Related Applications

[0001] This application claims priority to US Provisional Application 62/887019, filed August 15, 2019, the contents of which are hereby incorporated by reference in their entirety.

Field of the Invention

[0002] The present disclosure relates to aptamer-based sensors within nanoporous membranes that are resistant to fouling in biological and environmental media. Background

[0003] Electrochemical aptamer-based (E-AB) sensors have been utilized over the past several decades to detect for various analytes in a number of solutions. The presence of aptamers, either single or double stranded oligomers of deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), on the surface of an electrode can function to selectively bind targets in a biological solution. Aptamers are typically integrated into an electrode through one terminus, with the opposing terminus coupled to a redox reporter. Upon binding of a target molecule, the aptamer undergoes a conformational change that modulates the redox current and generates an electrochemical signal. The aptamer- target binding is reversible, as is the conformational change, which provides for a continuous label-free detection of the target.

[0004] While the real-time and continuous detection of a target molecule in a biological solution is of great value, the presence of other materials in biological samples can lead to fouling of the electrode. For example, non-specific bio-materials, such as proteins, cells, and oligonucleotides, in the biological fluid can become adsorbed at the electrode and negatively impact its ability to function.

[0005] Numerous approaches have been undertaken to attempt to prevent electrode fouling with varying results. Attempts have included pre-detection filtration, charged polymers on the surface to repel potential fouling agents and hydrogels on the electrode surface. However, these approaches offer the potential to interfere with the target reaching the aptamer successfully. Further, these approaches lack versatility as they can repel some biofouling agents, but not all. Thus a need for an anti-fouling design that allows for versatile biofouling prevention with unimpeded target access exists. Summary of the Invention

[0006] The present disclosure provides a fouling resistant electrochemical aptamer- based (E-AB) biosensor. The biosensor includes an electrode featuring a top surface of a film of nanoporous gold leaf (NPGL) affixed to an outer surface of a sensor body that forms an electrode body. The film may be affixed with one or more aptamers within at least one pore of the film. In certain aspects, the nanopores within the film may have a mean opening diameter of between about 7nm to about 45nm in size. In other aspects, the mean opening diameter is of between about 7 nm to about 30 nm in size. In further aspects, the mean opening diameter is of between about 15.5 nm and 40 nm in size.

[0007] In some aspects, the nanoporous gold film is between about lOnm and 1mm thick. In other aspects, the film is about 100nm thick.

[0008] The aptamers within the pores of the gold film may be single or double stranded oligonucleotides of DNA, RNA or PNA In certain aspects, the aptamers are single stranded. In certain aspects, the aptamers are of between about 15 to about 60 nucleic acids in length. [0009] In some aspects, the aptamers are affixed within the pores through a gold- sulfide bond provided by a thiol at the 5' terminus of the at least one aptamer.

[0010] In other aspects, the aptamers are affixed with a redox label at the 3' terminus of the through an amide link. In some aspects, the redox label is methylene blue. In other aspects, the redox label is selected from methylene blue, ferrocene, viologen, anthraquinone or any other quinones, daunomycin, organo-metallic redox labels, for example porphyrin complexes or crown ether cycles or linear ethers, ruthenium, bis- pyridine, tris-pyridine, bis-imidizole, cytochrome c, plastocyanin, and ethylenetetraacetic acid-metal complexes, or combinations thereof.

[0011] In some aspects, the aptamer binds a target ligand, which may cause a conformational change that changes the distance between the redox label and the nanoporous film. In some aspects, the target ligand is selected from an analyte, a small protein, a peptide, a metabolite, a hormone, a steroid, a nucleic acid oligomer, a saccharide, a cofactor, an enzyme, a metal or a carbohydrate.

