 7.0, or about 7.2, or about 7.4, or about 7.6, or about 7.8, or about 8.0, or about 8.5, or about 9.0.

In certain embodiments, the additive has a salt concentration of 0 mM. In some embodiments, the additive has a salt concentration of 50 mM to 150 mM. In other embodiments, the additive has a salt concentration of 100 mM to 140 mM. In further embodiments, the additive has a salt concentration of 130 mM to 137 mM.

In some embodiments, the additive has a salt concentration of about 0 mM to 150 mM, or about 0 mM to 140 mM, or about 0 mM to 130 mM, or about 0 mM to 120 mM, or about 0 mM to 110 mM, or about 0 mM to 100 Mm, or about 0 mM to about 75 mM, or about 0 mM to 50 mM, or about 0 mM to 25 mM, or about 25 mM to 150 mM, or about 25 mM to 140 mM, or about 25 mM to 130 mM, or about 25 mM to 120 mM, or about 25 mM to 110 mM, or about 25 mM to 100 Mm, or about 25 mM to about 75 mM, or about 25 mM to 50 mM, or about 50 mM to 150 mM, or about 50 mM to 140 mM, or about 50 mM to 130 mM, or about 50 mM to 120 mM, or about 50 mM to 110 mM, or about 50 mM to 100 mM, or about 50 mM to about 75 mM, or about 75 mM to 150 mM, or about 75 mM to 140 mM, or about 75 mM to 130 mM, or about 75 mM to 120 mM, or about 75 mM to 110 mM, or about 75 mM to 100 Mm, or about 100 to 150 mM, or about 100 mM to 140 mM, or about 100 mM to 130 mM, or about 100 mM to 120 mM, or about 100 mM to 110 mM, or about 110 mM to 150 mM, or about 110 mM to 140 mM, or about 110 mM to 130 mM, or about 110 mM to 120 mM, or about 120 mM to 150 mM, or about 120 mM to 140 mM, or about 120 mM to 130 mM, or about 130 mM to 150 mM, or about 130 mM to 140 mM, or about 140 mM to 150 mM.

In some embodiments, the salt is sodium (Na⁺), sodium chloride (NaCl), potassium (K⁺), potassium chloride (KCl), lithium (Li⁺), lithium chloride (LiCl), magnesium (Mg⁺²), magnesium chloride (MgCl₂), calcium (Ca⁺²), chloride (Cl⁻), phosphate (PO₄⁻³), nitrate (NO⁻), acetate (C₂H₃O₂⁻), carbonate (CO₃⁻²), bicarbonate (HCO₃⁻), or a combination thereof.

In certain embodiments, the additive is a buffer comprising a pH of 7 to 8. As an example, in some embodiments, the additive is a buffer comprising a pH of 7.4. In one embodiment, the additive is a PBS solution. In some embodiments, the PBS solution is a 10×PBS solution. In other embodiments, the additive is a phosphate buffer, a carbonate buffer, a MOPS buffer, a MOPSO buffer, a BES buffer, a TES buffer, a HEPES buffer, a DIPSO buffer, a MOBS buffer, or a TRIS buffer.

In some embodiments, the additive is formamide. In other embodiments, the additive is a surfactant. In some embodiments, the surfactant is SDS or Triton-X.

In some embodiments, the clean-up steps comprise adding a PBS solution to the electrochemical sensor. The PBS solution may be a 100 mM PBS solution. In other embodiments, the PBS solution is a 50 mM PBS solution. In some embodiments, the PBS solution is a 150 mM PBS solution. In some embodiments, the PBS solution has a concentration of about 50 mM, or about 100 mM, or about 125 mM, or about 150 mM.

