Reducing signal variability in electrochemical DNA detection

Abstract

The present disclosure provides for devices, compositions, and methods for improving signal variability in electrochemical detection, such as electrochemical detection with a biochip, such as a biochip including a gold electrode.

Description

[0001] REDUCING SIGNAL VARIABILITY IN ELECTROCHEMICAL DNA DETECTION

[0002] BACKGROUND OF THE DISCLOSURE

[0003] The detection of specific nucleic acids is an important tool for diagnostic medicine and molecular biology research. Gene probe assays currently play roles in identifying infectious organisms such as bacteria and viruses, in probing the expression of normal genes and identifying mutant genes such as oncogenes, in typing tissue for compatibility preceding tissue transplantation, in matching tissue or blood samples for forensic medicine, and for exploring homology among genes from different species. Ideally, a gene probe assay should be sensitive, specific and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis (for a review, see Abramson et al., Current Opinion in Biotechnology, 4:41-47 (1993)).

[0004] There is a variety of detection methods that have been developed to detect the product resulting from PCR amplification. Detection can be achieved directly by incorporating signal generating nucleotide analogues during the PCR reaction, or indirectly, e.g., such as by hybridization with nucleic acid probes having incorporated signal generating nucleotide analogues. In either approach, there is a need for having sensitive and stable nucleotide analogues, which ideally could be detected automatically. To that end, fluorescent tagging, with its advantages of high sensitivity and multicolor detection, had been developed and quickly came to dominate applications in nucleic acid sequencing and microarray expression analysis. A broad variety of fluorescent-tagged NTPs, dNTPs and ddNTPs are commercially available.

[0005] An alternative to fluorescence-based detection is electrochemical detection ("ECD"), which can be highly sensitive, rapid and amenable to inexpensive production in miniaturized ("lab-on-a-chip") formats. The term "electrochemical detection" is used to describe a range of detection techniques involving the application of an electric potential (via suitable electrodes) to a sample solution, followed by measurement of the resultant current. Electrochemical detection of a target in a sample can be carried out on a biochip. Generally, the electrode surface (such as those coated with a self-assembled monolayer (SAM)), has capture ligands which bind the target. In order to promote binding and sensitivity, SAM permeation layers containing electrolytic salts are dried on the SAM.

[0006] Electrochemical detection systems are valuable tools which are highly sensitive and can detect small amounts of target. Electrical and electrochemical monitoring of nucleic acid amplification requires no optical assistance so that the system can be simplified, downsized, and integrated into a small chip with the aid of complementary metal oxide semiconductor (CMOS)-compatible fabrication process, leading to the production of a scalable high-throughput analysis system in point-of-care applications. Signal variability is a critical issue in the field of electrochemical DNA sensing because it affects the reproducibility and reliability of the results. Inconsistent signals can lead to false positives or negatives, compromising the effectiveness of diagnostic tests and other applications that rely on precise DNA measurements. This is particularly problematic in clinical diagnostics, where accuracy is predominant. This variability can stem from various factors, such as inconsistent electrode surface conditions due to intrinsic challenges on making uniform electrode, and poor reproducibility of the sensing signals due to the complex electrolyte, impairing the accuracy and reliability of DNA detection.

[0007] BRIEF SUMMARY OF THE DISCLOSURE

[0008] In view of the foregoing, compositions and methods are provided which permit reliable electrochemical DNA sensing with consistent signals, permitting the precise quantification of patients' samples.

[0009] A first aspect of the present disclosure is a composition comprising hydrochloric acid, hydrogen peroxide, and water. In some embodiments, the hydrochloric acid prior to addition to the composition is concentrated hydrochloric acid (e.g., 12. IM). In some embodiments, the concentration of hydrochloric acid in aqueous solution is between about 0. IN and about 2.5N (i.e., between about 0. IM and about 2.5M). In some embodiments, the hydrogen peroxide is 10% to 50% w / w in water. In some embodiments, the hydrogen peroxide is 20% to 40% w / w in water. In some embodiments, a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 2.2M. In some embodiments, a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.5M. In some embodiments, the concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.0M. In some embodiments, a concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1.7M. In some embodiments, the concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1.0M. Herein, the composition may be used for treating an electrode, in particular a gold electrode, to obtain reduced signal variability as compared with an electrode that has not been treated.

[0010] A second aspect of the present disclosure is the use of a composition comprising hydrochloric acid, hydrogen peroxide, and water in treating an electrode. In some embodiments, the electrode is a gold electrode. In some embodiments, the electrode is treated to obtain reduced signal variability as compared with an electrode that has not been treated. In some embodiments, the hydrochloric acid prior to addition to the composition is concentrated hydrochloric acid (e.g., 12. IM). In some embodiments, the concentration of hydrochloric acid in aqueous solution is between about 0.1N and about 2.5N (i.e., between about 0.1M and about 2.5M). In some embodiments, the hydrogen peroxide is 10% to 50% w / w in water. In some embodiments, the hydrogen peroxide is 20% to 40% w / w in water. In some embodiments, a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 2.2M. In some embodiments, a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.5M. In some embodiments, the concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.0M. In some embodiments, a concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1 ,7M. In some embodiments, the concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1.0M.

[0011] A third aspect of the present disclosure is a method of preparing a treated gold electrode, comprising (a) obtaining a gold electrode; (b) incubating the obtained gold electrode with a composition comprising hydrochloric acid, hydrogen peroxide, and water for a predetermined amount of time to provide the treated gold electrode. In some embodiments, the predetermined amount of time ranges from between about 8 hours to about 24 hours. In some embodiments, the predetermined amount of time ranges from between about 12 hours to about 20 hours. In some embodiments, the hydrochloric acid prior to addition to the composition is concentrated hydrochloric acid (e.g., 12. IM). In some embodiments, the concentration of hydrochloric acid in aqueous solution is between about 0.1N and about 2.5N (i.e., between about 0.1M and about 2.5M). In some embodiments, the hydrogen peroxide is 10% to 50% w / w in water. In some embodiments, the hydrogen peroxide is 20% to 40% w / w in water. In some embodiments, a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 2.2M. In some embodiments, a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.5M. In some embodiments, the concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.0M. In some embodiments, a concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1.7M. In some embodiments, the method further comprises washing the electrode following the incubation with the composition. In some embodiments, the method further comprises forming a self-assembled monolayer on the surface of the treated gold electrode. Herein, the self-assembled monolayer may be formed on the surface of the treated gold electrode using a method as disclosed herein (e.g., as set forth in the sixth aspect below). In some embodiments, the method may further comprise incubating the electrode with a deposition solution comprising one or more capture probes and at least one carboxylic acid based small molecule selected from the group consisting of citrate, acetate, and formate, wherein the electrode is incubated with the deposition solution for a predetermined amount of time, which in particular embodiments can range from between about 10 minutes to about 30 minutes. In particular embodiments, the carboxylic acid based small molecule is citrate. In some embodiments, the treated gold electrode has a surface roughness (Ra) of between about HOnm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, the treated gold electrode has a surface roughness (Ra) of between about 120nm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, the treated gold electrode has a surface roughness (Ra) of between about 120nm to about 140nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, the method further comprises depositing an electrolyte layer on the formed selfassembled monolayer as disclosed herein. In some embodiments, the electrolyte layer comprises magnesium chloride. In some embodiments, the electrolyte layer further comprises one or more of a buffer, a stabilizer, and a non-ionic surfactant. In some embodiments, depositing an electrolyte layer is performed by applying an electrolyte solution as disclosed herein (e.g., in the seventh or in the tenth aspect below). In some embodiments, depositing an electrolyte layer on the formed self-assembled monolayer is performed by applying an electrolyte solution as disclosed herein (e.g., in the seventh or in the tenth aspect below). In some embodiments, the stabilizer is a sugar. In certain embodiments, the sugar is fructose or a non-reducing sugar selected from trehalose and sucrose.

[0012] A fourth aspect of the present disclosure is a gold surface having a surface roughness (Ra) of between about HOnm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope, wherein the gold surface is prepared by contacting the gold surface with a composition comprising hydrochloric acid, hydrogen peroxide, and water for a predetermined amount of time. In some embodiments, the predetermined amount of time ranges from between about 8 hours to about 24 hours. In some embodiments, the predetermined amount of time ranges from between about 12 hours to about 20 hours. In some embodiments, the hydrochloric acid prior to addition to the composition is concentrated hydrochloric acid (e.g., 12. IM). In some embodiments, the concentration of hydrochloric acid in aqueous solution is between about 0.1N and about 2.5N (i.e., between about 0.1M and about 2.5M). In some embodiments, the hydrogen peroxide is 10% to 50% w / w in water. In some embodiments, the hydrogen peroxide is 20% to 40% w / w in water. In some embodiments, a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 2.2M. In some embodiments, a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.5M. In some embodiments, the concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.0M. In some embodiments, a concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1 ,7M. In some embodiments, the concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1.0M. In some embodiments, the treated gold electrode has a surface roughness (Ra) of between about 120nm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, the treated gold electrode has a surface roughness (Ra) of between about 120nm to about 140nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, the gold surface further comprises a self-assembled monolayer. In some embodiments, the gold surface further comprises a plurality of capture probes. In some embodiments, the gold surface further comprises an electrolyte layer. In some embodiments, the capture probes may be attached to the gold electrode using a deposition solution disclosed herein. In some embodiments, the gold surface forms a gold electrode. In some embodiments, the gold surface (forming an electrode) is implemented on an analytical cartridge (e.g., a biochip cartridge disclosed herein) or on an analytical instrument. Herein, the electrode of the analytical cartridge or the analytical instrument may generate signals, which exhibit reduced signal variability as compared with an electrode that has not been treated.

[0013] A fifth aspect of the present disclosure is a deposition solution comprising one or more self-assembled monolayer species and at least one carboxylic acid based small molecule selected from the group consisting of citrate, acetate, and formate. In some embodiments, the one or more self-assembled monolayer species comprises a plurality of capture probes. Herein, using the deposition solutions of the present disclosure signal variability may be improved using a device including capture probes.

