Instant diagnosis method

Abstract

Disclosed herein is a method of biosensing a nucleic acid analyte in a sample, potentially a complex biological sample, and the use of such methods in diagnostic applications. Also disclosed herein are diagnostic methods using voltammetric biosensing devices comprising a porous substrate comprising a plurality of channels wherein at least a portion of the plurality of channels comprises a moiety, such as a capture probe, capable of binding to one or more nucleic acid analytes in a complex biological sample.

Description

Cross Reference to Related Applications

[0001] This application claims priority from Australian provisional patent application No. 2023900818 filed on 23 March 2023, the contents of which are incorporated herein in their entirety by this reference. TECHNICAL FIELD

[0002] The present invention relates generally to voltammetry for biosensing of nucleic acid analytes in complex biological samples and / or use of such methods in diagnostic applications. More particularly, the present invention relates to diagnostic methods using a voltammetric biosensing device comprising a porous substrate comprising a plurality of channels, wherein at least a portion of the plurality of channels comprises moieties, such as capture probes, capable of binding to one or more nucleic acid analytes in a complex biological sample. The one or more nucleic acid analytes can be biomarkers associated with an injury, condition or disease state, such as a brain injury, such as a traumatic brain injury. The methods of the present invention can optionally determine the concentration of the one or more nucleic acid analytes in the complex biological sample. BACKGROUND

[0003] There is currently a lack of suitable diagnostic tools that can be adapted and deployed in a convenient manner to assist in detecting one or more conditions in a subject over an appropriate time frame. Early detection of a condition can be important in determining appropriate treatment options (and reducing the risk of complications). It is therefore important to produce diagnostic tools that are capable of adapting to timely detection of appropriate biomarkers associated with a disease state or injury.

[0004] An example of an injury that can result in significant complications for a subject is a traumatic brain injury (TBI), which can be a brain injury caused by an external force. TBIs can be classified based on severity, ranging from mild to severe. TBIs are a leading cause of death and disability worldwide. TBIs can result in physical, cognitive, social, emotional, and behavioral symptoms, and outcomes can range from full recovery to permanent disability or death.

[0005] There is currently a lack of suitable diagnostic techniques that allow for accurate detection of a TBI and are capable of determining the severity of the TBI in an appropriately short time frame. Diagnostic techniques that have been explored in the context of TBIs range from imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) to detection and quantification of molecular markers from proteins to miRNAs, but these methods require time and resources and there are difficulties in accessing appropriate equipment and devices in a timely and cost-effective manner.

[0006] Among the diagnostic techniques available for TBI, the discovery of miRNA biomarkers associated with TBI, such as those described in PCT / US2015 / 051518, have shown particular promise. However, there remains a need for diagnostic tools that are capable of rapidly and sensitively detecting miRNAs (or other relevant nucleic acid biomarkers) in complex biological samples in a timely manner in a point-of-care diagnostic setting.

[0007] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these are prior art SUMMARY

[0008] Disclosed herein are voltammetric methods for biosensing nucleic acid analytes in complex biological samples and the use of such methods in diagnostic applications. More specifically, the present invention relates to diagnostic methods using a voltammetric biosensing device comprising a porous substrate having a plurality of channels, a portion of which contain capture probes capable of binding to one or more nucleic acid analytes and configured to detect one or more nucleic acid biomarkers in a complex biological sample. In some examples, the nucleic acid analytes that can be detected using the methods of the present invention are indicators of traumatic brain injury (TBI). Thus, the methods of the present invention can be used to diagnose TBI in complex biological samples such as blood and blood fractions based on detection of one or more nucleic acid analytes or biomarkers (e.g., miRNAs) associated with TBI, and optionally, to determine the severity of the TBI based on biomarker concentration and / or profile potentially simultaneously. However, it will be appreciated that the methods of the present invention can be used to biosense other nucleic acid analytes or biomarkers present in complex biological samples in a range of other diagnostic applications.