In some embodiments, the PBS solution is on the electrochemical sensor for 30 sec to 10 minutes. In other embodiments, the PBS solution is on the electrochemical sensor for 2 to 5 minutes. In certain embodiments, the PBS solution is on the electrochemical sensor for 3 minutes. In some embodiments, the PBS solution is on the electrochemical sensor for about 30 sec to 10 minutes, or about 30 sec to 8 minutes, or about 30 sec to 6 minutes, or about 30 sec to 5 minutes, or about 30 sec to 3 minutes, or about 30 sec to 2 minutes, or about 30 sec to 1 minute, or about 1 minute to 2 minutes, or about 1 minute to 3 minutes, or about 1 minute to about 5 minutes, or about 1 minute to 6 minutes, or about 1 minute 8 minutes, or about 1 minute to about 10 minutes, or about 2 minutes to 3 minutes, or about 2 minutes to 5 minutes, or about 2 minutes to 6 minutes, or about 2 minutes to 8 minutes or about 2 minutes to 8 minutes, or about 2 minutes to 10 minutes, or about 3 minutes to 5 minutes, or about 3 minutes to 6 minutes, or about 3 minutes to 8 minutes or about 3 minutes to 8 minutes, or about 3 minutes to 10 minutes, or about 5 minutes to 6 minutes, or about 5 minutes to 8 minutes or about 5 minutes to 8 minutes, or about 5 minutes to 10 minutes, or about 6 minutes to 8 minutes or about 6 minutes to 10 minutes, or about 8 minutes to 10 minutes.

In some embodiments, the clean-up steps further comprise removing the PBS solution from the electrochemical sensor. In some embodiments, the clean-up steps further comprise drying the electrochemical sensor. In some embodiments, the electrochemical sensor is dried at room temperature. In some embodiments, the electrochemical sensor is dried under atmospheric conditions. In other embodiments, the electrochemical sensor is dried under an inert gas atmosphere. In some embodiments, the inert gas is nitrogen.

In some embodiments, the clean-up steps described herein are repeated 2 to 10 times. In other embodiments, the clean-up steps described herein are repeated 3 to 5 times. In further embodiments, the cleanup steps described herein are repeated about 1 to 10 times, or about 1 to 8 times, or about 1 to 6 times, or about 1 to 5 times, or about 1 to 4 times, or about 1 to 3 times, or about 1 to 2 times, or about 2 to 10 times, or about 2 to 8 times, or about 2 to 6 times, or about 2 to 5 times, or about 2 to 4 times, or about 2 to 3 times, or about 3 to 10 times, or about 3 to 8 times, or about 3 to 6 times, or about 3 to 5 times, or about 3 to 4 times, or about 4 to 10 times, or about 4 to 8 times, or about 4 to 6 times, or about 4 to 5 times, or about 5 to 10 times, or about 5 to 8 times, or about 5 to 6 times, or about 6 to 10 times, or about 6 to 8 times, or about 8 to 10 times.

The present invention features a probe composition comprising an oligonucleotide and an indicator attached to a 5′ end of the oligonucleotide. The present invention may also feature a probe composition for detecting a single strand target oligonucleotide. In some embodiments, the probe is a reverse complement of the single strand target oligonucleotide. In some embodiments, the probe comprises an indicator attached to a 5′ end of the probe.

In some embodiments, the electrochemical sensors described herein can differentiate between hybridization rates of a target oligonucleotide and a probe bound to the sensor surface.

In some embodiments, the target oligonucleotide is DNA or mRNA. In some embodiments, the target oligonucleotide is from a pathogen. In some embodiments, the pathogen is a virus, bacteria, fungi, or prion. In some embodiments, the virus is the white spot syndrome virus. In some embodiments, the pathogen is a sexually transmitted disease including but limited to Herpes Simplex Virus, Syphilis, Gonorrhea, Trichomonas, Chlamydia. In other embodiments, the pathogen is influenza or a variant thereof (e.g., H1N1 and H5N1), or Covid-19.

In some embodiments, the electrochemical sensors described herein detect a target oligonucleotide sequence for a white spot syndrome virus envelope protein. In some embodiments, the envelope protein is VP24, VP26, VP28, or a combination thereof.