[0012] The electrical sensing between the aptamer and the gold film will pass the sensor body which can be operably connected to electrical measurement devices, such as a voltameter. In aspects, the sensor body is a good conductor of electricity. In some aspects, the sensor body is of a material selected from gold, a gold-coated metal, aluminum, copper, palladium, titanium, tungsten, silver, platinum, carbon (including graphite, nanotubes, graphene), mercury films, or an oxide-coated metal. In certain aspects, the sensor body is of a gold material. In further aspects, the film of nanoporous gold leaf is of between 9 and 18 karats of gold purity. In other aspects, the film is of between 9 and 18 karats of gold purity. In even further aspects, the film of nanoporous gold leaf is of 9 karat purity.

[0013] The present disclosure further provides for methods of using the E-AB sensors to detect and/or measure the presence of target ligands in a solution, such as a biological solution. In some aspects, the present disclosure provides for a method for measuring the presence of a target ligand in a biological solution by providing the biosensor electrode to a biological solution, wherein a change in signal in the biosensor electrode is proportional to the concentration of target ligand in the biological solution.

[0014] The present disclosure further provides methods of preparing the E-Ab sensors. In some aspects, the method includes: etching a film of gold in nitric acid to provide pores; affixing the film of gold to a planar gold electrode; affixing a single- stranded oligonucleotide aptamer to a pore within the film of gold by a gold-sulfide bond through a thiol affixed to the 5' terminus of the single-stranded oligonucleotide aptamer, wherein the 3' terminus of the single-stranded oligonucleotide aptamer is amide linked to a redox label; and modifying the electrode by incubation with 6-mercapto-l-hexanol.

Brief Description of the Drawings [0015] FIG. 1 is a graph showing percent signal retained after scanning 1 scan per minute for 15 minutes in Tris buffer.

[0016] FIG 2 is a graph showing percent change in signal, as measured by the change in peak current with respect to the initial current, with increasing ATP concentration in Tris buffer solution. [0017] FIG 3 is a graph showing percent signal retained after scanning 1 scan per minute for 15 minutes in Tris buffer (1-15), followed by 1 scan per minute for an additional 15 minutes in fetal bovine serum (16-30).

[0018] FIG 4 is a graph showing percent change in signal, as measured by the change in peak current with respect to the initial current, with increasing ATP concentration in fetal bovine serum

[0019] FIG 5 is an illustration showing a visual comparison of planar gold electrodes and nanoporous gold electrodes when placed in protein-containing media, such as serum.

[0020] FIG 6 is a graph comparing sensor stability in serum after lhr of aptamer modification to NPGL gold content.

[0021] FIG 7 is a graph showing 9K NPGL ATP sensor response to varying ATP concentrations compared to planar sensor.

[0022] FIG 8 is an illustration of one embodiment of making the aptamer-based nanoporous gold electrodes of the present invention. Detailed Description

[0023] The present disclosure concerns a modified sensor surface to provide improved target sensing with aptamer-based sensors. The present disclosure concerns electrochemical aptamer-based (E-AB) sensors on electrode interfaces that render the sensors resistant to fouling due to non-specific adsorption of contaminants. The present disclosure concerns providing an improved sensor interface that provides for detection of target analytes in complex media, including biological and environmental media, without requiring sample pretreatment. Typically, this class of sensor can be plagued by nonspecific adsorption of, for example, serum proteins when employed long term in complex media. The present sensor interface alleviates the challenges seen with prior strategies when considering the translatability of E-AB sensor devices into working devices.

[0024] The present disclosure concerns, in some aspects, a top surface of a biosensor electrode that is modified for improved target detection and biofouling resistance. The top surface provides an interface between a sensor body and the surrounding medium, such as a biological medium. The top surface may be affixed to a sensor body. In some aspects, the top surface is a gold leaf (GL) film. In other aspects, the top surface surrounds or encases the portion of the sensor body intended to be exposed or submerged in a biological medium.

[0025] In some aspects, the top surface of the sensor is a nanoporous gold leaf film. The top surface is affixed to a sensor body through known means. In some instances, the top surface is affixed to the sensor body through physical forces, such as similar surface energy between the top surface and the sensor body or through van der Waal forces. The presence of nanopores on the top surface allows for a higher surface area when utilized in a solution, such as a biological solution as discussed herein.