[0014] A sixth aspect of the present disclosure is a method of preparing a self-assembled monolayer on an electrode, wherein the method comprises incubating the electrode with a deposition solution comprising one or more capture probes and at least one carboxylic acid based small molecule selected from the group consisting of citrate, acetate, and formate, wherein the electrode is incubated with the deposition solution for a predetermined amount of time. In some embodiments, the predetermined amount of time ranges from between about 10 minutes to about 30 minutes. In some embodiments, the carboxylic acid based small molecule is citrate. In some embodiments, the electrode is a gold electrode. In some embodiments, the gold electrode has a surface roughness (Ra) as disclosed herein, e.g., of between about HOnm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, the one or more selfassembled monolayer species comprises a plurality of capture probes. Herein, using the deposition solutions of the present disclosure signal variability may be improved. In some embodiments, the method comprises prior to incubating the electrode with a deposition solution, treating the electrode with a composition disclosed herein (e.g., as described in aspect 1 above). In some embodiments, the gold electrode is treated by contacting the gold electrode with a composition comprising hydrochloric acid, hydrogen peroxide, and water for a predetermined amount of time. In some embodiments, the hydrochloric acid prior to addition to the composition is concentrated hydrochloric acid (e.g., 12. IM). In some embodiments, the concentration of hydrochloric acid in aqueous solution is between about 0.1N and about 2.5N (i.e., between about 0.1M and about 2.5M). In some embodiments, the hydrogen peroxide is 10% to 50% w / w in water. In some embodiments, the hydrogen peroxide is 20% to 40% w / w in water. In some embodiments, a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 2.2M. In some embodiments, a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.5M. In some embodiments, the concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.0M. In some embodiments, a concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1 ,7M. In some embodiments, the concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1 ,0M. In some embodiments, the method further comprises depositing an electrolyte layer on the formed self-assembled monolayer. In some embodiments, the electrolyte layer comprises magnesium chloride. In some embodiments, the electrolyte layer further comprises one or more of a buffer, a stabilizer, and a non-ionic surfactant. In some embodiments, depositing an electrolyte layer on the formed self-assembled monolayer is performed by applying an electrolyte solution as disclosed herein (e.g., in the seventh or in the tenth aspect below). In some embodiments, the stabilizer is a sugar. In certain embodiments, the sugar is fructose or a non-reducing sugar selected from trehalose and sucrose.

[0015] A seventh aspect of the present disclosure is an electrolyte solution comprising a buffer, magnesium chloride, a stabilizer, a non-ionic surfactant, and water. In some embodiments, the stabilizer is a sugar. In certain embodiments, the sugar is fructose or a non-reducing sugar selected from trehalose and sucrose. In some embodiments, the electrolyte solution has a pH ranging from about 7.5 to about 8.5 In some embodiments, the electrolyte solution has a pH ranging from about 7.7 to about 8.3. In some embodiments, the amount of magnesium chloride in the electrolyte solution ranges from about 600mM to about 700mM. In some embodiments, the amount of buffer in the electrolyte solution ranges from about 600mM to about 700mM. In some embodiments, the amount of non-ionic surfactant in the electrolyte solution ranges from between about 2.5% to about 7% by total volume of the electrolyte solution. In some embodiments, the non-ionic surfactant is selected from a group consisting of ethoxylated alcohols, alkyl polyglycosides, ethylene oxide / propylene oxide block copolymers, and acetylenic diol-based surfactants, particularly ethoxylated acetylenic diols (such as, e.g., Dynol™ 604). In certain embodiments, the electrolyte solution comprises a buffer, magnesium chloride, a stabilizer, a non-ionic surfactant, and water, wherein the electrolyte solution does not include a zwitterionic surfactant. In some embodiments, the stabilizer is a sugar. In certain embodiments, the sugar is fructose or a non-reducing sugar selected from trehalose and sucrose.

[0016] An eighth aspect of the present disclosure is the use of an electrolyte solution (e.g., an electrolyte solution as disclosed in the seventh or tenth aspect) in the preparation of an electrolyte layer, wherein the electrolyte solution comprises a buffer, magnesium chloride, a stabilizer, a non-ionic surfactant, and water. In some embodiments, the electrolyte solution is an electrolyte solution as disclosed herein (e.g., in the seventh aspect above or the tenth aspect below). Herein, the electrolyte layer prepared using an electrolyte compositions of the present disclosure may provide improved electron transfer between a label (e.g., a ferrocene or osmium label) and the electrode. In some embodiments, the stabilizer is a sugar. In certain embodiments, the sugar is fructose or a non-reducing sugar selected from trehalose and sucrose.

[0017] A ninth aspect of the present disclosure is a method of preparing an electrolyte layer comprising dispensing the electrolyte solutions disclosed herein (e.g., as disclosed in the seventh aspect above or the tenth aspect below) onto an electrode, wherein the electrode comprises one or more capture probes. In some embodiments, the dispensed electrolyte solution is permitted to dry passively. In some embodiments, the dispensed electrolyte solution is actively dried. Herein, the electrolyte layer prepared using an electrolyte compositions of the present disclosure may provide improved electron transfer between a label (e.g., a ferrocene or osmium label) and the electrode.

[0018] A tenth aspect of the present disclosure is an electrolyte solution comprising a buffer, magnesium chloride, a stabilizer, a zwitterionic surfactant, a non-ionic surfactant, and water. In some embodiments, the electrolyte solution further comprises a sugar. In some embodiments, the sugar is fructose or a non-reducing sugar selected from trehalose and sucrose. In some embodiments, the zwitterionic surfactant is a sultaine, a betaine, a phospholipid, or a sphingolipid. In some embodiments, the zwitterionic surfactant is a betaine (e.g., alkyl dimethyl betaine and cocodimethyl amidopropyl betaine), an amine oxide (e.g., a Cs to Cis, or C12 to Cis amine oxides), and sulfo and hydroxy betaines, such as N-alkyl-N,N-dimethylammino-l -propane sulfonate where the alkyl group can be Csto Cis. In some embodiments, the zwitterionic surfactant is 3-(decyldimethylammonio)propanesulfonate inner salt. In some embodiments, the electrolyte solution has a pH ranging from about 7.5 to about 8.5. In some embodiments, the electrolyte solution has a pH ranging from about 7.7 to about 8.3. In some embodiments, the amount of magnesium chloride in the electrolyte solution ranges from about 600mM to about 700mM. In some embodiments, the amount of buffer in the electrolyte solution ranges from about 600mM to about 700mM. In some embodiments, the amount of non-ionic surfactant in the electrolyte solution ranged from between about 2.5% to about 7% by total volume of the electrolyte solution. In some embodiments, the non-ionic surfactant is selected from a group consisting of ethoxylated alcohols, alkyl polyglycosides, ethylene oxide / propylene oxide block copolymers, and acetylenic diol-based surfactants, particularly ethoxylated acetylenic diols (such as, e.g., Dynol™ 604). In some embodiments, the stabilizer is a sugar. In certain embodiments, the sugar is fructose or a nonreducing sugar selected from trehalose and sucrose.

[0019] An eleventh aspect of the present disclosure is the use of an electrolyte solution (e.g., an electrolyte solution as disclosed in the tenth aspect) in the preparation of an electrolyte layer, wherein the electrolyte solution comprises a buffer, magnesium chloride, a stabilizer, a non-ionic surfactant, a zwitterionic surfactant, and water. In some embodiments, the zwitterionic surfactant is a sultaine, a betaine, a phospholipid, or a sphingolipid. In some embodiments, the zwitterionic surfactant is a betaine (e.g., alkyl dimethyl betaine and cocodimethyl amidopropyl betaine), an amine oxide (e.g., a Cs to Cis, or C12 to Cis amine oxides, and sulfo and hydroxy betaines, such as N-alkyl-N,N- dimethylammino-1 -propane sulfonate where the alkyl group can be Cs to Cis. In some embodiments, the zwitterionic surfactant is 3-(decyldimethylammonio)propanesulfonate inner salt. Herein, the electrolyte layer may provide improved electron transfer between a label (e.g., a ferrocene or osmium label) and the electrode. In some embodiments, the stabilizer is a sugar. In certain embodiments, the sugar is fructose or a non-reducing sugar selected from trehalose and sucrose.

[0020] A twelfth aspect of the present disclosure is a biochip cartridge comprising a substrate comprising a printed circuit board comprising an electro wetting grid of electrodes forming a droplet pathway; an array of detection electrodes accessible to the droplet pathway, each comprising a self-assembled monolayer including one or more capture probes; and a plurality of interconnections from the electro wetting grid and the detection electrodes, wherein each electrode of the electro wetting grid of electrodes is a gold electrode each having a surface roughness (Ra) of between about 1 lOnm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, the treated gold electrode has a surface roughness (Ra) of between about 120nm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, the treated gold electrode has a surface roughness (Ra) of between about 120nm to about 140nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, each gold electrode is prepared by contacting each of the gold electrodes with a composition comprising hydrochloric acid, hydrogen peroxide, and water for a predetermined amount of time (e.g., a composition as disclosed in the first aspect). In some embodiments, the self-assembled monolayer comprising the one or more capture probes is prepared by depositing a deposition solution as disclosed herein comprising one or more self-assembled monolayer species and at least one carboxylic acid based small molecule selected from the group consisting of citrate, acetate, and formate onto the electrode. Herein, using the deposition solutions of the present disclosure signal variability may be improved. In some embodiments, the self-assembled monolayer further comprises an electrolyte layer. In some embodiments, the electrolyte layer is prepared by applying an electrolyte solution (e.g., an electrolyte solution as disclosed in the seventh or tenth aspect), wherein the electrolyte solution comprises a buffer, magnesium chloride, a stabilizer, a non-ionic surfactant, and water. In some embodiments, the electrolyte solution is an electrolyte solution as disclosed herein (e.g., in the seventh aspect or the tenth aspect). In some embodiments, the stabilizer is a sugar. In certain embodiments, the sugar is fructose or a non-reducing sugar selected from trehalose and sucrose.

[0021] A thirteenth aspect of the present disclosure is a biochip cartridge comprising a substrate comprising a printed circuit board comprising an electro wetting grid of electrodes forming a droplet pathway; an array of detection electrodes accessible to the droplet pathway, each comprising a self-assembled monolayer including one or more capture probes; and a plurality of interconnections from the electro wetting grid and the detection electrodes; wherein the self-assembled monolayer comprising the one or more capture probes is prepared by depositing a solution comprising one or more self-assembled monolayer species and at least one carboxylic acid based small molecule selected from the group consisting of citrate, acetate, and formate onto the electrode; and wherein the self-assembled monolayer further comprises an electrolyte layer comprising MgCb. In some embodiments, each detection electrode of the electro wetting grid of electrodes is a gold electrode each having a surface roughness (Ra) of between about 1 lOnm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, the gold electrode has a surface roughness (Ra) of between about 120nm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, the gold electrode has a surface roughness (Ra) of between about 120nmto about 140nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, each gold electrode is prepared by contacting each of the gold electrodes with a composition comprising hydrochloric acid, hydrogen peroxide, and water for a predetermined amount of time (e.g., a composition as disclosed in the first aspect). In some embodiments, the self-assembled monolayer comprising the one or more capture probes is prepared by depositing a deposition solution as disclosed herein comprising one or more self-assembled monolayer species and at least one carboxylic acid based small molecule selected from the group consisting of citrate, acetate, and formate onto the electrode. In some embodiments, the one or more self-assembled monolayer species comprises a plurality of capture probes. Herein, using the deposition solutions of the present disclosure signal variability may be improved. In some embodiments, the electrolyte layer is prepared by applying an electrolyte solution (e.g., an electrolyte solution as disclosed in the seventh or tenth aspect), wherein the electrolyte solution comprises a buffer, magnesium chloride, a stabilizer, a non-ionic surfactant, and water. In some embodiments, the electrolyte solution is an electrolyte solution as disclosed herein (e.g., in the seventh aspect or the tenth aspect). In some embodiments, the stabilizer is a sugar. In certain embodiments, the sugar is fructose or a non-reducing sugar selected from trehalose and sucrose.