The present invention features an electrochemical sensor comprising a probe attached to a surface of the electrochemical sensor and a back-filler additive. In some embodiments, the back-filler additive fills any space on the surface of the electrochemical sensor not occupied by the probe.

In some embodiments, the electrochemical sensors described herein are able to detect a single strand target oligonucleotide sequence that is complementary to the probe attached to a said sensor. In some embodiments, the target oligonucleotide is a DNA oligonucleotide or an RNA oligonucleotide.

The present invention may further feature a method of producing an electrochemical sensor for detecting a single-stranded target oligonucleotide. In some embodiments, the method comprises preparing a gold substrate. In some embodiments, the method comprises adhering disulfide terminated oligonucleotides to the gold substrate. In some embodiments, the method comprises reducing the disulfide, causing a thiol to bind directly to a surface of the gold substrate. In some embodiments, the method comprises removing excess thiol and oligonucleotides. In some embodiments, the method comprises adding a back-filler additive to the surface of the gold substrate. In some embodiments, the method comprises rinsing the gold substrate with water and drying with nitrogen.

EXAMPLE

The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

Example 1: Preparing a Gold Electrode

1.0 Mechanical Polishing using Pads: Place three polishing pads on polishing pad holders. The first polishing pad is used to remove bulk contamination from the gold electrode. Place enough DI water onto the pad for the pad to become wet, but not soaked. Water should not drip of the pad if turned on its side. Rub the top of the gold disk electrode(s) on the polishing pad in figure eight and circular motions for 10 seconds. Make sure to keep the top of the gold disk electrode(s) completely flat when rubbing them on the polishing pads. Polishing rate should be close to two rotations per second.

Rinse the electrode(s) by spraying the sides and body of the electrode directly with DI water until the surface and body of the electrode(s) appear visibly dean. Flick off excess DI water by tapping it gently. The second polishing pad uses alumina powder to polish the gold surface by removing additional contamination as well as a thin layer of gold. Place a small quantity, approximately 500 mg, or alumina powder onto the second polishing pad. Add DI water onto the pad until it is moist, and the alumina forms a thick paste.

Rub, by applying moderate pressure, the top of the gold disk electrode(s) on the polishing pad through the alumina powder in figure eight and circular motions for three minutes. Rinse the electrode by spraying the sides and body of the electrode directly with DI water until the surface and body of the electrode appear visibly clean. Wipe the electrode's sides/body with an IPA wipe.

The third polishing pad is used to remove any excess alumina contamination from the gold electrode. Place enough DI water onto the third pad to allow it to become wet, but not enough to have any standing water. Rub the top of the gold disk electrode(s) on the polishing pad in figure eight and circular motions for 30 seconds. Rinse the electrode(s) by spraying the sides and body of the electrode(s) directly with DI water until the surface and body of the electrode(s) appear visibly clean. Dry the electrode with an IPA wipe. Rinse the electrode(s) with DI water. Remove excess DI water by gently flicking the electrode(s). Place the rinsed electrode, gold side facing up, in a rack and allow to air dry.

2.0 Electrochemically Cleaning using Linear Sweep Voltammetry: Prepare a solution of 0.5 M H₂SO₄ solution. Electrochemically clean the electrodes using linear sweep voltammetry (LSV). Place the working electrode, reference electrode, and counter electrode in a 10 mL aliquot of the 0.5 M H₂SO₄ solution. Attach electrodes to a potentiostat using the appropriate connectors.

Compare resulting voltammogram against standard voltammogram, looking for differences. Exact currents will be different due to differences in actual exposed gold area. Differences constitute extra peaks, shifted peaks, or distorted peaks. Once electrodes are deemed clean, remove from 0.5 M H₂SO₄, and rinse with DI water. Replace reference and counter electrodes and discard 0.5 M H₂SO₄ appropriately. Dispose of H2SO4 by neutralizing. Air dry the electrodes.