[0026] In some aspects, the gold employed in the gold leaf film and/or electrode disclosed herein is of a certain level of purity or karat. In some aspects, the karat of the gold is of between 7 and 24, including 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23. In some aspects the gold is 9 karat. In other aspects, the gold is of between 9 and 18 karat.

[0027] In some aspects, the top surface is a nanoporous gold leaf film that has a thickness of between lOnm and 1mm, including about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, and 900nm. In some aspects, the gold leaf has a thickness of about 100nm.

[0028] In some aspects, the nanoporous gold leaf top surface provides pores with opening diameter sizes ranging from about 7nm to about 45nm or of about 7nm to about 30nm, including about 8nm, 9nm, 10nm, 11nm, 12nm, 13nm, 14nm, 15nm, 16nm, 17nm, 18nm, 19nm, 20nm, 21nm, 22nm, 23nm, 24nm, 25nm, 26nm, 27nm, 28nm, and 29nm.

In some aspects, the pores are of between about 15.5nm and 45.0nm.

[0029] In some aspects, the nanoporous gold leaf top surface is prepared by obtaining a small piece of gold foil that is placed in concentrated nitric acid to allow for etching. It will be appreciated that the length of time allowed for etching may affect the final pore size, as can the concentration of nitric acid. In some aspects, the nitric acid may be at a concentration of between about 0.5M and 10M, including about 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, and 9M. Typically, for a leaf of about 100nm thickness, an etching time of between 10 and 20 minutes may be sufficient, including about 11, 12, 13, 14, 15, 16, 17, 18, and 19 mins. Following etching, the gold foil may be stored in deionized water for a period of about 15mins to cease the etching process. The foil may then be placed in fresh water with the sensor body. Water is removed, allowing the foil to cover the sensor body. Foil that is not covering the sensor body may then be removed, thereby leaving a top surface of nanoporous gold leaf affixed to the sensor body.

[0030] In some aspects, the present disclosure concerns a sensor body. In some aspects, the sensor body is of an electro-conductive material, such that any electrical signal generated by the aptamer can conduct through the sensor body and to a measuring or detecting device connected thereto. The sensor body may be of any suitable conducting material for electrochemical sensing, including, for example: gold or any gold- coated metal or material, aluminum, copper, palladium, titanium, tungsten, silver, platinum, carbon (including graphite, nanotubes, graphene), mercury films, oxide- coated metals, semiconductor materials, and any other conductive material.

[0031] The sensor body may be in the form of any required or desired shape or size. For example, the sensor body may be a disc, cylinder, wire, sphere, paddle, rectangle, strip, array, screen printed or other. Those skilled in the art will appreciate that a narrow sensor body, such as a thin wire, may in some aspects provide further less invasive advantages to in vivo applications.

[0032] In some aspects, the sensor body is in electrical communication with a measuring or detecting device, as well as ancillary components such as power supplies or connectors thereto. Such communication may include wires or other electrically conductive elements that connect the sensor body to detectors, measuring devices, controllers, power supplies, voltage regulators, and other control elements which operate the sensing element. The sensor body may further be connected to structures designed and arranged to house or support the sensor body during operation to retain such in place to ensure proper operation.

[0033] In some aspects, the sensor body is part of an overall biosensor electrode sensing system to detect and/or measure the presence of a target ligand or molecule in a medium, such as a biological medium. The sensing system, in addition to the sensor body that can function as a biosensor electrode, may also include further elements, such as a reference electrode, a counter electrode, a voltage and/or current source, control elements and a means for reading or detecting changes in electrical conduction through the sensor body. The sensor body and/or other electrodes may in some instances be configured for various electrochemical observation techniques, including potentiometry, amperometry, and voltammetry, such as differential pulse voltammetry, cyclic voltammetry, alternating current voltammetry, and square wave voltammetry. In some aspects, the sensing system may further include controllers to provide currents and or voltages in working and/or reference electrodes within the proper operating parameters. Further components may include readout circuitry, data collection, and data storage components.