[0009] The objective of this invention is, in particular, to assist the clinical decision-making process and ultimately contribute to significantly improved management of TBI symptoms, and where possible, improved clinical and surgical outcomes, by enabling faster detection of TBI-related biomarkers. To achieve this objective, the inventors have developed a TBI diagnostic assay based on nucleic acid biomarkers (i.e., microRNA) that can be performed in “patient-friendly” settings, such as in field hospitals or on the sidelines. While integrating certain diagnostic technologies into benchtop and handheld “point-of-care diagnostic” devices has indeed enabled patient assessment and / or diagnosis of a range of conditions in “patient-friendly” settings to some extent (e.g., diagnostic tests based on lateral flow antibodies), the need for sample pretreatment or preparation to transform complex biological samples from their collected form into an analytically suitable form has been a barrier to the adoption of point-of-care diagnostic platforms for nucleic acid-based biomarkers such as TBI. In this respect, prior to this invention, diagnostic assays and methods configured to detect nucleic acid analytes were not well-suited for direct administration to complex biological samples or for minimal sample preparation.

[0010] In this paper, methods for detecting nucleic acid analytes, such as nucleic acid biomarkers, in complex biological samples can utilize devices comprising fabricated porous substrates, such as porous substrates comprising silicon (pSi). The porous substrate may contain channels (e.g., at the micrometer and / or nanometer scales) with controllable dimensions relative to depth and / or cross-section. The porous substrate can be stabilized (e.g., to reduce degradation, chemical reactions, and / or improve electrochemical stability, such as via thermal (hydrogen) carbonization), followed by the introduction of a capture probe or a capture probe precursor, such as the introduction of functional groups that can be modified to become capture probes, or the introduction of functional groups on at least one surface of the porous substrate (e.g., within at least a portion of multiple channels) to act as anchoring points to connect and immobilize one or more capture probes. Stabilization may involve exposing the porous substrate to high temperatures for a period of time.

[0011] When performing the method of the present invention, one or more nucleic acid analytes, such as DNA, mRNA, or miRNA analytes, can bind to one or more capture probes disposed in at least a portion of a plurality of channels in a porous substrate. The one or more nucleic acid analytes can be detected by electrochemical measurement using appropriate means, such as a potentiostat. By using a composition containing one or more analytes at known concentrations, calibration curves, graphs, and data can be obtained from the electrochemical measurement results.

[0012] Porous substrates can form part of an electrochemical sensor in which, within at least a portion of multiple channels, the binding of one or more analytes to immobilized complementary trapping probes causes at least partial blockage, which in turn hinders redox substances (e.g., [Fe(CN)6)). 3- / 4-diffusion into the electrochemically active layer (e.g., electrochemical transducer), resulting in a decrease in peak current intensity, which can be detected and quantified. For sensors deployed and / or exposed to biological samples, signals from any channel blockage can also be affected by biological contamination mechanisms, for example, where contaminants attach to a portion of an electrode (e.g., an electrode derived from or based on a porous substrate as described herein) via non-specific adsorption. Contaminants can potentially reduce the area of the electrode surface available for one or more analytes, resulting in a decrease in, for example, differential pulse voltammetry signals. In this regard, controlling and selecting the dimensions of the plurality of channels can eliminate or reduce problems associated with biological contamination, thereby improving the ability to detect nucleic acid analytes in complex biological samples. Without being bound by any theory, this can be due, at least in part, to the dimensions of the channels being at a scale such that proteins and / or other potential biological molecules that cause contamination (such as those that can be present in a complex biological sample containing the target analytes) are unable to enter or progress within the channels, while the target analytes are at a scale meaning they are small enough and mobile to reach the capture probes disposed within at least a portion of the plurality of channels. Thus, an advantage of the methods of the present invention can be the ability to directly detect the presence of one or more nucleic acid analytes in a biologically complex medium, while biological contamination is minimal or reduced. The ability to detect nucleic acid analytes in complex biological media (e.g., blood) using the methods of the present invention can reduce the need to handle samples prior to testing and / or minimize human manipulation that can lead to contamination, degradation, and / or error. As described above, the ability to perform diagnostics on complex biological samples such as blood or serum can also allow for testing to be performed in the field, thereby shortening the time frame in which a diagnosis can be made. This can also reduce the need to transport one or more samples to a separate environment / facility for testing and / or analysis, which potentially shortens the time to obtain any results, and also potentially allows for a diagnosis to be determined (and acted upon) in a shorter time frame, thereby mitigating deleterious effects on the subject.