Example 2: Making an Electrochemical Sensor

Make the desired concentration of the working probe by diluting the probe with 10 mM PBS. Let the probe and PBS solution sit for approximately 15 minutes. Add probe and PBS solution to clean electrode(s). Cover the electrodes with an opaque covering to protect from light exposure and contaminants. Let the probe and PBS solution sit on the electrode(s) for 30 minutes. Add TCEP and to the electrode(s) with the probe solution still on the electrode(s). Let the TCEP and probe solution react on the electrode(s) for 60 minutes. Flick off the probe and TCEP solutions from the electrode(s). Add mercaptohexanol (MCH) and TCEP solution to the electrode(s). Let the MCH and TCEP solution sit on the electrode(s) and react for one hour. Rinse the MCH and TCEP solution off with DI water for 15 seconds, then use nitrogen compressed gas to dry the electrode(s). Store the electrode(s) under an opaque cover to protect the electrode(s) from UV light degradation.

Embodiments

The following embodiments are intended to be illustrative only and not to be limiting in any way.

Embodiment 1: A method of producing an electrochemical sensor for detecting a target oligonucleotide, the method comprising: (a) mixing disulfide terminated oligonucleotides having a free thiol moiety at a 3′ end with a gold substrate; and (b) subsequent to (a) introducing to the gold substrate a composition for reducing thiol moieties of the oligonucleotides, thereby causing the oligonucleotides to bind to a surface of the gold substrate.

Embodiment 2: The method of embodiment 1 further comprising removing excess thiol and oligonucleotides.

Embodiment 3: The method of embodiment 1 or embodiment 2 further comprising adding a back-filler additive to the surface of the gold substrate, the back filler binds to a portion of space on the gold substrate not occupied by the oligonucleotides.

Embodiment 4: The method of embodiment 3, wherein the back-filler additive is organic.

Embodiment 5: The method of embodiment 3, wherein the back-filler additive comprises a thiol moiety at a first end.

Embodiment 6: The method of embodiment 5, wherein the thiol moiety binds to the surface of the gold substrate.

Embodiment 7: The method of embodiment 3, wherein the back-filer additive further comprises a carbon chain linked to the thiol moiety at the first end of the back-filler additive.

Embodiment 8: The method of embodiment 7, wherein the carbon chain is a hydrocarbon chain.

Embodiment 9: The method of embodiment 7 or embodiment 8, wherein the carbon chain is linear.

Embodiment 10: The method of embodiment 7 or embodiment 8, wherein the carbon chain is branched.

Embodiment 11: The method of embodiment 3, wherein the back-filler additive comprises a hydrocarbon chain linked to a thiol moiety.

Embodiment 12: The method of embodiment 3, wherein the back-filler additive is mercaptohexanol.

Embodiment 13: The method of any of embodiments 3-12, wherein the back-filler additive is nonreactive.

Embodiment 14: The method of embodiment 3 further comprising rinsing the gold substrate with water and drying with nitrogen.

Embodiment 15: The method of any one of embodiments 1-15, wherein the oligonucleotides are single-stranded oligonucleotide probes, said probes comprising an indicator attached to a 5′ end of the probe.

Embodiment 16: The method of any one of embodiments 1-15, wherein the oligonucleotides are single-stranded oligonucleotide probes, said probes comprising an indicator attached to a 5′ end of the probe.

Embodiment 17: The method of embodiment 16, wherein the indicator comprises methylene blue, methylene violet, Ruthenium hexamine, or ferrocene.

Embodiment 18: The method of any one of embodiments 15-17, wherein the single-stranded oligonucleotide probes are a reverse complement of the single-stranded target oligonucleotide.

Embodiment 19: The method of any one of embodiments 1-18, furthering comprising preparing the gold substrate, wherein preparing the gold substrate comprises electrochemically cleaning a surface of the gold substrate.