[0034] In some aspects, the sensor body is encased or covered by or partially covered by the top surface. In some aspects, the top surface is porous or semi-porous. In other aspects, the top surface features nanopores or a nanoporous network throughout the depth of the top surface. In some aspects, the nanoporous gold leaf pores contain one or more aptamers.

[0035] In some aspects, the present disclosure concerns materials that provide for the top surface of the sensor body. In some aspects, the top surface is a nanoporous material capable of transmitting or conducting an electrochemical signal, such as a signal generate by a conformational change in the aptamer due to target binding. In some aspects, the top surface may be of gold, such as a gold leaf. In other aspects, the top surface is a nanoporous material capable of conducting or transmitting an electrochemical signal. In some aspects, the top surface is a nanoporous gold leaf film affixed to a gold electrode with aptamers residing within the pores of the top surface.

[0036] In some aspects of the present disclosure, the top surface of the sensor body includes aptamers within nanopores or nanoporous channels therein. Aptamers are known in the art and may be specific for almost any target, for example being generated by systematic evolution of ligands by exponential enrichment (SELEX) methodologies (see, e.g., Wu et al., Anal. Chem. 91: 15335-15344, 2019). Aptamers may include a sequence or string of oligonucleotides, such as DNA aptamers, RNA aptamers, and aptamers comprising non-natural nucleic acids may be used, as well as hybrids of the foregoing and including polymers, such as PNA. In some aspects, the oligonucleotides may be capable of forming "stem-loop" or "hairpin" structures (also referred to "stem- loop" or "hairpin", or simply "stem-loops" or "hairpins"), with an electroactive label to detect hybridization events. Typical aptamers are about 15-60 bases in length, including 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 5, 52, 53, 54, 55, 56, 57, 58, and 59mers. However, aptamers of any size may also be used. Extant aptamers known in the art include those capable of binding target species such as doxorubicin, ATP, lysozyme, thrombin, HIV trans-acting responsive element, hemin, interferon, vascular endothelial growth factor, prostate specific antigen, dopamine, and cocaine. [0037] In some aspects the aptamer is affixed to the pore surfaces of the top surface through a covalent interaction. In some aspects, the aptamer is bound through a thiol conjugation, alkanethiol, cyclic disulfide, dithiothreitol, diothiols, adenosine or phosphorothioated adenosines. In certain aspects, the aptamer is a single-stranded oligomer, such as ssDNA or RNA, affixed at the 5' terminus with a thiol functional group. The thiol can react with the gold of the top surface and provide a gold-sulfide bond to affix aptamers within the pores. (See, generally, Odeh et al., Molecules 25(1):3 (2020). Martinez-Jothar et al., J. Control Release 28: 101-09 (2018), and Danesh et al., Int. J. Pharm. 489: 311-17, (2015)). In one aspect, an aptamer solution can be pre-treated with a phosphine, such as with tris-(2-carboxyethyl) phosphine or dithiothreitol, for a period of time between about 30 mins to 5 hours, including about 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240, 255, 270, and 285 mins, in order to break any disulfide bonds. While some of the single-stranded oligomer aptamers may be physisorbed to the gold surface, a vast majority of the aptamers on the surface are chemisorbed and anchored through a gold-sulfide bond. [0038] In some aspects of the invention, the aptamer features a terminal label. In some aspects, the aptamer is a single stranded oligomer, such as ssDNA or RNA affixed at the 3' terminus. A redox label can be provided as an ester which can react with the amine at the 3' terminus to link the two together. In some aspects, the redox label is a methylene blue succinimide ester connected to the amine-terminated ssDNA or RNA. [0039] In some aspects, the terminal label may be of a redox material, such that a change in the distance between the label and the gold leaf causes a detectable change in electron transfer between the two. As such, when a ligand binds to the aptamer, the physical shape changes and the redox label distance changes, causing a detectable change in electron transfer and the resistance created between the two. [0040] In some aspects, the redox label is capable of electron transfer to or from the electrode, optionally with direct transfer to the top surface and then to the sensor body. With sufficient proximity and accessibility of a redox label to the electrode, an electrical signal, e.g. current, voltage, or other measurable electrical interaction, will occur between the redox label and the electrode. The redox label may be positioned such that binding of the target ligand to the aptamer causes a measurable change in the electrical signal generated by the redox label. In some aspects, the redox label is positioned at the terminus of the aptamer, for example as depicted in Fig. 8. In other aspects, the redox label is present on a separate polynucleotide strand that binds to the aptamer in the absence of target species and that is displaced by binding of the target species to the aptamer (see, Xiao et al., J. Am. Chem. Soc., 127: 17990-91 (2005)). In some aspects, the redox labels may be configured for "turn-off" wherein the redox signal is decreased by the binding of the target ligand. In other aspects, the redox labels may be configured for "turn-on" signaling wherein the redox signal is increased by the binding of the target ligand. The placement of such sensing label can be selected using known methods of designing electrochemical sensors.