[0022] BRIEF DESCRIPTION OF THE FIGURES

[0023] For a general understanding of the features of the disclosure, reference is made to the drawings. In the drawings, reference numerals have been used throughout to identify identical elements. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. FIG. 1A provides a schematic showing the working principle of electrochemical detection on an electrode, such as a gold electrode, in accordance with one embodiment of the present disclosure.

[0024] FIG. IB illustrates a gold electrode including a capture probe, where the capture probe is hybridized to a single stranded nucleic acid molecule (e.g., a single strand of a target DNA molecule) and a signal probe. The signal probe is itself conjugated to one or more labels, such as one or more ferrocene labels (or, in the alternative, one or more osmium labels).

[0025] FIG. 2 depicts a device including an electrode 10, a monolayer including a plurality of capture probes 11, and an electrolyte layer 12, in accordance with one embodiment of the present disclosure.

[0026] FIG. 3 depicts a device 1 including an electrode 10, a monolayer including a plurality of capture probes 11, wherein the plurality of capture probes 11 are substantially uniformly distributed on the surface of the electrode 10. Negative charged small molecules 20 are illustrated which, when present in a deposition solution, are believed to facilitate the substantially uniform distribution of the capture probes when the deposition solution is deposited onto the gold electrode 10 (which may be treated or untreated with one of the treatment compositions of the present disclosure).

[0027] FIG. 4 depicts the surface morphology of gold electrodes observed by atomic force microscopy before (top) and after (bottom) the treatment with an acidic aqueous solution of hydrochloric acid and hydrogen peroxide (HH). Comparative analysis of the atomic force microscopy (AFM) images revealed a significant reduction in surface roughness (for instance, Ra 143 nm) following the HH treatment, indicating that the process effectively smoothed the electrode surface. This result suggests that the HH treatment can modify the topography of the gold electrode, potentially improving its surface properties for applications requiring enhanced uniformity and reduced variability. Electrochemical signals were measured using the potentiostat to evaluate the impact of HH solution treatment on signal variability.

[0028] FIG. 5 illustrates the relationship between the electrochemical signals (ferrocene and osmium (Os)), and the surfaces treated with HH solution to non-treated controls. A coefficient of variation (CV) was selected as a metric to evaluate the signal variability because it is believed to normalize the standard deviation relative to the mean, offering a dimensionless measure of variability. It is believed that this makes it particularly effective for comparing datasets with different scales of mean values as it highlights the relative consistency of the data rather than just the absolute dispersion. In signal evaluations, a lower CV indicates that variability is minimal relative to the signal strength ensuring more reliable results. FIG. 5 demonstrates that electrodes treated with HH solution exhibited significantly lower signal variability with a coefficient of variation (CV) of 11.1% for the ferrocene signal compared to 68% for the non-treated control. Similarly, the Os signal, corresponding to the capture probe attached to the electrode surface, showed a reduced CV after treatment. These findings suggest that the HH solution "smoothed" the gold electrode surface leading to more uniform capture probe attachment (Os signal) and, consequently, a more stable sandwich assay performance (ferrocene signal). FIG. 5 clearly demonstrates how surface treatment directly impacts signal consistency and overall assay reliability.

[0029] FIG. 6 illustrates the relationship between the electrochemical signals (ferrocene and Os) and the condition of deposition solution with (left column) and without (right column) citrate by means of ePlex machine. The citrate addition into a deposition solution resulted in a lower CV for both signals, with the ferrocene signals (top) showing a CV of 8.65% compared to 26.3% for the deposition solution without citrate. Similarly, the Os signal (bottom), which corresponds to the capture probe attached to the electrode surface, also displayed reduced variability. These findings suggest that citrate improved capture probe uniformity by inducing repulsive interactions and passivating specific facets of the gold surface. The graph highlights how the addition of citrate into the deposition solution enhances signal consistency.

[0030] FIG. 7 compares the ferrocene signal variability on gold electrodes using existing and modified electrolyte compositions. In this example, the modified electrolyte comprised MgCh, Tris buffer, a non-ionic surfactant, and trehalose. The modified electrolyte showed a marked improvement, with the ferrocene signal variability reduced to a CV of 14.9% compared to 23% for the original composition. This improvement is likely due to the modified electrolyte stabilizing the hybridization process, resulting in better overall assay reliability. The data underscores the importance of electrolyte composition in reducing variability and optimizing system performance.

[0031] FIG. 8 illustrates the combined effect of citrate treatment and modified electrolyte composition on ferrocene from gold electrodes (left). While the modified electrolyte alone significantly reduced signal variability (middle), combining it with citrate treatment further enhanced performance. This combined approach not only reduced signal variability but also increased signal intensity, indicating a synergistic effect. The citrate treatment likely improved capture probe uniformity through repulsive interactions and surface passivation, while the hybridization was stabilized by the modified electrolyte. Together, these methods optimized both signal consistency and strength, demonstrating the value of integrating surface and electrolyte modifications for improved assay performance.

[0032] FIG. 9 depicts a comparison of the surface spreading behavior of the electrolyte of the present disclosure which comprises MgCh, Tris buffer, a non-ionic surfactant, and trehalose with (left) and without (right) the addition of a zwitterionic (ZI) compound. In the presence of ZI, the buffer exhibits lower surface tension, resulting in enhanced spreading and more uniform coverage across the detection surface. It is believed that this uniformity not only reduces signal variability but also improves manufacturability by facilitating consistent surface treatment during production. In contrast, the modified electrolyte alone shows poor spreading, leading to uneven coverage. FIG. 10 depicts a comparison of the signal performance across gold pads under three electrolyte conditions: the modified electrolyte of the present disclosure which comprises MgCh, Tris buffer, a non-ionic surfactant, and trehalose with (MgCh-Tris + ZI), without (MgCh-Tris) ZI, and an existing electrolyte. Both MgCh-Tris + ZI and MgCh-Tris achieved similarly low signal variability (17.2% and 12.25%, respectively), confirming that the addition of ZI did not compromise reproducibility, whereas the existing electrolyte exhibited consistently higher variability, underscoring the advantage of the MgCh-Tris-based formulations.

[0033] DETAILED DESCRIPTION

[0034] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0035] As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0036] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of' or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0037] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary. As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0038] As used herein, the term "about" means encompassing plus or minus 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. For example, about 90% refers to a range encompassing between 81% and 99%.

[0039] As used herein, the terms "detect", "detecting”, or "detection" refer to an act of determining the existence or presence of one or more targets (e.g., microorganism nucleic acids, amplicons, etc.) in a sample. In some embodiments, target detection occurs when the amplicon forms a hybridization complex with the complimentary signal and capture probe.

[0040] As used herein, the phrase "detection system" refers to a method that facilitates the visualization of PCR-amplified DNA products. Examples of suitable detection systems include systems that depend on detection of color, radioactivity, fluorescence, chemiluminescence or electrochemical.

[0041] As used herein, the phrases "electrochemical system" or "electrochemical detection system" refer to a system that determines the presence and / or quantity of a redox analyte through measurements of electrical signal in a solution between a working electrode and a counter electrode, such as induced by a redox reaction or electrical potential from the release or absorption of ions. In some embodiments, the redox reaction refers to the loss of electrons (oxidation) or gain of electrons (reduction) that a material undergoes during electrical stimulation such as applying a potential. Redox reactions take place at the working electrode, and which, for chemical detection, is typically constructed from an inert material such as platinum or carbon. In some embodiments, the potential of the working electrode is measured against a reference electrode, which is typically a stable, well-behaved electrochemical halfcell such as silver / silver chloride. In some embodiments, the electrochemical system can be used to support many different techniques for determining the presence and concentration of the target biomolecules including, but not limited to, various types of voltammetry, amperometry, potentiometry, coulometry, conductometry, and conductimetry such as AC voltammetry, differential pulse voltammetry, square wave voltammetry, electrochemical impedance spectroscopy, anodic stripping voltammetry, cyclic voltammetry, and fast scan cyclic voltammetry. In some embodiments, the electrochemical system may further include one or more negative control electrode and a positive control electrode. In the context of the invention, a single electrochemical system may be used to detect and quantify more than one type of target analyte. The use of electrochemical systems is described in more detail in U.S. Patent Nos. 9,957,553, 9,498,778, 9,557,295, 8,501,921, 6,600,026, 6,740,518 and U.S. Application Publication Nos. 2016 / 0129437, 2018 / 0095100, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0042] As used herein, the term "surfactant" refers to a surface active agent that is used to lower the surface tension between liquids or a liquid and a solid. Surfactants can act as detergents, wetting agents, emulsifiers, foaming agents, or dispersants.

[0043] As used herein, the term "non-ionic surfactant" refers to an amphiphilic surface-active agent that possesses both a hydrophilic head group and a hydrophobic tail, characterized in that the hydrophilic head group carries no net electrical charge at a neutral pH. Non-ionic surfactants do not dissociate into ions in an aqueous solution and rely primarily on hydrogen bonding (e.g., via ether oxygens or hydroxyl groups) to interact with water. These agents generally rely on hydrogen bonding (e.g., via ether linkages or hydroxyl groups) to achieve solubility in aqueous media. The term encompasses compounds capable of lowering static and / or dynamic surface tension without dissociating into ions. In some embodiments, the non-ionic surfactant is selected from the group consisting of ethoxylated alcohols, alkyl polyglycosides, ethylene oxide / propylene oxide block copolymers, and acetylenic diol-based surfactants. In certain embodiments, the non-ionic surfactant is or comprises an ethoxylated acetylenic diol. This may be characterized by a dimeric structure comprising two hydrophilic head groups and two hydrophobic tails connected by an acetylenic linker. This structure provides superior wetting capabilities and low foam generation compared to traditional linear non-ionic surfactants. A specific example of a suitable non-ionic surfactant for use in the present disclosure is Dynol™ 604 (2,5,8, 1 l-tetramethyl-6-dodecyn-5,8-diol ethoxylate).