Embodiment 20: The method of embodiment 19, wherein electrochemically cleaning the surface of the gold substrate increases the amount of oligonucleotides bound to the surface of the gold compared to an unclean surface.

Embodiment 21: The method of any one of embodiments 1-20, further comprising clean-up steps, wherein the clean-up steps comprise adding an additive to the gold substrate to break interactions between the oligonucleotides and stabilize the electrochemical sensor.

Embodiment 22: The method of embodiment 21, wherein the clean-up steps comprise adding an additive to disrupt the interactions between adjacent oligonucleotides.

Embodiment 23: The method of embodiment 22, wherein the additive is a buffer, a salt solution, or a combination thereof.

Embodiment 24: The method of embodiment 23, wherein the salt solution comprises sodium (Na⁺), sodium chloride (NaCl), potassium (K⁺), potassium chloride (KCl), lithium (Li⁺), lithium chloride (LiCl), magnesium (Mg⁺²), magnesium chloride (MgCl₂), calcium (Ca⁺²), chloride (Cl⁻), phosphate (PO₄⁻³), nitrate (NO⁻), acetate (C₂H₃O₂⁻), carbonate (CO₃⁻²), bicarbonate (HCO₃⁻), or a combination thereof.

Embodiment 25: The method of any of embodiments 22-24, wherein the additive has a salt concentration of 0 mM.

Embodiment 26: The method of any of embodiments 22-24, wherein the additive has a salt concentration of 50 mM to 150 mM.

Embodiment 27: The method of any of embodiments 22-24, wherein the additive has a salt concentration of 100 mM to 140 mM.

Embodiment 28: The method of any of embodiments 22-24, wherein the additive has a salt concentration of 130 mM to 137 mM.

Embodiment 29: The method of embodiment 23, wherein the buffer comprises a phosphate buffer, a carbonate buffer, a MOPS buffer, a MOPSO buffer, a BES buffer, a TES buffer, a HEPES buffer, a DIPSO buffer, a MOBS buffer, a PBS buffer, or a TRIS buffer.

Embodiment 30: The method of any of embodiments 22-29, wherein the additive has a pH of 7 to 7.4.

Embodiment 31: The method of any of embodiments 22-29, wherein the additive is a buffer comprising a pH of 7 to 7.4.

Embodiment 32: The method of embodiment 22, wherein the additive is a PBS solution.

Embodiment 33: The method of embodiment 32, wherein the PBS solution is a 10×PBS solution.

Embodiment 34: The method of embodiment 22, wherein the additive comprises formamide.

Embodiment 35: The method of embodiment 22, wherein the additive comprises a surfactant.

Embodiment 36: The electrochemical sensor or embodiment 35, wherein the surfactant is SDS or Triton-X.

Embodiment 37: The method of any one of embodiments 21-36, wherein the clean-up steps comprises adding a PBS solution to the electrochemical sensor.

Embodiment 38: The method of embodiment 37, wherein the PBS solution is a 100 mM PBS solution.

Embodiment 39: The method of embodiment 37 or embodiment 38, wherein the PBS solution is on the electrochemical sensor for 30 seconds to 10 minutes.

Embodiment 40: The method of any one of embodiments 37-39, wherein the PBS solution is on the electrochemical sensor for 2 to 5 minutes.

Embodiment 41: The method of any one of embodiments 37-40. wherein the PBS solution is on the electrochemical sensor for 3 minutes.

Embodiment 42: The method of any one of embodiments 21-41, wherein the clean-up steps further comprises removing the PBS solution from the electrochemical sensor.

Embodiment 43: The method of any one of embodiments 21-42, wherein the clean-up steps further comprises drying the electrochemical sensor.

Embodiment 44: The method of embodiment 43. wherein the electrochemical sensor is dried at room temperature.

Embodiment 45: The method of embodiment 43 or embodiment 44, wherein the electrochemical sensor is dried under atmospheric conditions.