[0041] In some aspects, the redox label is methylene blue. In other aspects, the redox sensor is selected from known redox labels including Exemplary redox labels include methylene blue, ferrocene, viologen, anthraquinone or any other quinones, daunomycin, organo-metallic redox labels, for example porphyrin complexes or crown ether cycles or linear ethers, ruthenium, bis-pyridine, tris-pyridine, bis-imidizole, cytochrome c, plastocyanin, and ethylenetetraacetic acid-metal complexes, or combinations thereof.

[0042] In some aspects, the redox label is affixed to the aptamer and then the aptamer-label unit is affixed to the top surface, such as with a carbon-chain linker or directly with a thymine. In some aspects, the aptamers affix to the top surface by incubation at a with an aptamer solution at a concentration of between about 100nM and 500nM, including about 200nm, 300nm and 400nm, for a period of between about 1 and 5 hours, including about 2, 3 and 4 hours. In further aspects, the incubation can occur at a temperature of between about 20 and 45 °C, including about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, and 43 °C.

[0043] In some aspects, the top surface is further affixed with carbon chain materials to prevent non-specific binding and/or to provide a buffer between the redox label and the electrode/sensor body. In some aspects, the carbon chain comprises 6-mercapto-1- hexanol or 6-aminohexanol. The carbon-chains can be bound by incubation of a concentration of between about 100 nM and 500 nm, including about 200, 300 and 400 nm for a period of between about 30 mins and 2 hrs, including 45, 60, 75, 90, and 105 mins. The bound carbon chains may prevent non-specific adsorption of the aptamers onto the electrode surface, thus providing for more reliable signal measurements.

[0044] In some aspects, the aptamers bind a target ligand. The structures of some aptamers for known ligands are known in the art, while further aptamers for other ligands can be readily identified though known methods in the art including systematic evolution of ligands by exponential enrichment (SELEX). In some aspects, the aptamer may be an aminoglycoside-binding aptamer, a cocaine-binding aptamer, any small molecule- binding aptamer, a thrombin-binding aptamer, a platelet derived growth factor-binding aptamer, a neuropeptide Y-binding aptamer, any protein-binding aptamer, an inorganic ion-binding aptamer or a DNAzyme binding aptamer.

[0045] In some aspects, the target ligand may be any inorganic or organic molecule, for example: a small molecule drug, a metabolite, a hormone, a peptide, a protein, a carbohydrate, a nucleic acid, an analyte, a steroid, a nucleic acid oligomer, a saccharide, a cofactor, an enzyme, a metal, or any other composition of matter. The target ligand may be a therapeutic drug, an antibiotic, an illicit drug, an antibiotic agent, or a chemotherapeutic drug. The target ligand may include a naturally-occurring factor, such as a hormone, metabolite, growth factor, neurotransmitter, or similar. The target ligand may be any other species of interest, for example, species such as pathogens {including pathogen induced or derived factors), toxins, nutrients, and pollutants. [0046] The target ligand hinds the aptamer and the aptamer assumes a conformation such that the redox label is in proximity to the sensor body and/or top surface, causing the flow or Faradic current or other measurable electronic interactions. In a sensor comprising a plurality of recognition elements, the bulk dynamics of target binding and dissociation and the resulting electronic interactions with the substrate create a measurable electronic signal that is proportional to the concentration of the target species in the sample solution.