[0013] It would also be an advantage if measurements could be obtained from a wide range of samples (potentially derived from different sources), and provided in different forms. This would make sample testing flexible (e.g., not requiring a particular form and / or purity and / or concentration of sample). The type or form of sample can include a liquid sample obtained from a subject, such as a sample in the form of blood and / or serum, which can be used directly, or after dilution in a suitable solvent such as a buffer solution or saline. In this regard, minimal or no sample preparation can be required.

[0014] Another possible advantage of the methods disclosed herein can not only be to detect the presence of one or more nucleic acid analytes in a complex biological sample to determine the presence of a medical condition, such as TBI, but also to quantify the amount of one or more nucleic acid analytes to potentially determine the severity of the condition (e.g. by developing a calibration curve using appropriate standards and identifying threshold values associated with different stages of severity). This can assist triage efforts and / or determine the most appropriate treatment regime for the subject. Such determination potentially can occur within a short timeframe, thereby allowing for a rapid assessment of the situation and allowing for appropriate treatment steps to be taken, whilst reducing and / or minimising delays.

[0015] Thus, in a first aspect, the present application provides a method of detecting at least one biomarker (e.g. at least one nucleic acid analyte) in a complex biological sample, the method comprising: (i) introducing the complex biological sample into an electrochemical biosensor comprising a porous substrate comprising a plurality of channels, wherein: - at least a portion of the plurality of channels has an average depth of about 1.0 pm to about 6.0 pm; and / or - at least a portion of the plurality of channels has an average diameter or cross-section in the range of about 15 nm to about 40 nm, wherein one or more capture probes capable of specifically binding to at least the biomarker, e.g. at least one nucleic acid analyte, to form a probe-analyte complex are disposed on at least one surface of at least a portion of the plurality of channels; and (ii) optionally determining whether at least one biomarker, e.g. at least one nucleic acid analyte, is present in the sample based on measurements of peak current obtained from differential pulse voltammograms before and after introducing the complex biological sample into the electrochemical biosensor, wherein a change in peak current detected after introducing the complex biological sample into the electrochemical biosensor is indicative of the presence of at least one biomarker, e.g. at least one nucleic acid analyte, in the complex biological sample, and no change in peak current detected after introducing the complex biological sample into the electrochemical biosensor is indicative of the absence of at least one biomarker, e.g. at least one nucleic acid analyte, above a threshold level in the complex biological sample.

[0016] In one example, the capture probes are capable of specifically binding to at least one nucleic acid analyte to form a probe-analyte complex, wherein the presence of the probe-analyte complex within the channel causes at least partial steric hindrance and / or charge blockage of the channel compared to channels in which the probe-analyte complex is not present.

[0017] In one example, at least a portion of the plurality of channels has an average depth ranging from about 1.0 μιη to about 6.0 μιη, and at least a portion of the plurality of channels has an average diameter or cross-section ranging from about 15 nm to about 40 nm. For example, at least a portion of the plurality of channels has an average depth ranging from about 1.0 μιη to about 6.0 μιη, and an average diameter or cross-section ranging from about 15 nm to about 40 nm.

[0018] In one example, at least 10% of the plurality of channels has an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 15% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 20% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 25% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 30% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 35% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 40% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 45% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 50% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη.

[0019] Alternatively, or in addition to at least 10% of the plurality of channels having an average depth in the range of about 1.0 pm to about 6.0 pm as described above, at least 10% of the plurality of channels can have an average diameter or cross-section in the range of about 15 nm to about 40 nm. For example, at least 15% of the plurality of channels can have an average diameter or cross-section in the range of about 15 nm to about 40 nm. For example, at least 20% of the plurality of channels can have an average diameter or cross-section in the range of about 15 nm to about 40 nm. For example, at least 25% of the plurality of channels can have an average diameter or cross-section in the range of about 15 nm to about 40 nm. For example, at least 30% of the plurality of channels can have an average diameter or cross-section in the range of about 15 nm to about 40 nm. For example, at least 35% of the plurality of channels can have an average diameter or cross-section in the range of about 15 nm to about 40 nm. For example, at least 40% of the plurality of channels can have an average diameter or cross-section in the range of about 15 nm to about 40 nm. For example, at least 45% of the plurality of channels can have an average diameter or cross-section in the range of about 15 nm to about 40 nm. For example, at least 50% of the plurality of channels can have an average diameter or cross-section in the range of about 15 nm to about 40 nm.