Embodiment 46: The method of embodiment 43 or embodiment 44, wherein the electrochemical sensor is dried under an inert gas atmosphere.

Embodiment 47: The method of embodiment 46, wherein the inert gas is nitrogen.

Embodiment 48: The method of any one of embodiments 21-47, wherein the clean-up steps are repeated 2 to 10 times.

Embodiment 49: The method of any one of embodiments 21-48, wherein the clean-up steps repeated 3 to 5 times.

Embodiment 50: A method of producing an electrochemical sensor for detecting a target oligonucleotide, the method comprising: (a) mixing disulfide terminated oligonucleotides having a free thiol moiety at a 3′ end with to a gold substrate; (b) subsequent to (a) introducing to the gold substrate a composition for reducing the thiol moieties of the oligonucleotides, thereby causing the oligonucleotides to bind to a surface of the gold substrate: (c) removing excess thiol and oligonucleotides from the surface of the gold substrate; (d) adding a back-filler additive to the surface of the gold substrate, the back filler binds to a portion of space on the gold substrate not occupied by the oligonucleotides; and (e) rinsing the gold substrate with water and drying with nitrogen.

Embodiment 51: The method of embodiment 50, wherein the back-finer additive is organic.

Embodiment 52: The method of embodiment 50, wherein the back-filler additive comprises a thiol moiety at a first end.

Embodiment 53: The method of embodiment 52, wherein the thiol moiety binds to the surface of the gold substrate.

Embodiment 54: The method of embodiment 50, wherein the back-filler additive further comprises a carbon chain linked to the thiol moiety at the first end of the back-filler additive

Embodiment 55: The method of embodiment 54, wherein the carbon chain is a hydrocarbon chain.

Embodiment 56: The method of embodiment 54 or embodiment 55, wherein the carbon chain is linear.

Embodiment 57: The method of embodiment 54 or embodiment 55, wherein the carbon chain is branched.

Embodiment 58: The method of embodiment 50, wherein the back-filler additive comprises a hydrocarbon chain linked to a thiol moiety.

Embodiment 59: The method of embodiment 50, wherein the back-filler additive is mercaptohexanol.

Embodiment 60: The method of any of embodiments 50-59, wherein the back-finer additive is nonreactive.

Embodiment 81: The method of any one of embodiments 50-60, wherein the target oligonucleotides are single stranded target oligonucleotides.

Embodiment 62: The method of any one of embodiments 50-61, wherein the oligonucleotides are single-stranded oligonucleotide probes, said probes comprising an indicator attached to a 5′ end of the probe.

Embodiment 63: The method of embodiment 62, wherein the indicator comprises methylene blue, methylene violet, Ruthenium hexamine, or ferrocene.

Embodiment 64: The method of any one of embodiments 61-83, wherein the single-stranded oligonucleotide probes are a reverse complement of the single-stranded target oligonucleotide.

Embodiment 65: The method of any one of embodiments 50-64, furthering comprising preparing the gold substrate, wherein preparing the gold substrate comprises electrochemically cleaning a surface of the gold substrate.

Embodiment 68: The method of embodiment 65, wherein electrochemically cleaning the surface of the gold substrate increases the amount of oligonucleotides bound to the surface of the gold compared to an unclean surface.

Embodiment 67: The method of any one of embodiments 50-66, further comprising clean-up steps, wherein the clean-up steps comprise adding an additive to the gold substrate to break interactions between the oligonucleotides and stabilize the electrochemical sensor.

Embodiment 68: The method of embodiment 67, wherein the clean-up steps comprise adding an additive to disrupt the interactions between adjacent oligonucleotides.

Embodiment 69: The method of embodiment 67 or embodiment 68, wherein the additive is a buffer, a salt solution, or a combination thereof.