[0047] Methods of using

[0048] In some aspects, the present invention includes methods of using the E-AB sensors described herein. The top surface affixed to the sensor body can function as an electrode, particularly with the sensor body being operably linked to power sources and/or power measuring devices. The electrode can be submerged or contacted with a biological fluid and changes in the electrical current detected. As depicted in FIG 5, the presence of the aptamers within the pores allows for small ligands to freely associate, yet larger items in the solution cannot enter the pore due to size restriction. As also depicted in FIG 5, a planar electrode surface can trap larger items when ligand binds to the aptamer, which can lead to fouling, particularly when proteins are trapped to the surface of the electrode. With aptamers located within the pores, the proteins cannot access a vicinity near the aptamer and then cannot be trapped, thereby avoiding fouling the electrode.

[0049] The present disclosure provides an approach that takes advantage of the nanoscale structure of nanoporous gold electrodes fabricated from gold leaf. Sensors are fabricated using nanoporous gold leaf electrodes to provide high active sensing surface area while burying most of the sensor surface area within the porous network (FIG 5). The nanoporous electrode provides size exclusion which prevents proteins, like those commonly found in media like serum, sweat, etc., from adsorbing to the majority of the working surface area of the sensor. When compared to sensors fabricated on a planar electrode, the nanoporous E-AB sensors show comparable quantitative response to the addition of target analyte (FIGs 2 and 4), while displaying maintained baseline signal fidelity when the electrodes are employed in complex media (FIGs 1 and 3). Conversely, planar electrodes typically display a loss in signal fidelity over the same time scales.

[0050] One of the benefits of the present invention comes from the enhanced surface area of the NPGL electrode when compared to a planar electrode. Nearly, if not all, of a planar electrode's surface area is exposed to the biological solution with no barrier or protection against non-specific adsorptions of proteins found in the sample solution onto the electrode surface. The NPGL electrodes, on the other hand, provide both the benefit of size exclusion as well as a much larger active surface area. These two benefits stem from the network of nanoscale pores and ligaments formed during the NPGL fabrication process. With the size difference between the aptamers and proteins commonly found to foul electrode surfaces like fibrinogen, approximately 15.5nm and 45.0nm, respectively, the pores provide an excellent filtration system for this type of application.

[0051] Further, prior methods can only detect electroactive species that have a formal potential that falls within the potential window of the solvent being used. In some aspects, the E-AB system of the present disclosure system is applicable to virtually any small molecule/protein of interest because the signal is relatively fixed at ~-0.28V. It is understood that such value can vary with other redox probes, such as ferrocene or anthraquinones. In addition, the signal provided by the top surface of the electrode in the present disclosure comes from a redox label on the sensor itself rather than relying on the electrochemistry of the analyte of interest. Typically, the electrode can be interrogated by voltammetry, amperometry, coulometry or electrical impedance spectroscopy. Other prior methods rely on an added redox label rather than one that is contained in the aptamer itself. Some prior methods also incorporate additional non- sensing single stranded DNA, which complicates the sensing platform.

[0052] In summary, the present disclosure provides a sensor interface that can be used in conjunction with E-AB sensors to detect analytes of interest in untreated media, like serum, while alleviating challenges with nonspecific fouling of the electrode surface. Examples

[0053] Example 1 -

[0054] A small piece of gold foil was cut from a stock piece. This small piece was then transferred into a container of concentrated nitric acid and allowed to etch for a set amount of time (~14 minutes). After that, the piece was transferred over to a container of deionized water using a glass slide and left in there for 15 minutes to stop any further etching from occurring. The piece was then transferred once more to a container with water and a planar gold electrode to be modified and the water was removed from the container until the etched gold foil covered the gold electrode surface. From there, the gold foil that was not directly in contact with the gold electrode was wiped away, leaving a small circle of nanoporous gold leaf on top of the original gold electrode.