[0020] In some examples, the porous substrate comprises, consists essentially of, or consists of an electrically conductive or semiconductive material. In some examples, the porous substrate comprises, consists essentially of, or consists of a material selected from the group consisting of platinum, gold, silver, cobalt, gallium, indium, tin, vanadium, zirconium, copper, aluminum, iridium, palladium, rhodium, silicon, zinc, iron, steel, brass, carbon, graphite, alloys, oxides, or metal compounds of these elements, and mixtures thereof. For example, the porous substrate can comprise, consist essentially of, or consist of silicon.

[0021] In some examples, a layer comprising, consisting essentially of, or consisting of carbon can be disposed on at least a portion of the porous substrate.

[0022] In one embodiment, the porous substrate comprises silicon, wherein the silicon comprises at least one layer that has been modified by at least one of thermal and / or chemical treatment.

[0023] In another embodiment, the porous substrate, optionally comprising, consisting essentially of, or consisting of silicon, comprises, consists essentially of, or consists of a monolayer comprising one or more of: ■one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more functional groups capable of reacting with amine groups; and / or ■ one or more functional groups capable of forming a covalent bond with a complementary functional group on one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more carboxylic acid groups.

[0024] In another embodiment, the porous substrate optionally comprising silicon, consisting essentially of silicon, or consisting of silicon comprises a layer comprising one or more of: ■ one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more functional groups capable of reacting with an amine group; and / or ■ one or more functional groups capable of forming a covalent bond with a complementary functional group on one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more carboxylic acid groups.

[0025] In another embodiment, the porous substrate optionally comprising silicon, consisting essentially of silicon, or consisting of silicon comprises at least one layer comprising one or more of: ■ one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more functional groups capable of reacting with an amine group; and / or ■ one or more functional groups capable of forming a covalent bond with a complementary functional group on one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more carboxylic acid groups.

[0026] In another embodiment, the porous substrate optionally comprising silicon, consisting essentially of silicon, or consisting of silicon does not comprise, consists essentially of, or consists of a plurality of layers comprising one or more of: ■ one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more functional groups capable of reacting with an amine group; and / or ■one or more functional groups capable of forming a covalent bond with a complementary functional group on one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more carboxylic acid groups.

[0027] In some examples disclosed herein, the one or more capture probes are capable of specifically binding to one or more nucleic acid analytes. In some examples, the porous substrate comprises a plurality of capture probes, each of which is capable of specifically binding to one or more nucleic acid analytes.

[0028] The at least one nucleic acid analyte can be a biomarker. For example, the nucleic acid can be selected from the group consisting of: a DNA analyte, a messenger RNA (mRNA) analyte, a ribosomal RNA (rRNA) analyte, a transfer RNA (tRNA) analyte, a microRNA (miRNA) analyte, a short interfering RNA (siRNA) analyte, a plasmid DNA (pDNA), and mixtures thereof. In one example, the nucleic acid analyte is a miRNA.

[0029] The capture probes can be selected to suit the nucleic acid analyte to be detected. For example, one or more of the at least one capture probe can be a DNA capture probe or a peptide nucleic acid probe (PNA) probe. In one example, the at least one capture probe is a DNA capture probe. In one example, the at least one capture probe is a PNA probe. In any example disclosed herein, the capture probes can have modified chemical properties, such as to improve stability, improve binding affinity, and / or reduce off-target effects of the capture probes. Modified chemical properties of DNA and PNA probes are known in the art and are contemplated herein.

[0030] The method can be used to detect and diagnose a nucleic acid analyte or biomarker associated with a condition, disease, or injury. Accordingly, the at least one nucleic acid analyte can be associated with a condition, disease state, or injury. In one particular example, the at least one nucleic acid analyte is associated with a brain injury, optionally a traumatic brain injury (TBI), and the method is used to diagnose a brain injury (e.g., a TBI). However, other medical indications are described and contemplated herein.

[0031] In one particular example, the method comprises detecting the presence or absence of one or more miRNAs associated with a TBI in a complex biological sample (e.g., blood, a blood fraction, or a diluted blood solution). For example, the one or more miRNAs can be selected from the group consisting of miR-142-3p, miR-196b-5p, let-7f-5p, miR-150-5p, miR-196b-5p, and combinations thereof.