[0044] As used herein, the term "zwitterionic surfactant" refers to an amphiphilic surfactant molecule having no net charge that includes a hydrophobic group and one or more hydrophilic groups, as well as two moieties of opposite formal charges.

[0045] OVERVIEW

[0046] The present disclosure provides for devices, compositions, and methods for improving signal variability in electrochemical detection, such as electrochemical detection with a biochip, such as a biochip including a gold electrode.

[0047] Electrochemical detection technology is based on the principles of competitive DNA hybridization and electrochemical detection in which there is immobilization of a single stranded DNA segment (ss- DNA) onto an electrode (e.g., a gold electrode) and the measurement of changes in electrical parameters caused by the hybridization in presence of a reporter molecule attached to the complimentary ss-DNA present in the test sample. FIG. 1 A is an annotated schematic showing the basic principles of electrochemical detection technology.

[0048] In these electrochemical systems, the target amplicons are typically mixed with labeled signal probes (e.g., ferrocene-labeled signal probes) that are complementary to the specific targets, such as specific target nucleic acid molecules (see FIGS. 1A and IB). Target nucleic acid molecules hybridize to the complementary sequence of the signal and capture probes, which are bound to electrodes (e.g., gold electrodes or gold-plated electrodes) as shown in FIG. IB. The presence of each target is determined by voltammetry which generates specific electrical signals from the labeled signal probe (see FIG. 1A). The use of microfluidic systems in the electrochemical detection of target analytes is described in more detail in U.S. Pat. Nos. 10,005,080, 9,557,295, 8,501,921, 6,600,026, and 6,740,518, the disclosures of which are herein incorporated by reference in their entireties.

[0049] Electrochemical detection and electrochemical detection systems may be further described in reference to the following non-limiting example. In some embodiments, target nucleic acids are extracted from samples and amplified (e.g., using polymerase chain reaction (PCR)). In some embodiments, the resulting double-stranded DNA is then digested (e.g., by an exonuclease) to create a single-stranded DNA. In some embodiments, the single stranded amplicons are first annealed to labeled signal probes, e.g., ferrocene-conjugated signal probes (see FIGS. 1A and IB). In some embodiments, the resultant complexes maintain an amplicon-overhang that is complimentary to separate capture probes, which are bound to an electrode, such as a gold electrode (see FIGS. 1A and IB). In some embodiments, the signal probe-amplicon complexes bind to the capture probes, immobilizing the label, e.g., ferrocene, at the electrode as part of a three-nucleotide complex (see FIGS. 1A and IB). With this assembly, redox cycling, e.g., iron redox cyclic, is induced by an applied potential at the electrode, and the resultant faradaic current is assessed voltammeterically against the background capacitative current (see FIG. 1A). By probing this relationship at each electrode, the skilled artisan can rapidly assess which amplicons (and source pathogens, in turn) are present or absent.

[0050] DEVICE INCLUDING A TREATED ELECTRODE AND METHODS OF PREPARING TREATED ELECTRODES

[0051] The present disclosure provides for devices 1 that include a substrate having a plurality of electrodes. One such electrode 10 is depicted in FIG. 2. The term "electrode" as used herein refers to a composition, which, when connected to an electronic device, is able to sense a current or charge and convert it to a signal. Alternatively, an electrode can be defined as a composition which can apply a potential to and / or pass electrons to or from species in the solution. In some embodiments, the electrodes are gold electrodes. In some embodiments, the electrode is a gold electrode. In some embodiments, the electrodes are formed on a substrate. The substrate can comprise a wide variety of materials, as will be appreciated by those in the art. In some embodiments, the substrate comprises a printer circuit board. Printed circuit board materials are those that comprise an insulating substrate that is coated with a conducting layer and processed using lithography techniques, particularly photolithography techniques, to form the patterns of electrodes and interconnects (sometimes referred to in the art as interconnections or leads). Other non-limiting examples of suitable substrates include fiberglass, teflon, ceramics, glass, silicon, mica, plastic (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethanes, Teflon™, and derivatives thereof, etc.), GETEK (a blend of polypropylene oxide and fiberglass), etc.

[0052] In some embodiments, and as described further herein, each electrode (such as a gold electrode) includes a self-assembled monolayer including one or more species, such as one or more capture probes (described further herein). In some embodiments, each electrode includes an interconnection which is attached to the electrode at one end and is ultimately attached to a device that can control the electrode. In this manner, and in some embodiments, each electrode is independently addressable. In some embodiments, the substrate can be part of a larger device comprising a detection chamber that exposes a given volume of sample to the detection electrode. Generally, the detection chamber ranges from about InL to ImL, such as from about lOpL to 500pL. As will be appreciated by those in the art, depending on the experimental conditions and assay, smaller or larger volumes may be used. In some embodiments, the detection chamber and electrode are part of a cartridge that can be placed into a device comprising electronic components (an AC / DC voltage source, an ammeter, a processor, a readout display, temperature controller, light source, etc.). In this embodiment, the interconnections from each electrode are positioned such that upon insertion of the cartridge into the device, connections between the electrodes and the electronic components are established.

[0053] Gold electrodes may be prepared according to methods known in the art. In some embodiments, high purity gold is utilized, and it may be deposited on a surface via vacuum deposition processes (sputtering and evaporation) or solution deposition (electroplating or electroless processes). In those embodiments where electroplating is utilized, the substrate must initially comprise a conductive material. For instance, if fiberglass circuit boards are utilized, they are frequently provided with a conductive foil, such as a copper foil. In some embodiments, depending on the substrate, an adhesion layer is introduced between the substrate and the gold electrode in order to ensure good mechanical stability. In some embodiments, an adhesion layer comprises a metal, such as chromium, titanium, titanium / tungsten, tantalum, nickel, or palladium, may be first deposited (e.g., by sputtering and evaporation, electroplating, etc.). In some embodiments, the adhesion layer is from about 100 A thick to about 25 microns (1000 microinches). In embodiments where the adhesion metal is electrochemically active, the electrode metal must be coated at a thickness that prevents "bleed-through." On the other hand, if the adhesion metal is not electrochemically active, the electrode metal may be thinner. In some embodiments, the electrode metal is deposited at a thicknesses ranging from about 500 A to about 5 microns (i.e., about 2 microinches to about 200 microinches), such as from about 0.75 microns to about 2 microns (i.e., about 30 microinches to about 80 microinches), such as between about 1 micron to about 2 microns (i.e., about 40 microinches to about 80 microinches), such as between about 1.25 microns to about 2 microns (i.e., about 50 microinches to about 80 microinches), such as between about 1 micron to about 1.75 microns (i.e., about 40 microinches to about 70 microinches). In general, the electrode metal is deposited to make electrodes ranging in size from about 5 microns to about 5 mm in diameter, such as from about 100 to about 250 microns.

[0054] Table 1: Non-limiting examples of suitable treatment compositions.

[0055] In some embodiments, the electrodes are treated by contacting the electrodes with a treatment composition for a predetermined amount of time. In some embodiments, the treatment composition comprises hydrochloric acid and hydrogen peroxide. In other embodiments, the treatment composition comprises hydrochloric acid, hydrogen peroxide, and water. In some embodiments, the hydrochloric acid prior to addition to the composition is concentrated hydrochloric acid (e.g., 12. IM). In some embodiments, the concentration of hydrochloric acid in aqueous solution is between about 0.1N and about 2.5N (i.e., between about 0.1M and about 2.5M). In some embodiments, the hydrogen peroxide is 10% to 50% w / w in water. In other embodiments, the hydrogen peroxide is 20% to 40% w / w in water. In yet other embodiments, the hydrogen peroxide is 25% to 35% w / w in water. In other embodiments, the hydrogen peroxide is about 25% w / w in water, such as about 26% w / w in water, such as about 27% w / w in water, such as about 28% w / w in water, such as about 29% w / w in water, such as about 30% w / w in water, such as about 31% w / w in water, such as about 32% w / w in water, such as about 33% w / w in water, such as about 34% w / w in water, such as about 35% w / w in water, etc. A non-limiting treatment composition comprises hydrochloric acid (cone. 37% w / w, 12.1 M), hydrogen peroxide (30% w / w in water, 9.8 M), and water (distilled, deionized). Other non-limiting examples of treatment compositions are set forth in Table 1.

[0056] In some embodiments, the treatment composition is incubated with the electrode for a time period ranging from between about 8 hours to about 24 hours, such as between about 10 hours to about 22 hours, such as between about 12 hours to about 20 hours. In some embodiments, the treatment composition is incubated with the electrode for a time period of about 12 hours, such as about 13 hours, such as about 14 hours, such as about 15 hours, such as about 6 hours, such as about 7 hours, such as about 18 hours, such as about 19 hours, such as about 20 hours, etc. In some embodiments, a treatment composition comprising hydrochloric acid, hydrogen peroxide (30% w / w in water), and water (distilled, deionized) is incubated with an electrode for between about 14 hours and about 18 hours. Following treatment of the electrode with the treatment composition for the predetermined amount of time, the treated electrode may be washed one or more times (such as three times) with distilled, deionized water. The treated electrode may then be further processed, e.g., modified to include a selfassembled monolayer including one or more components. The roughness of an electrode surface may be measured by atomic force microscopy (AFM), which measures the height variation of the electrode surface (see, e.g., FIG. 4). In some embodiments, electrode surfaces treated in accordance with the present disclosure have a surface roughness (Ra) of about HOnm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope, such as a surface roughness (Ra) of about 120nm to about 150nm, such as a surface roughness (Ra) of about 120nm to about 140nm. In contrast, untreated electrodes, i.e., those not treated with a treatment composition in accordance with the methods described herein, have a surface roughness (Ra) of about 150nm to about 200nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope. In some embodiments, the surface roughness measurements are made using atomic force microscopy instrument with an EBD NCHR High-density carbon tip (about 7nm radius).

[0057] Without wishing to be bound by any particular theory, it is believed that a treated electrode surface (versus an untreated electrode surface) facilitates a more uniform or consistent distribution of selfassembled monolayer components, such as capture probes, conjugated to, adhered to, or adsorbed onto the surface of the treated electrode. Again, and without wishing to be bound by any particular theory, it is believed that a more uniform or consistent distribution of capture probes on a treated electrode's surface provides better access to the capture probes, such as by signal probes and or single stranded target nucleic acid molecules (see FIG. IB).