[0055] Once nanoporous gold leaf (NPGL) electrodes are fabricated and cleaned, the electrodes then go through a series of incubation steps in order to produce a functioning sensor. The first step is incubating the NPGL electrodes in a 200nM aqueous solution of aptamer for four hours. These aptamers consist of a ssDNA that has been modified on both the 5' and 3' end. The 5' end was modified with a thiol linker which enabled immobilization of the ssDNA onto the gold surface. The 3' end, on the other hand, was modified with a methylene blue redox label. This label was covalently bound to the ssDNA by reacting methylene blue succinimide ester with the amine-terminated ssDNA. In one embodiment, the aptamer solution was preferably pretreated with tris-(2-carboxyethyl) phosphine for at least one hour in order to break any disulfide bonds. While some of the ssDNA aptamers may be physisorbed to the gold surface at this point, a vast majority of the aptamers on the surface are chemisorbed and anchored to the surface through a gold-sulfide bond.

[0056] After the four hour incubation step, the NPGL electrodes were removed from the aptamer solution and rinsed with distilled water in order to remove any physisorbed aptamers from the surface. The electrodes were then placed in a 30mM aqueous solution of 6-mercapto-1-hexanol for one hour. This second incubation step helps to prevent non- specific adsorption of the aptamers onto the electrode surface, thus leading to more reliable signal measurements. The electrodes were then rinsed once more with distilled water to remove any excess 6-mercapto-l-hexanol and then placed in a buffer solution for one hour. This final incubation period in the buffer solution allowed the newly fabricated sensors to equilibrate in a solution of similar ionic strength and pH to that of the sample solution in which the sensors will be employed. An illustration of the basic steps of this method are shown in FIG 8.

[0057] The sensor was then tested for its rate of signal loss. The sensor of the present disclosure was placed in a solution of Tris buffer for 15 minutes alongside a planar gold electrode and the percentage of signal loss over the period of time was calculated. As shown in FIG 1, the NPGL electrode provided improved signal retention over the time course.

[0058] The NPGL sensor was next tested against a planar gold electrode for ligand response. An aptamer that binds ATP was utilized for both and the electrode were placed in a Tris buffer and titrated over time with an ATP solution. The NPGL showed better signal change over the planar electrode as the concentration of ATP increased (see FIG 2).

[0059] The NPGL sensor was then tested for signal loss in serum. The sensor and a planar gold electrode were submerged in Tris buffer for 15 minutes and then moved to serum for a further 15 minutes. The planar electrode showed slightly worse signal loss in Tris, but significantly lost signal almost immediately when placed in serum. The NPGL signal in serum remained good, with a less than 20% loss of signal after the full half hour (see FIG 3).

[0060] The NPGL sensor was then tested in serum with ATP ligand titration to determine the responsiveness of the sensor. Again a planar gold electrode was used for comparison. The NPGL sensor showed a marked improvement in detecting the ATP over the planar electrode throughout (see FIG 4). [0061] The quality of the gold for the NPGL was next examined with three electrodes prepared, one with 9K gold and one with 12K gold and one with 18K gold. The three were assessed for stability in fetal bovine serum after an initial 15 minutes in Tris buffer. The 9K NPGL showed best signal retention, although the 18K was still outperforming the planar electrode (FIG 6). The 9K NPGL was then titrated with ATP in serum and signal change determined. The NPGL showed good sensitivity with high consistency over repeated experiments (FIG 7).

[0062] The present disclosure accordingly provides:

[0063] 1. A biosensor electrode comprising a top surface of a film of nanoporous gold leaf (NPGL) affixed to an outer surface of a sensor body to form an electrode body, wherein the film is affixed with one or more aptamers within at least one pore of the film.

[0064] 2. A biosensor electrode as set forth in the preceding clause, wherein the pores of the film have a mean opening diameter of between about 7nm to about 45nm in size.

[0065] 3. A biosensor electrode as set forth in any preceding clause, wherein the mean opening diameter is of between about 7 nm to about 30 nm in size.