Embodiment 70: The method of embodiment 69, wherein the salt solution comprises sodium (Na⁺), sodium chloride (NaCl), potassium (K⁺), potassium chloride (KCl), lithium (Li⁺), lithium chloride (LiCl), magnesium (Mg⁺²), magnesium chloride (MgCl₂), calcium (Ca⁺²), chloride (Cl⁻), phosphate (PO₄⁻³), nitrate (NO⁻), acetate (C₂H₃O₂⁻), carbonate (CO₃⁻²), bicarbonate (HCO₃⁻), or a combination thereof.

Embodiment 71: The method of any of embodiments 67-70, wherein the additive has a salt concentration of 0 mM.

Embodiment 72: The method of any of embodiments 67-70, wherein the additive has a salt concentration of 50 mM to 150 mM.

Embodiment 73: The method of any of embodiments 67-70, wherein the additive has a salt concentration of 100 mM to 140 mM.

Embodiment 74: The method of any of embodiments 67-70, wherein the additive has a salt concentration of 130 mM to 137 mM.

Embodiment 75: The method of embodiment 69, wherein the buffer comprises a phosphate buffer, a carbonate buffer, a MOPS buffer, a MOPSO buffer, a BES buffer, a TES buffer, a HEPES buffer, a DIPSO buffer, a MOBS buffer, a PBS buffer, or a TRIS buffer.

Embodiment 76: The method of any of embodiments 67-75, wherein the additive has a pH of 7 to 7.4.

Embodiment 77: The method of any of embodiments 67-75, wherein the additive is a buffer comprising a pH of 7 to 7.4.

Embodiment 78: The method of embodiment 87 or embodiment 68, wherein the additive is a PBS solution.

Embodiment 79: The method of embodiment 78, wherein the PBS solution is a 10×PBS solution.

Embodiment 80: The method of embodiment 67 or embodiment 68, wherein the additive comprises formamide.

Embodiment 81: The method of embodiment 67 or embodiment 68, wherein the additive comprises a surfactant.

Embodiment 82: The electrochemical sensor of embodiment 35, wherein the surfactant is SDS or Triton-X.

Embodiment 83: The method of any one of embodiments 67-82, wherein the clean-up steps comprises adding a PBS solution to the electrochemical sensor.

Embodiment 84: The method of embodiment 83, wherein the PBS solution is a 100 mM PBS solution.

Embodiment 85: The method of embodiment 83 or embodiment 84, wherein the PBS solution is on the electrochemical sensor for 30 seconds to 10 minutes.

Embodiment 88: The method of any one of embodiments 83-85, wherein the PBS solution is on the electrochemical sensor for 2 to 5 minutes.

Embodiment 87: The method of any one of embodiments 83-85, wherein the PBS solution is on the electrochemical sensor for 3 minutes.

Embodiment 88: The method of any one of embodiments 67-87, wherein the clean-up steps further comprises removing the PBS solution from the electrochemical sensor.

Embodiment 89: The method of any one of embodiments 67-88, wherein the clean-up steps further comprises drying the electrochemical sensor.

Embodiment 90: The method of embodiment 89, wherein the electrochemical sensor is dried at room temperature.

Embodiment 91: The method of embodiment 89 or embodiment 90, wherein the electrochemical sensor is dried under atmospheric conditions.

Embodiment 92: The method of embodiment 89 or embodiment 90, wherein the electrochemical sensor is dried under an inert gas atmosphere.

Embodiment 93: The method of embodiment 92, wherein the inert gas is nitrogen.

Embodiment 94: The method of any one of embodiments 67-93, wherein the clean-up steps are repeated 2 to 10 times.

Embodiment 95: The method of any one of embodiments 67-94 wherein the clean-up steps repeated 3 to 5 times.

As used herein, the term “about” refers to plus or minus 10% of the referenced number.

Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of” or “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of” or “consisting of” is met.