[0058] The comparatively improved surface uniformity of treated electrodes versus untreated electrodes has been shown herein to lead to a reduction in signal variability (see, e.g., FIG. 5). Indeed, measurements made using electrodes, e.g., gold electrodes, which have been treated in accordance with the methods described herein demonstrate that those electrodes that have been treated with the disclosed treatment compositions have comparatively reduced signal variability as compared with those electrodes that have not been treated (see, e.g., FIG. 5).

[0059] In some embodiments, the device is a printed circuit board with gold electrodes as the working electrode, along with reference and counter electrode. In some embodiments, an external potentiostat controls the voltage between working electrode (where the ferrocene or osmium reaction occurs) and the reference electrode. In some embodiments, by sweeping the potential across the oxidation and reduction ranges of ferrocene or osmium, the system can detect the redox peaks. As ferrocene or osmium labels oxidize and reduce, electrons are transferred between the gold electrode and the ferrocene molecules, generating a current that the potentiostat records. For instance, mean, standard deviation, and CV measurements made with a device 1 including a treated gold electrode 10 (including a capture probe 11 (see FIG. 2) having a signal -probe amplicon complex bound thereto, where the signal probe includes a ferrocene label) are about 6.2nA to about 7.2nA (mean), about 0.70nA to about 0.80nA (std dev), and about 9% to about 16% (CV), respectively. In comparison, mean, standard deviation, and CV measurements made with a device 1 including an untreated gold electrode 10 (including a capture probe 11 having a signal -probe amplicon complex bound thereto, where the signal probe includes a ferrocene label) are about 2.5nA to about 3.5nA (mean), about InA to about 1.8nA (std dev), and about 40% to about 90% (CV), respectively. Unexpectedly, signal variability is improved when a treated electrode is utilized as compared with an untreated electrode.

[0060] By way of another example, mean, standard deviation, and CV measurements made with device 1 including a treated gold electrode 10 (including a capture probe 11 having a signal -probe amplicon complex bound thereto, where the capture probe includes an osmium label) are about 1.2nA to about 2nA (mean), about 0.25nA to about 0.35nA (std dev), and about 15% to about 20% (CV), respectively. In comparison, mean, standard deviation, and CV measurements made with a device 1 including an untreated gold electrode 10 (including a capture probe 11 having a signal -probe amplicon complex bound thereto, where the capture probe includes an osmium label) are about 0.5nA to about InA (mean), about 0.5nA to about 0.9nA (std dev), and about 40% to about 90% (CV), respectively. Unexpectedly, signal variability is improved when a treated electrode is utilized as compared with an untreated electrode.

[0061] Based on the foregoing data, and without wishing to be bound by any particular theory, it is believed that the enhanced surface "smoothness" minimized the variability in the distribution of the capture probe, leading to more uniform hybridization and reduced signal variability (see, e.g., FIG. 5).

[0062] ELECTRODES INCLUDING A PLURALITY OF CAPTURE PROBES

[0063] As described herein, the electrodes 10 (treated or untreated) may include a plurality of capture probes 11 conjugated to, adhered to, and / or adsorbed onto the surface of the treated or untreated electrode. Following the preparation of the electrode (e.g., gold electrode, which may be treated or untreated) a self-assembled monolayer may be formed on the surface of the treated electrode. The terms "monolayer" or "self-assembled monolayer" refer to a relatively ordered assembly of molecules spontaneously chemisorbed on a surface, in which the molecules are oriented approximately parallel to each other and roughly perpendicular to the surface. Each of the molecules includes a functional group that adheres to the surface, and a portion that interacts with neighboring molecules in the monolayer to form the relatively ordered array.

[0064] In some embodiments, the self-assembled monolayer includes a plurality of capture probes. In other embodiments, the self-assembled monolayer includes two or more different species. For instance, the self-assembled monolayer may include (i) a plurality of capture probes; and (ii) one or more additional species. In some embodiments, the self-assembled monolayer may include (i) a plurality of capture probes of a first species; and (ii) one or more plurality of capture probes of additional species (i.e., of a second, third, fourth, ... species). In some embodiments, the capture probes are modified either at the 3' end or the 5' end with a disulfide linker for covalent attachment to an electrode surface. Such disulfide linkers and methods of attachment are described in U.S. Pat. No. 6,753,143 and U.S. Pat. No. 7,820,391, each of which is incorporated by reference herein in their entireties. For instance, capture probes can be adhered to electrodes or other substrate surfaces directly or indirectly, covalently, or noncovalently, using a variety of well-known techniques. See, e.g., Ch. 13, Chemically Modified Electrodes, Martin and Foss, pp. 403-442, Laboratory Techniques in Electroanalytical Chemistry; 2d Ed., Kissinger and Heineman, Eds., MARCEL DEKKER, INC. (1996); Biochip Technology, Cheng and Kricka, Eds. George H. Buchanan Printing Company, Bridgeport, N.J. (2001).

[0065] In some embodiments, a deposition solution including one or more self-assembled monolayer species (e.g., the plurality of capture probes and / or one or more additional species) is prepared which may then be introduced to the surface of an electrode. In some embodiments, the deposition solution is introduced to an electrode treated with one of the treatment compositions of the present disclosure. In other embodiments, the deposition solution is introduced to an untreated electrode.

[0066] In some embodiments, the deposition solution includes one or more carboxylic acid based small molecules such as, but not limited to, citrate, acetate, and / or formate. For instance, between about 15nL to about 25nL of the carboxylic acid based small molecule is introduced to a solution including the capture probe and permitted to incubate for a time period ranging from between about 3 minutes to about 30 minutes, such as between about 3 minutes to about 20 minutes, such as about 3 minutes to about 10 minutes, such as about 3 minutes to about 5 minutes. In other embodiments, about 15nL, such as about 16nL, such as about 17nL, such as about 18nL, such as about 19nL, such as about 20nL, such as about 21nL, such as about 22nL, such as about 23nL, such as about 24nL, or such as about 25nL of the carboxylic acid based small molecule is introduced to a solution including the capture probe and permitted to incubate for a time period ranging from between about 3 minutes to about 30 minutes, such as between about 3 minutes to about 20 minutes, such as about 3 minutes to about 10 minutes, such as about 3 minutes to about 5 minutes.

[0067] In some embodiments, the electrode surface is exposed to the deposition solutioning including the selfassembled monolayer species, such as by solution deposition, gas phase deposition, microcontact printing, spray deposition, deposition using neat components, etc.

[0068] Applying the Deposition Solution

[0069] In some embodiments, a drop deposition technique is used to add the monolayer forming species to the surface of the electrode. Drop deposition techniques are well known for making "spot" arrays. Such a technique is performed to add a different composition (i.e., a different monolayer forming species) to the surface of each electrode. Alternatively, the self-assembled monolayer species may be identical for each electrode, and this may be accomplished using a drop deposition technique or the immersion of the entire substrate or a surface of the substrate into a solution.

[0070] In some embodiments, spotting the deposition solution onto the electrodes is performed using any number of known spotting systems, generally by placing the boards on an X-Y table, preferably in a humidity chamber. The size of the spotting drop will vary with the size of the electrodes on the boards and the equipment used for delivery of the solution; for example, for 250pM size electrodes, a 30 nanoliter drop is used. The volume should be sufficient to cover the electrode surface completely. The drop is incubated at room temperature for a period of time (sec to overnight, with 5 minutes preferred) and then the drop is removed by rinsing in a Milli-Q water bath. The boards are then optionally treated with a second deposition solution, generally comprising insulator in organic solvent, preferably acetonitrile, by immersion in a 45°C. bath. After 30 minutes, the boards are removed and immersed in an acetonitrile bath for 30 seconds followed by a milli-Q water bath for 30 seconds. The boards are dried under a stream of nitrogen. Preferably, only the water rinse is employed.

[0071] In some embodiments, a salt buffer is dispensed (such as between about IpL to about ImL) onto the gold electrode, such as through a pipette or dispensing machine or apparatus and dried, such as under vacuum. In some embodiments, the dispensed salt buffer is rehydrated with a mixture of target and signal probes and used as a hybridization solution and an electrolyte for signal detection.

[0072] It is believed that by adding a carboxylic acid based small molecule to any deposition solution including the self-assembled monolayer species, the spacing between adjacent capture probes of the plurality of capture probes conjugated to, adhered to, or adsorbed onto the surface of the electrode may be better controlled. Indeed, measurements made using devices 1 including capture probes 11 which were prepared using the deposition solutions of the present disclosure have comparatively reduced signal variability as compared with devices including capture probes prepared according to prior art methods (see, e.g., FIG. 6). For instance, mean, standard deviation, and CV measurements made with a device 1 including a gold electrode 10 including a plurality of capture probes 11 prepared in the presence of a carboxylic acid based small molecule (including signal-probe amplicon complexes bound to the capture probes, where the signal probes includes a ferrocene label) are about 200nA to about 400nA (mean), about 20nA to about 50nA (std dev), and about 8% to about 15% (CV), respectively. In comparison, mean, standard deviation, and CV measurements made with a device 1 including a gold electrode 10 including a plurality of capture probes 11 prepared without the use of a carboxylic acid based small molecule (including a signal-probe amplicon complexes bound to the capture probes, where the signal probes includes a ferrocene label) are about lOOnA to about 300nA (mean), about 30nA to about 70nA (std dev), and about 20% to about 40% (CV), respectively. Unexpectedly, signal variability is improved using a device including capture probes where were prepared using the deposition solutions of the present disclosure.

[0073] For instance, mean, standard deviation, and CV measurements made with a device 1 including a gold electrode 10 including a plurality of capture probes 11 prepared in the presence of a carboxylic acid based small molecule (including signal-probe amplicon complexes bound to the capture probes, where the capture probes includes an osmium label) are about 20nA to about 30nA (mean), about 3.5nA to about 7nA (std dev), and about 15% to about 20% (CV), respectively. In comparison, mean, standard deviation, and CV measurements made with a device 1 including a gold electrode 10 including a plurality of capture probes 11 prepared without the use of a carboxylic acid based small molecule (including signal-probe amplicon complexes bound to the capture probes, where the capture probes includes an osmium label) are about lOnA to about 20nA (mean), about 3.5nA to about 7nA (std dev), and about 20% to about 40% (CV), respectively. Unexpectedly, signal variability is improved using a device including capture probes where were prepared using the deposition solutions of the present disclosure.