[0066] 4. A biosensor electrode as set forth in any preceding clause, wherein the mean opening diameter is of between about 15.5 nm and 40 nm in size. [0067] 5. A biosensor electrode as set forth in any preceding clause, wherein the film is between about lOnm and 1mm thick.

[0068] 6. A biosensor electrode as set forth in any preceding clause, wherein the film is about 100nm thick. [0069] 7. A biosensor electrode as set forth in any preceding clause, wherein the at least-one aptamer is single stranded.

[0070] 8. A biosensor electrode as set forth in any preceding clause, wherein the at least one aptamer is affixed within the at least one pore through a gold-sulfide bond provided by a thiol at the 5' terminus of the at least one aptamer.

[0071] 9. A biosensor electrode as set forth in any preceding clause, wherein a redox label is affixed to the 3' terminus of the at least one aptamer through an amide link.

[0072] 10. A biosensor electrode as set forth in any preceding clause, wherein the redox label is methylene blue. [0073] 11. A biosensor electrode as set forth in any preceding clause, wherein the at least one aptamer comprises a redox label.

[0074] 12. A biosensor electrode as set forth in any preceding clause, wherein the redox label is selected from the group consisting of methylene blue, ferrocene, viologen, anthraquinone or any other quinones, daunomycin, organo-metallic redox labels, for example porphyrin complexes or crown ether cycles or linear ethers, ruthenium, bis- pyridine, tris-pyridine, bis-imidizole, cytochrome c, plastocyanin, and ethylenetetraacetic acid-metal complexes, or combinations thereof.

[0075] 13. A biosensor electrode as set forth in any preceding clause, wherein the at least one aptamer binds a target ligand. [0076] 14. A biosensor electrode as set forth in any preceding clause, wherein the target ligand is selected from the group consisting of an analyte, a small protein, a peptide, a metabolite, a hormone, a steroid, a nucleic acid oligomer, a saccharide, a cofactor, an enzyme, a metal or a carbohydrate. [0077] 15. A biosensor electrode as set forth in any preceding clause, wherein the at least one aptamer is of between about 15 to about 60 nucleic acids in length.

[0078] 16. A biosensor electrode as set forth in any preceding clause, wherein the sensor body is comprised of a material selected from the group consisting of gold, a gold- coated metal, aluminum, copper, palladium, titanium, tungsten, silver, platinum, carbon (including graphite, nanotubes, graphene), mercury films, or an oxide-coated metal.

[0079] 17. A biosensor electrode as set forth in any preceding clause, wherein the sensor body is gold.

[0080] 18. A biosensor electrode as set forth in any preceding clause, wherein the sensor body is operably connected to a voltameter.

[0081] 19. A biosensor electrode as set forth in any preceding clause, wherein the film of nanoporous gold leaf is of between 9 and 18 karats of gold purity.

[0082] 20. A biosensor electrode of any preceding clause, wherein the film of nanoporous gold leaf is of 9 karat purity. [0083] 21. A method for measuring the presence of a target ligand in a biological solution comprising providing a biosensor electrode as set forth in any preceding clause to a biological solution, wherein a change in signal in the biosensor electrode is proportional to the concentration of target ligand in the biological solution.

[0084] 21. A method of preparing a biosensor electrode as set forth in any preceding clause, comprising:

(a) etching a film of gold in nitric acid to provide pores;

(b) affixing the film of gold to a planar gold electrode; (c) affixing a single-stranded oligonucleotide aptamer to a pore within the film of gold by a gold-sulfide bond through a thiol affixed to the 5' terminus of the single-stranded oligonucleotide aptamer, wherein the 3' terminus of the single-stranded oligonucleotide aptamer is amide linked to a redox label; and (d) modifying the electrode by incubation with 6-mercapto-l-hexanol.

[0085] While the invention has been described in connection with the specific embodiments thereof, it will be understood that it is capable of further modification. Furthermore, this application is intended to cover any variations, uses, or adaptations of the invention, including such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains, and as fall within the scope of the appended claims.

[0086] All publications, including patents, published patent applications and non- patent literature referenced herein are incorporated by reference in their entirety.