[0032] In some examples, the method comprises determining the severity of the condition, disease state, or injury by determining the concentration of at least one biomarker (e.g., at least one nucleic acid analyte) in the sample. For example, the disease state or injury can be a brain injury, such as a TBI, and the concentration of at least one nucleic acid analyte associated with the brain injury (e.g., TBI) is determined for determining the severity of the brain injury in the subject providing the sample. According to this example, the nucleic acid analyte will be associated with the brain injury (e.g., TBI) and present at a concentration that is correlated to the severity of the injury.

[0033] As described herein, the methods of the present application can be performed on a complex biological sample or medium. In some instances herein, the terms and phrases “complex biological sample,” “sample,” and “medium” can be used interchangeably. In some examples, the sample can be a liquid sample. In one example, the liquid sample is a bodily fluid. Bodily fluids can include, but are not limited to, one or more of the following: whole blood, fractionated blood, serum, plasma, urine, saliva, sweat, spinal fluid, mucus (e.g., nasal discharge and sputum), amniotic fluid, aqueous humor, vitreous humor, breast milk, cerebrospinal fluid (CSF), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine, and exhaled breath condensate. In other examples, the liquid sample is a cell lysate, cell culture medium, and mixtures thereof. In certain examples, the method can be used to directly detect at least one nucleic acid analyte in a complex biological sample. In other examples, the complex biological sample can be subjected to one or more treatments (e.g., dilution, lysis, fractionation) prior to detection with a porous substrate or a device comprising a porous substrate. In some examples disclosed herein describing a complex biological sample, one or more of the sample can be obtained from a mammalian subject, such as a human. In one example, the sample is obtained from a subject, optionally a human subject, and optionally comprises or consists of whole blood, serum, plasma, urine, saliva, sweat, spinal fluid, sputum, a cell lysate, and mixtures thereof.

[0034] In other examples of the method according to the present application for detecting a nucleic acid analyte associated with an allergen or a contaminant, the complex biological sample can be a biological sample or an organic sample selected from, but not limited to, the following: a soil sample, a water sample, a plant material, a food or beverage sample, an agricultural product, a surface debris, a surface swab, or any combination thereof. In some examples disclosed herein, the complex biological sample can be obtained in liquid form, or treated into liquid form, for contact with a porous substrate.

[0035] According to any example disclosed herein, the method of the present application is capable of detecting at least one nucleic acid analyte in a complex biological sample at a concentration of about 1 pM or less.

[0036] The electrochemical sensor can comprise one or more electrodes. For example, the electrochemical sensor can comprise: - a working electrode comprising a porous substrate described herein; - a reference electrode; and - a counter electrode.

[0037] In one example, an insulating material is disposed on at least a portion of the electrode.

[0038] In one example, the electrochemical sensor is capable of scanning a range of potentials and measuring current. In one example of the method, the electrochemical sensor comprises a potentiostat.

[0039] In one example, the change in peak current is normalized via the relationship of equation (1): (1)

[0040] wherein is the normalized current change, is the peak current value measured after incubation in the buffer for a first time period, and I n is the peak current value measured after incubation in the target solution at a given concentration for a second time period compared to a control sample.

[0041] In one example, the first time period and / or the second time period is independently about 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, or 20 minutes or less. For example, the first time period and / or the second time period is independently about 60 minutes or less. For example, the first time period and / or the second time period is independently about 50 minutes or less. For example, the first time period and / or the second time period is independently about 40 minutes or less. For example, the first time period and / or the second time period is independently about 30 minutes or less. For example, the first time period and / or the second time period is independently about 20 minutes or less. In some examples, the first time period and the second time period are the same. In other examples, the first time period and the second time period are different.

[0042] In one example, the method comprises determining the concentration of the nucleic acid analyte detected in the complex biological sample based on the level of decrease in peak current intensity. In some examples, the method further comprises comparing the concentration of the at least one nucleic acid analyte to a calibration curve.

[0043] In one particular example, the complex biological sample is: - from a human; and / or - a liquid sample; and / or - selected from the group consisting of: whole blood, serum, plasma, urine, saliva, sweat, spinal fluid, sputum, cell lysate, cell culture media, and mixtures thereof; and / or - obtained from a subject suspected of having a brain injury (e.g., TBI).