[0074] ELECTROLYTE LAYER

[0075] With reference to FIG. 2, an electrolyte layer 12 may be disposed onto the capture probes 11. In some embodiments, the electrolyte layer 12 is prepared by depositing an electrolyte solution on the electrode 10 including the capture probes 11; and permitting the deposited electrolyte solution to dry. In some embodiments, the electrode 10 is treated with a treatment composition in accordance with the claimed invention. In other embodiments, the electrode 10 is not treated with a treatment composition in accordance with the claimed invention. In some embodiments, the capture probes 11 are prepared / deposited using a solution including a carboxylic acid based small molecule such as described herein (see FIG.8). In other embodiments, the capture probes 11 are not prepared / deposited using a solution including a carboxylic acid based small molecule. In some embodiments, an electrolyte solution is dispensed (such as between about IpL to about ImL) onto the gold electrode, such as through a pipette or dispensing machine or apparatus and dried, such as under vacuum. In some embodiments, the dispensed electrolyte solution is rehydrated with a mixture of target and signal probes and used as a hybridization solution and an electrolyte for signal detection. In some embodiments, the electrolyte solution comprises a buffer, magnesium chloride, a stabilizer, a non-ionic surfactant, and water. In some embodiments, the buffer is selected from TRIS buffer, HEPES buffer, and phosphate buffer. In certain embodiments, the buffer is TRIS buffer. In some embodiments, the stabilizer is a sugar. In some embodiments, the sugar is fructose or a non-reducing sugar selected from trehalose and sucrose. In some embodiments, the non-ionic surfactant is selected from a group consisting of ethoxylated alcohols, alkyl polyglycosides, ethylene oxide / propylene oxide block copolymers, and acetylenic diol-based surfactants, particularly ethoxylated acetylenic diols (such as, e.g., Dynol™ 604). Alternatively, alkyl sulfates such as sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other salts of organosulfates may be used as surfactant.

[0076] In some embodiments, the electrolyte solution further includes a zwitterionic surfactant. In some embodiments, the zwitterionic surfactant can be, for example, a sultaine, a betaine, a phospholipid, or a sphingolipid.

[0077] In some embodiments, zwitterionic surfactants include betaines, including alkyl dimethyl betaine and cocodimethyl amidopropyl betaine, amine oxides (for example Cs to Cis, or Ci2to Cis amine oxides), and sulfo and hydroxy betaines, such as N-alkyl-N,N-dimethylammino-l -propane sulfonate where the alkyl group can be Csto Cis. Suitable amine oxides include alkyl dimethyl amine oxide or alkyl amido propyl dimethyl amine oxide.

[0078] In some embodiments, the zwitterionic surfactant can be a sulfobetaine (SB), amidosulfobetaine (ASB) or octylbenzyl amido sufobetaine. In some embodiments, the zwitterionic surfactant can be, for example, 3 -((3 -cholamidopropyl)dimethylammonio)-l -propanesulfonate (CHAPS), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, phosphatidylserine, phosphatidylethanolamine, phosphotidylcholine, or sphingomyelin.

[0079] In some embodiments, the zwitterionic surfactant can be an aminosulfobetaine or aromatic aminosulfobetaine.

[0080] In some embodiments, the zwitterionic surfactant is 3-(dimethyl(3-(4-octylbenzamido)propyl) ammonio) propane- 1 -sulfonate (ASB-C80).

[0081] Some non-limiting examples of zwitterionic surfactants include 3-(N,N-Dimethyltetradecylammonio) propanesulfonate (SB3 - 14), 3 -(4-Heptyl)phenyl-3 -hydroxypropyl)dimethylammoniopropanesulfonate (C7BzO), 3-(decyldimethylammonio) propanesulfonate inner salt (SB3-10), 3-(dodecyldimethyl- ammonio) propanesulfonate inner salt (SB3-12), 3-(N,N-dimethyloctadecylammonio) propanesulfonate (SB3-18), 3-(N,N-dimethyl-octylammonio) propanesulfonate inner salt (SB3-8), 3-(N,N- dimethylpalmitylammonio) propanesulfonate (SB3-16), 3-[N,N-dimethyl(3-myristoylaminopropyl) ammonio]propane-sulfonate (ASB-14), CHAPS, CHAPSO, acetylated lecithin, alkyl(C 12-30) dialkylamine-N-oxide apricotamidopropyl betaine, babassuamidopropyl betaine, behenyl betaine, bis 2 -hydroxy ethyl tallow glycinate, Cl 2- 14 alkyl dimethyl betaine, canolamidopropyl betaine, capric / caprylic amidopropyl betaine, capryloamidopropyl betaine, cetyl betaine, 3-[(Cocamidocthyl) dimethylammonio]-2-hydroxypropanesulfonate, 3-[(Cocamidocthyl)dimethyl-ammonio]propane- sulfonate, cocamidopropyl betaine, cocamidopropyl dimethylamino-hydroxypropyl hydrolyzed collagen, N-[3-cocamido)-propyl]-N,N-dimethyl betaine, potassium salt, cocamidopropyl hydroxysultaine, cocamidopropyl sulfobetaine, cocaminobutyric acid, cocaminopropionic acid, cocoamphodipropionic acid, coco-betaine, cocodimethylammonium-3 -sulfopropylbetaine, cocoiminodiglycinate, cocoiminodipropionate, coco / olcamidopropyl betaine, cocoyl sarcosinamide DEA, DEA-cocoamphodipropionate, dihydroxyethyl tallow glycinate, dimethicone propyl PG- betaine, N,N-dimethyl-N-lauric acid-amidopropyl-N-(3-sulfopropyl)-ammonium betaine, N,N- dimethyl-N-myristyl-N-(3-sulfopropyl)-ammonium betaine, N,N-dimethyl-N-palmityl-N-(3- sulfopropyl)-ammonium betaine, N,N-dimethyl-N-stearamidopropyl-N-(3-sulfopropyl)-ammonium betaine, N,N-dimethyl-N-stearyl-N-(3-sulfopropyl)-ammonium betaine, N,N-dimethyl-N- tallow-N- (3-sulfopropyl)-ammonium betaine, disodium caproamphodiacetate, disodium caproampho- dipropionate, disodium capryloamphodiacetate, disodium capryloamphodipropionate, disodium cocoamphodiacetate, disodium cocoamphodipropionate, disodium isostearoamphodipropionate, disodium laureth-5 carboxyamphodiacetate, disodium lauriminodipropionate, disodium lauroampho- diacetate, disodium lauroamphodipropionate, disodium octyl b-iminodipropionate, disodium olcoamphodiacetate, disodium olcoamphodipropionate, disodium PPG-2-isodeceth-7 carboxyamphodiacetate, disodium soyamphodiacetate, disodium stearoamphodiacetate, disodium tallamphodipropionate, disodium tallowamphodiacetate, disodium tallowiminodipropionate, disodium wheatgermamphodiacetate, N,N-distearyl-N-methyl-N-(3 -sulfopropyl)-ammonium betaine, crucamidopropyl hydroxysultaine, ethylhexyl dipropionate, ethyl hydroxymethyl oleyl oxazoline, ethyl PEG- 15 cocamine sulfate, hydrogenated lecithin, hydrolyzed protein, isostearamidopropyl betaine, 3-[(Lauramidocthyl)dimethylammonio]-2-hydroxypropane-sulfonate, 3-[(Lauramidocthyl) dimethylammonio]propanesulfonate, lauramido-propyl betaine, lauramidopropyl dimethyl betaine, lauraminopropionic acid, lauroamphodipropionic acid, lauroyl lysine, lauryl betaine, lauryl hydroxysultaine, lauryl sultaine, linolcamidopropyl betaine, lysolecithin, milk lipid amidopropyl betaine, myristamidopropyl betaine, octyl dipropionate, octyliminodipropionate, n-octyl-N,N- dimethyl-3 -ammonio- 1 -propanesulfonate, n-decyl-N,N-dimethyl-3 -ammonio- 1 -propanesulfonate, n- dodecyl-N,N-dimethyl-3-ammonio-l-propane-sulfonate, n-tetradecyl-N,N-dimethyl-3 -ammonio- 1- propanesulfonate, n-hexadecyl-N,N-dimethyl-3 -ammonio- 1 -propanesulfonate, n-octadecyl-N,N- dimethyl-3-ammonio-l-propane-sulfonate, oleamidopropyl betaine, oleyl betaine, 4,4(5H)- oxazoledimethanol, 2-(heptadecenyl) betaine, palmitamidopropyl betaine, palmitamine oxide, PMAL- C6, PMAL-C12, PMAL-C16, ricinolcamidopropyl betaine, ricinoleamidopropyl betaine / IPDI copolymer, sesamidopropyl betaine, sodium Cl 2- 15 alkoxypropyl iminodipropionate, sodium caproamphoacetate, sodium capryloamphoacetate, sodium capryloamphohydroxypropyl sulfonate, sodium capryloamphopropionate, sodium carboxymethyl tallow polypropylamine, sodium cocaminopropionate, sodium cocoamphoacetate, sodium cocoamphohydroxypropyl sulfonate, sodium cocoamphopropionate, sodium dicarboxyethyl cocophosphoethyl imidazoline, sodium hydrogenated tallow dimethyl glycinate, sodium isostearoamphopropionate, sodium lauriminodipropionate, sodium lauroamphoacetate, sodium oleoamphohydroxypropylsulfonate, sodium oleoamphopropionate, sodium stearoamphoacetate, sodium tallamphopropionate, soyamidopropyl betaine, stearyl betaine, 3- [(Stearamidoethyl)dimethylammonio] -2 -hydroxypropanesulfonate, 3-[(Stearamidoethyl)-dimethyl- ammonio]propanesulfonate, tallowamidopropyl hydroxysultaine, tallowamphopoly-carboxypropionic acid, trisodium lauroampho PG-acetate phosphate chloride, undecylenamidopropyl betaine, and wheat germamidopropyl betaine.

[0082] In some embodiments, the zwitterionic surfactant is selected from SB 3-10, SB 3-12, SB 3-14, ASB- 14, ASB-16, or ASB-C80.

[0083] In some embodiments, the amount of zwitterionic surfactant in the electrolyte solution ranges from between about 1% to about 10% by total volume of the electrolyte solution. In other embodiments, the amount of zwitterionic surfactant in the electrolyte solution ranges from between about 2% to about 5% by total volume of the electrolyte solution.

[0084] Among the suitable nonionic surfactants are condensation products of Q-C30 alcohols with sugar or starch polymers. These compounds can be represented by the formula (S)n-O-R, wherein S is a sugar moiety such as glucose, fructose, mannose, and galactose; n is an integer of from about 1 to about 1000, and R is Cs-Cso alkyl. Examples of suitable Cs-Cso alcohols from which the R group may be derived include decyl alcohol, cetyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, oleyl alcohol, and the like. Specific examples of these surfactants include decyl polyglucoside and lauryl polyglucoside.