Claims

1. A method of producing an electrochemical sensor for detecting a target oligonucleotide, the method comprising:

a) mixing disulfide terminated oligonucleotides having a free thiol moiety at a 3′ end with a gold substrate; and

b) subsequent to (a) introducing to the gold substrate a composition for reducing thiol moieties of the oligonucleotides, thereby causing the oligonucleotides to bind to a surface of the gold substrate.

2. The method of claim 1 further comprising removing excess thiol and oligonucleotides.

3. The method of claim 2 further comprising adding a back-filler additive to the surface of the gold substrate, the back filler binds to a portion of space on the gold substrate not occupied by the oligonucleotides.

4. The method of claim 3, wherein the back-filler additive is organic.

5. The method of claim 3, wherein the back-filler additive comprises a thiol moiety at a first end which binds to the surface of the gold substrate.

6. The method of claim 3, wherein the back-filler additive further comprises a carbon chain linked to the thiol moiety at the first end of the back-filler additive.

7. The method of claim 3, wherein the back-filler additive is mercaptohexanol.

8. The method of claim 3, wherein the back-filler additive is nonreactive.

9. The method of claim 3 further comprising rinsing the gold substrate with water and drying with nitrogen.

10. The method of claim 1, further comprising preparing the gold substrate, wherein preparing the gold substrate comprises electrochemically cleaning a surface of the gold substrate.

11. The method of claim 1, further comprising clean-up steps, wherein the clean-up steps comprise adding an additive to the gold substrate.

12. The method of claim 1, wherein the additive is a buffer, a salt solution, or a combination thereof.

13. The method of claim 12, wherein the salt solution comprises sodium (Na+), sodium chloride (NaCl), potassium (K+), potassium chloride (KCl), lithium (Li+), lithium chloride (LiCl), magnesium (Mg+2), magnesium chloride (MgCl2), calcium (Ca+2), chloride (Cl−), phosphate (PO4−3), nitrate (NO−), acetate (C2H3O2−), carbonate (CO3−2), bicarbonate (HCO3−), or a combination thereof.

14. The method of claim 12, wherein the buffer comprises a phosphate buffer, a carbonate buffer, a MOPS buffer, a MOPSO buffer, a BES buffer, a TES buffer, a HEPES buffer, a DIPSO buffer, a MOBS buffer, a PBS buffer, or a TRIS buffer.

15. The method of claim 12, wherein the additive has a pH of 7 to 7.4.

16. The method of claim 12, wherein the additive comprises a surfactant.

17. A method of producing an electrochemical sensor for detecting a target oligonucleotide, the method comprising:

a) mixing disulfide terminated oligonucleotides having a free thiol moiety at a 3′ end with to a gold substrate;

b) subsequent to (a) introducing to the gold substrate a composition for reducing the thiol moieties or the oligonucleotides, thereby causing the oligonucleotides to bind to a surface of the gold substrate;

c) removing excess thiol and oligonucleotides from the surface of the gold substrate; and

d) adding a back-filler additive to the surface of the gold substrate, the back filler binds to a portion of space on the gold substrate not occupied by the oligonucleotides; and

e) rinsing the gold substrate with water and drying with nitrogen.

18. The method of claim 17, wherein the back-filler additive is organic.

19. The method of claim 17, wherein the back-filler additive comprises a thiol moiety at a first end that binds to the surface of the gold substrate.

20. The method of claim 17, wherein the back-filler additive further comprises a carbon chain linked to the thiol moiety at the first end of the back-filler additive.

21. The method of claim 17, wherein the back-filler additive is nonreactive.

22. The method of claim 17, furthering comprising preparing the gold substrate, wherein preparing the gold substrate comprises electrochemically cleaning a surface of the gold substrate.

23. The method of claim 17, further comprising clean-up steps, wherein the clean-up steps comprise adding an additive to the gold substrate.

24. The method of claim 23, wherein the additive is a buffer, a salt solution, or a combination thereof.

25. The method of claim 24, wherein the additive has a pH of 7 to 7.4.