[0044] Further, while diagnostic applications in humans are expressly contemplated, diagnostic applications in non-human animals are also contemplated, such as, but not limited to: livestock, companion animals, working animals, and laboratory animals. Thus, the subject in which a condition, disease, or injury is to be detected can be any subject. Thus, the porous substrate can be tailored to detect one or more specific biomarkers associated with a particular species and / or target condition.

[0045] In one example, the at least one nucleic acid analyte is detected, and / or the concentration thereof is quantified, within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 15 minutes, or about 10 minutes of introducing the sample into the electrochemical biosensor. For example, the nucleic acid analyte can be detected, and / or the concentration thereof quantified, within about 60 minutes of introducing the sample into the electrochemical biosensor. For example, the nucleic acid analyte can be detected, and / or the concentration thereof quantified, within about 50 minutes of introducing the sample into the electrochemical biosensor. For example, the nucleic acid analyte can be detected, and / or the concentration thereof quantified, within about 40 minutes of introducing the sample into the electrochemical biosensor. For example, the nucleic acid analyte can be detected, and / or the concentration thereof quantified, within about 30 minutes of introducing the sample into the electrochemical biosensor. For example, the nucleic acid analyte can be detected, and / or the concentration thereof quantified, within about 20 minutes or less of introducing the sample into the electrochemical biosensor.

[0046] In a second aspect, the present application provides a porous substrate for use in the method of the first aspect, the porous substrate comprising a plurality of channels, wherein: - at least a portion of the plurality of channels has an average depth of about 1.0 pm to about 6.0 pm; and / or - at least a portion of the plurality of channels has an average diameter or cross-section in the range of about 15 nm to about 40 nm, wherein one or more capture probes capable of specifically binding at least a biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex are disposed on at least one surface of at least a portion of the plurality of channels.

[0047] In one example, the capture probes are capable of specifically binding at least one nucleic acid analyte to form a probe-analyte complex, wherein the presence of the probe-analyte complex within a channel results in at least partial steric and / or charge blockage of the channel compared to a channel in which the probe-analyte complex is not present.

[0048] In one example, at least a portion of the plurality of channels has an average depth ranging from about 1.0 μιη to about 6.0 μιη, and at least a portion of the plurality of channels has an average diameter or cross-section ranging from about 15 nm to about 40 nm. For example, at least a portion of the plurality of channels has an average depth ranging from about 1.0 μιη to about 6.0 μιη, and an average diameter or cross-section ranging from about 15 nm to about 40 nm.

[0049] In one example, at least 10% of the plurality of channels has an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 15% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 20% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 25% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 30% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 35% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 40% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 45% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, at least 50% of the plurality of channels can have an average depth ranging from about 1.0 μιη to about 6.0 μιη.

[0050] Alternatively, or in addition to at least 10% of the plurality of channels having an average depth in a range of about 1.0 pm to about 6.0 pm as described above, at least 10% of the plurality of channels can have an average diameter or cross-section in a range of about 15 nm to about 40 nm. For example, at least 15% of the plurality of channels can have an average diameter or cross-section in a range of about 15 nm to about 40 nm. For example, at least 20% of the plurality of channels can have an average diameter or cross-section in a range of about 15 nm to about 40 nm. For example, at least 25% of the plurality of channels can have an average diameter or cross-section in a range of about 15 nm to about 40 nm. For example, at least 30% of the plurality of channels can have an average diameter or cross-section in a range of about 15 nm to about 40 nm. For example, at least 35% of the plurality of channels can have an average diameter or cross-section in a range of about 15 nm to about 40 nm. For example, at least 40% of the plurality of channels can have an average diameter or cross-section in a range of about 15 nm to about 40 nm. For example, at least 45% of the plurality of channels can have an average diameter or cross-section in a range of about 15 nm to about 40 nm. For example, at least 50% of the plurality of channels can have an average diameter or cross-section in a range of about 15 nm to about 40 nm.