[0085] Other suitable nonionic surfactants include the condensation products of alkylene oxides with fatty acids (i.e., alkylene oxide esters of fatty acids). These materials have the general formula RCO(X)nOH, wherein R is a C10-C30 alkyl, X is -OCH2CH2- (derived from ethylene oxide) or -OCH2CHCH3- (derived from propylene oxide), and n is an integer from about 1 to about 200.

[0086] Yet other suitable nonionic surfactants are the condensation products of alkylene oxides with fatty acids (i.e., alkylene oxide diesters of fatty acids) having the formula RCO(X)nOOCR, wherein R is a C10-C30 alkyl, X is -OCH2CH2- (derived from ethylene oxide) or -OCH2CHCH3- (derived from propylene oxide), and n is an integer from about 1 to about 200. Yet other nonionic surfactants are the condensation products of alkylene oxides with fatty alcohols (i.e., alkylene oxide ethers of fatty alcohols) having the general formula R(X)nOR', wherein R is C10-C30 alkyl, n is an integer from about 1 to about 200, and R' is H or a C10-C30 alkyl.

[0087] Still other nonionic surfactants are the compounds having the formula RCO(X)nOR' wherein R and R' are C10-C30 alkyl, X is -OCH2CH2- (derived from ethylene oxide) or -OCH2CHCH3- (derived from propylene oxide), and n is an integer from about 1 to about 200. Examples of alkylene oxide-derived nonionic surfactants include ceteth-1, ceteth-2, ceteth-6, ceteth-10, ceteth-12, ceteraeth-2, ceteareth-6, ceteareth-10, ceteareth-12, steareth-1, steareth-2, stearteth-6, steareth-10, steareth-12, PEG-2 stearate, PEG4 stearate, PEG6 stearate, PEG- 10 stearate, PEG- 12 stearate, PEG-20 glyceryl stearate, PEG-80 glyceryl tallowate, PPG- 10 glyceryl stearate, PEG-30 glyceryl cocoate, PEG-80 glyceryl cocoate, PEG-200 glyceryl tallowate, PEG-8 dilaurate, PEG- 10 distearate, and mixtures thereof. Still other useful nonionic surfactants include polyhydroxy fatty acid amides disclosed, for example, in U.S. Pat. Nos. 2,965,576, 2,703,798, and 1,985,424, which are incorporated herein by reference.

[0088] In some embodiments, the amount of non-ionic surfactant in the electrolyte solution ranges from between about 0.01% to about 0.1% by total volume of the electrolyte solution.

[0089] In some embodiments, the electrolyte solution can include a mixture of two or more surfactants in any ratio. In some embodiments, a ratio of an amount of non-ionic surfactant to zwitterionic surfactant in the electrolyte solution ranges from between about 0.005:1 to about 2:1. In some embodiments, the ratio nonionic surfactant to zwitterionic surfactant is from about 0.01:1 to about 5:1. In other embodiments, the ratio is from about 0.025:1 to about 4:1, such as from about 0.05:1 to about 3:1. In yet other embodiments, the ratio is from about 0.06: 1 to about 2.5: 1, and such as from about 0.01 : 1 to about 2:1.

[0090] In some embodiments, the pH of the electrolyte solution ranges from between about 7.5 to about 8.5, such as from about 7.6 to about 8.4, such as from, about 7.7 to about 8.3, such as from about 7.8 to about 8.2, or such as from about 7.9 to about 8.0. In some embodiments, the pH of the electrolyte solution is about 7.7, such as about 7.8, such as about 7.9, such as about 8.0, such as about 8.1, such as about 8.3, or such as about 8.3.

[0091] In some embodiments, the amount of buffer in the electrolyte solution ranges from about 600mM to about 700mM, such as about from 620mM to about 680mM, such as about from 630mM to about 670mM, such as from about 640mM to about 660mM. In some embodiments, the amount of buffer in the electrolyte solution is about 635mM, such as about 640mM, such as about 645mM, such as about 650mM, such as about 655mM.

[0092] In some embodiments, the amount of magnesium chloride (MgCb)in the electrolyte solution ranges from about 600mM to about 700mM, such as about from 620mM to about 680mM, such as about from 630mM to about 670mM, or such as from about 640mM to about 660mM. In some embodiments, the amount of magnesium chloride in the electrolyte solution is about 635mM, such as about 640mM, such as about 645mM, such as about 650mM, or such as about 655mM. In some embodiments, the amount of sugar (including non-reducing sugar) in the electrolyte solution ranges from about 0.16mM to about 0.28mM, such as about from 0.17mM to about 0.27mM, such as about from 0.18mM to about 0.26mM, such as from about 0.19mM to about 0.25mM, such as about from 0.20mM to about 0.24mM, or such as about from 0.21mM to about 0.23mM. In some embodiments, the amount of sugar (including nonreducing sugar) in the electrolyte solution is about 0.20mM, such as about 0.21mM, such as about 0.22mM, such as about 0.22mM, or such as about 0.24mM. In some embodiments, the amount of surfactant comprises between about 2.5% to about 7% by total volume of the electrolyte solution, such as between about 3% to about 5% or such as between about 3.5% to about 4%. It is believed that the inclusion of an electrolyte layer prepared using one of the electrolyte compositions of the present disclosure in a device (see FIG. 2) permits improved electron transfer between a label (e.g., a ferrocene or osmium label) and the electrode (e.g., a gold electrode). This improved electron transfer is believed to improve signal variability as compared with devices that do not include an electrolyte layer prepared using one of the electrolyte compositions of the present disclosure (see, e.g., FIG. 7). Without wishing to be bound by any particular theory, it is believed that the disclosed electrolyte facilitates improved hybridization, such as permitting stable electron transfer. For instance, mean, standard deviation, and CV measurements made with a device 1 including a gold electrode 10 including a plurality of capture probes 11 and further including an electrolyte layer 12 prepared using one of the electrolyte compositions of the present disclosure (including signal-probe amplicon complexes bound to the capture probes, where the signal probes includes a ferrocene label) are about 70nA to about 150nA (mean), about 8nA to about 20nA (std dev), and about 10% to about 20% (CV), respectively. In comparison, mean, standard deviation, and CV measurements made with a device 1 including a gold electrode 10 including a plurality of capture probes 11 and further including an electrolyte layer 12, but not an electrolyte layer prepared using one of the electrolyte compositions of the present disclosure (including a signal-probe amplicon complexes bound to the capture probes, where the signal probes includes a ferrocene label) are about 150nA to about 250nA (mean), about 40nA to about 90nA (std dev), and about 20% to about 50% (CV), respectively. Unexpectedly, signal variability is improved with devices utilizing an electrolyte layer prepared in accordance with the present disclosure (see, e.g., FIG. 7). For instance, mean, standard deviation, and CV measurements made with a device 1 including a gold electrode 10 including a plurality of capture probes 11 and further including an electrolyte layer 12 prepared using one of the electrolyte compositions of the present disclosure (including signal-probe amplicon complexes bound to the capture probes, where the capture probes includes an osmium label) are about lOnA to about 20nA (mean), about 3nA to about 8nA (std dev), and about 20% to about 35% (CV), respectively.

[0093] EXAMPLES

[0094] To assess whether additional improvements could be achieved beyond the optimized electrolyte formulation, a zwitterionic (ZI) surfactant was introduced into the system. Following capture probe immobilization and the sequential addition of target and signal probes, the modified electrolyte containing the surfactant was applied during the detection process. The electrochemical measurements revealed that the level of signal variability was essentially unchanged compared to the baseline optimized electrolyte, suggesting that the reproducibility of the assay was already maximized under the modified conditions. Although no measurable reduction in variability was observed, the introduction of the ZI surfactant produced a distinct improvement in surface behavior. The surfactant lowered the surface energy of the electrolyte, enabling the droplet to spread more evenly across the gold electrode surface (FIG. 9). This uniform spreading reduced the occurrence of incomplete wetting, edge effects, or localized film irregularities that often arise during fabrication. As a consequence, the dispensing of the electrolyte process became more robust, less sensitive to operator handling, and more tolerant of small fluctuations in environmental or processing parameters. From a manufacturing perspective, these improvements are particularly important. By ensuring reproducible spreading and consistent coverage, the ZI surfactant simplified device assembly and facilitated scale-up, ultimately enhancing manufacturability without altering the core electrochemical performance. Thus, while the surfactant did not contribute directly to further reducing signal variability, it provided a significant process-level advantage by stabilizing the coating behavior and supporting reliable mass production.

[0095] For instance, mean, standard deviation, and CV measurements made with a device 1 including a gold electrode 10 including a plurality of capture probes 11 and further including an electrolyte layer 12 prepared using one of the electrolyte compositions of the present disclosure (including signal-probe amplicon complexes bound to the capture probes, where the signal probes includes a ferrocene label) are about 150nA to about 250nA (mean), about 20nA to about 40nA (std dev), and about 10% to about 20% (CV), respectively. In comparison, mean, standard deviation, and CV measurements made with a device 1 including a gold electrode 10 including a plurality of capture probes 11 and further including an electrolyte layer 12, but not an electrolyte layer prepared using one of the electrolyte compositions of the present disclosure (including a signal-probe amplicon complexes bound to the capture probes, where the signal probes includes a ferrocene label) are about 200nA to about 450nA (mean), about 60nA to about lOOnA (std dev), and about 20% to about 50% (CV), respectively, (see, e.g., FIG. 10).

[0096] Although the present disclosure has been described with reference to several illustrative embodiments, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More particularly, reasonable variations and modifications are possible in the component parts and / or arrangements of the subject combination arrangement within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the disclosure. In addition to variations and modifications in the component parts and / or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

CLAIMS1. A composition comprising hydrochloric acid, hydrogen peroxide, and water.

2. The composition of claim 1, wherein the hydrochloric acid prior to addition to the composition is concentrated hydrochloric acid.

3. The composition of claim 2, wherein the concentration of the hydrochloric acid in the composition ranges from between about 0. IM and about 2.5M.

4. The composition of any one of claims 1 to 3, wherein the hydrogen peroxide is 10% to 50% w / w in water.

5. The composition of claim 4, wherein the hydrogen peroxide is 20% to 40% w / w in water.

6. The composition of any one of claims 1 to 5, wherein a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 2.2M.

7. The composition of any one of claims 1 to 5, wherein a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.5M.

8. The composition of any one of claims 1 to 5, wherein a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.0M.

9. The composition of any one of claims 1 to 5, wherein a concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1.7M.

10. The composition of any one of claims 1 to 5, wherein a concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1.0M.

11. Use of the composition of any one of claims 1 to 10 in treating an electrode.

12. The use of claim 11, wherein the electrode is a gold electrode.

13. A method of preparing a treated gold electrode, comprising a. obtaining a gold electrode; b. incubating the obtained gold electrode with the composition of any one of claims 1 to 10 for a predetermined amount of time to provide the treated gold electrode.

14. The method of claim 13, wherein the predetermined amount of time ranges from between about 8 hours to about 24 hours.

15. The method of claim 13, wherein the predetermined amount of time ranges from between about 12 hours to about 20 hours.

16. The method of any one of claims 13 to 15 further comprising washing the electrode following the incubation with the composition of any one of claims 1 to 10.

17. The method of any one of claims 13 to 16 further comprising the step of forming a self-assembled monolayer on the surface of the treated gold electrode.

18. The method of any one of claims 13 to 17, wherein the treated gold electrode has a surface roughness (Ra) of between about HOnm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope.

19. The method of any one of claims 13 to 17, wherein the treated gold electrode has a surface roughness (Ra) of between about 120nm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope.

20. The method of any one of claims 13 to 17, wherein the treated gold electrode has a surface roughness (Ra) of between about 120nm to about 140nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope.

21. A gold surface having a surface roughness (Ra) of between about 1 lOnm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope, wherein the gold surface is prepared by contacting the gold surface with a composition comprising hydrochloric acid, hydrogen peroxide, and water for a predetermined amount of time.

22. The gold surface of claim 21, wherein the predetermined amount of time ranges from between about 8 hours to about 24 hours.

23. The gold surface of claim 21, wherein the predetermined amount of time ranges from between about 12 hours to about 20 hours.

24. The gold surface of any one of claims 21 to 23, wherein the hydrochloric acid prior to addition to the composition is concentrated hydrochloric acid.

25. The gold surface of claim 24, wherein the concentration of the hydrochloric acid in the composition ranges from between about 0. IM and about 2.5M.

26. The gold surface of any one of claims 21 to 25, wherein the hydrogen peroxide is 10% to 50% w / w in water.

27. The gold surface of any one of claims 21 to 25, wherein the hydrogen peroxide is 20% to 40% w / w in water.

28. The gold surface of any one of claims 21 to 27, wherein a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 2.2M.

29. The gold surface of any one of claims 21 to 27, wherein a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.5M.

30. The gold surface of any one of claims 21 to 27, wherein a concentration of the hydrochloric acid in the composition ranges from between about 0.3M to about 1.0M.

31. The gold surface of any one of claims 21 to 27, wherein a concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1.7M.

32. The gold surface of any one of claims 21 to 27, wherein a concentration of the hydrogen peroxide in the composition ranges from between about 0.2M to about 1.0M.

33. The gold surface of any one of claims 21 to 32, wherein the treated gold electrode has a surface roughness (Ra) of between about 120nm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope.

34. The gold surface of any one of claims 21 to 32, wherein the treated gold electrode has a surface roughness (Ra) of between about 120nm to about 140nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope.

35. The gold surface of any one of claims 21 to 35, wherein the gold surface further comprises a self-assembled monolayer.

36. The gold surface of any one of claims 21 to 36, wherein the gold surface further comprises a plurality of capture probes.

37. A detection system comprising the gold surface of any one of claims 21 to 36.

38. A deposition solution comprising one or more self-assembled monolayer species and at least one carboxylic acid based small molecule selected from the group consisting of citrate, acetate, and formate.

39. The deposition solution of claim 38, wherein the one or more self-assembled monolayer species comprises a plurality of capture probes.

40. A method of preparing a self-assembled monolayer on an electrode, wherein the method comprises incubating the electrode with a deposition solution comprising one or more capture probes and at least one carboxylic acid based small molecule selected from the group consisting of citrate, acetate, and formate, wherein the electrode is incubated with the deposition solution for a predetermined amount of time.

41. The method of claim 40, wherein the predetermined amount of time ranges from between 10 minutes to about 30 minutes.

42. The method of claim 40 to 41, wherein the carboxylic acid based small molecule is citrate.

43. The method of any one of claims 40 to 42, wherein the electrode is a gold electrode.

44. The method of claim 43, wherein the gold electrode has a surface roughness (Ra) of between about 1 lOnm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope.

45. The method of claim 44, wherein the gold electrode is treated by contacting the gold electrode with a composition comprising hydrochloric acid, hydrogen peroxide, and water for a predetermined amount of time.

46. The method of any one of claims 40 to 45 further comprising depositing an electrolyte layer on the formed self-assembled monolayer.

47. The method of claim 46, wherein the electrolyte layer comprises magnesium chloride.

48. The method of claim 46, wherein the electrolyte layer further comprises one or more of a buffer, a stabilizer, and a non-ionic surfactant.

49. An electrolyte solution comprising a buffer, magnesium chloride, a stabilizer, a non-ionic surfactant, an optional zwitterionic surfactant, and water.

50. The electrolyte solution of claim 49 further comprising a sugar stabilizer.

51. The electrolyte solution of claim 50, wherein the sugar stabilizer is fructose or a non-reducing sugar selected from trehalose and sucrose.

52. The electrolyte solution of any one of claims 49 to 51, wherein the electrolyte solution has a pH ranging from about 7.5 to about 8.

553. The electrolyte solution of any one of claims 49 to 51, wherein the electrolyte solution has a pH ranging from about 7.7 to about 8.

354. The electrolyte solution of any one of claims 49 to 53, wherein the amount of magnesium chloride in the electrolyte solution ranges from about 600mM to about 700mM.

55. The electrolyte solution of any one of claims 49 to 53, wherein the amount of buffer in the electrolyte solution ranges from about 600mM to about 700mM.

56. The electrolyte solution of any one of claims 49 to 53, wherein the amount of the non-ionic surfactant in the electrolyte solution ranges from between about 2.5% to about 7% by total volume of the electrolyte solution.

57. Use of the electrolyte solution of any one of claims 49 to 56 in the preparation of an electrolyte layer.

58. A method of preparing an electrolyte layer comprising dispensing the electrolyte solution of any one of claims 49 to 56 onto an electrode, wherein the electrode comprises one or more capture probes.

59. The method of claim 58, wherein the dispensed electrolyte solution is permitted to dry passively.

60. The method of claim 58, wherein the dispensed electrolyte solution is actively dried.

61. A biochip cartridge comprising: a substrate comprising: a printed circuit board comprising an electrowetting grid of electrodes forming a droplet pathway;an array of detection electrodes accessible to the droplet pathway, each comprising a selfassembled monolayer including one or more capture probes; and a plurality of interconnections from the electro wetting grid and the detection electrodes, wherein each electrode of the electro wetting grid of electrodes is a gold electrode each having a surface roughness (Ra) of between about HOnm to about 150nm when measured in an area of about 80pm x about 80pm (measured in air) with an atomic force microscope.

62. The biochip cartridge of claim 61, wherein each gold electrode is prepared by contacting each of the gold electrodes with a composition comprising hydrochloric acid, hydrogen peroxide, and water for a predetermined amount of time.

63. The biochip cartridge of any one of claim 61 to 62, wherein the self-assembled monolayer comprising the one or more capture probes is prepared by depositing a solution comprising one or more self-assembled monolayer species and at least one carboxylic acid based small molecule selected from the group consisting of citrate, acetate, and formate onto the electrode.

64. A biochip cartridge comprising: a substrate comprising a printed circuit board comprising an electrowetting grid of electrodes forming a droplet pathway; an array of detection electrodes accessible to the droplet pathway, each comprising a selfassembled monolayer including one or more capture probes; and a plurality of interconnections from the electro wetting grid and the detection electrodes; wherein the self-assembled monolayer comprising the one or more capture probes is prepared by depositing a solution comprising one or more self-assembled monolayer species and at least one carboxylic acid based small molecule selected from the group consisting of citrate, acetate, and formate onto the electrode; and wherein the self-assembled monolayer further comprises an electrolyte layer comprising MgCh.

65. An electrolyte solution comprising a buffer, magnesium chloride, a stabilizer, a non-ionic surfactant, a zwitterionic surfactant, and water.

66. The electrolyte solution of claim 65 further comprising a sugar stabilizer.

67. The electrolyte solution of claim 66, wherein the sugar stabilizer is fructose or a non-reducing sugar selected from trehalose and sucrose.

68. The electrolyte solution of any one of claims 64 to 67, wherein the electrolyte solution has a pH ranging from about 7.5 to about 8.

569. The electrolyte solution of any one of claims 64 to 67, wherein the electrolyte solution has a pH ranging from about 7.7 to about 8.

370. The electrolyte solution of any one of claims 64 to 69, wherein the zwitterionic surfactant is a sultaine, a betaine, a phospholipid, or a sphingolipid.

71. The electrolyte solution of any one of claims 64 to 69, wherein the zwitterionic surfactant is a betaine, an amine oxide, a sulfobetaine, an amidosulfobetaine, and a hydroxy betaine.

72. The electrolyte solution of claim 71, wherein the zwitterionic surfactant is sulfobetaine.

73. The electrolyte solution of claims 71, wherein the zwitterionic surfactant is amidosulfobetaine.

74. The electrolyte solution of any one of claims 64 to 69, wherein the zwitterionic surfactant is selected from the group consisting of 3-((3-cholamidopropyl)dimethylammonio)-l- propanesulfonate (CHAPS), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, phosphatidylserine, phosphatidylethanolamine, phosphotidylcholine, and sphingomyelin.

75. The electrolyte solution of any one of claims 64 to 74, wherein a ratio of an amount of non-ionic surfactant in the electrolyte solution to an amount of zwitterionic surfactant in the electrolyte solution ranges from 0.005: 1 to about 2:1.

76. The electrolyte solution of claim 75, wherein the ratio ranges from 0.025:1 to 4: 1.

77. The electrolyte solution of any one of claims 64 to 76, wherein the amount of zwitterionic surfactant in the electrolyte solution ranges from between about 1% to about 10% by total volume of the electrolyte solution.

78. The electrolyte solution of any one of claims 64 to 76, wherein the amount of zwitterionic surfactant in the electrolyte solution ranges from between about 2% to about 5% by total volume of the electrolyte solution.

79. Use of the electrolyte solution of any one of claims 64 to 78 in the preparation of an electrolyte layer.

80. A method of preparing an electrolyte layer comprising dispensing the electrolyte solution of any one of claims 64 to 78 onto an electrode, wherein the electrode comprises one or more capture probes.

81. The method of claim 80, wherein the dispensed electrolyte solution is permitted to dry passively.

82. The method of claim 80, wherein the dispensed electrolyte solution is actively dried.

83. An electrolyte solution comprising a buffer, magnesium chloride, a stabilizer, a non-ionic surfactant, and water, wherein the electrolyte solution does not include a zwitterionic surfactant.

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