[0051] In some examples, the porous substrate comprises, consists essentially of, or consists of an electrically conductive or semiconductive material. In some examples, the porous substrate comprises, consists essentially of, or consists of a material selected from the group consisting of platinum, gold, silver, cobalt, gallium, indium, tin, vanadium, zirconium, copper, aluminum, iridium, palladium, rhodium, silicon, zinc, iron, steel, brass, carbon, graphite, alloys, oxides, or metal compounds of these elements, and mixtures thereof. For example, the porous substrate can comprise, consist essentially of, or consist of silicon.

[0052] In some examples, a layer comprising, consisting essentially of, or consisting of carbon can be disposed on at least a portion of the porous substrate.

[0053] In some examples disclosed herein, one or more capture probes are capable of specifically binding to one or more nucleic acid analytes. In some examples, the porous substrate comprises a plurality of capture probes, each of which is capable of specifically binding to one or more nucleic acid analytes.

[0054] The at least one nucleic acid analyte can be a biomarker. For example, the nucleic acid can be selected from the group consisting of: a DNA analyte, a messenger RNA (mRNA) analyte, a ribosomal RNA (rRNA) analyte, a transfer RNA (tRNA) analyte, a microRNA (miRNA) analyte, a short interfering RNA (siRNA) analyte, a plasmid DNA (pDNA), and mixtures thereof. In one example, the nucleic acid analyte is a miRNA.

[0055] The capture probes can be selected to suit the nucleic acid analyte to be detected. For example, one or more of the at least one capture probe can be a DNA capture probe or a peptide nucleic acid probe (PNA) probe. In one example, the at least one capture probe is a DNA capture probe. In one example, the at least one capture probe is a PNA probe. In any of the examples disclosed herein, the capture probes can have modified chemical properties, such as to improve stability, improve binding affinity, and / or reduce off-target effects of the capture probes. Modified chemical properties of DNA and PNA probes are known in the art and are contemplated herein.

[0056] Accordingly, the present application provides a porous substrate as described herein for use in detecting at least one nucleic acid analyte. Exemplary types of nucleic acid analytes are described herein and can be selected from the group consisting of: a DNA analyte, a messenger RNA (mRNA) analyte, a ribosomal RNA (rRNA) analyte, a transfer RNA (tRNA) analyte, a microRNA (miRNA) analyte, a short interfering RNA (siRNA) analyte, a plasmid DNA (pDNA), and mixtures thereof.

[0057] As described herein, it is contemplated that the porous substrate of the present application can be used to detect a nucleic acid analyte or biomarker associated with a condition, disease, or injury. Accordingly, the at least one nucleic acid analyte can be associated with a condition, disease state, or injury. In one specific example, the at least one nucleic acid analyte is associated with a brain injury, optionally a traumatic brain injury (TBI). However, other medical indications are described and contemplated herein.

[0058] In one specific example, the porous substrate comprises one or more capture probes capable of specifically binding one or more miRNAs associated with TBI in a complex biological sample (e.g., blood, a blood fraction, or a diluted blood solution). For example, the one or more miRNAs can be selected from the group consisting of miR-142-3p, miR-150-5p, miR-196b-5p, let-7f-5p, miR-196b-5p, and combinations thereof. The porous substrate of the present disclosure can be used to detect nucleic acid analytes in a range of samples and sample types. However, in one example, the sample is a liquid sample. Alternatively, the sample has been prepared or treated into a liquid form. The liquid sample can comprise or consist of a biological sample or medium, such as a complex biological sample or medium. In one example, the liquid sample is a bodily fluid. Bodily fluids can include, but are not limited to, one or more of the following: whole blood, fractionated blood, serum, plasma, urine, saliva, sweat, spinal fluid, mucus (e.g., nasal discharge and sputum), amniotic fluid, aqueous humor, vitreous humor, breast milk, cerebrospinal fluid (CSF), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine, and exhaled breath condensate.

[0059] In other examples, the liquid sample is a cell lysate, a cell culture medium, and mixtures thereof. In certain examples, the porous substrate can be used to directly detect at least one nucleic acid analyte in a liquid sample. In other examples, the liquid sample can be subjected to one or more treatments (e.g., dilution, lysis, fractionation) prior to detection with the porous substrate or a device comprising the porous substrate. In some examples disclosed herein that describe biological liquid samples, the sample can be obtained from a mammalian subject, such as a human. In one example, the sample is obtained from a subject, optionally a human subject, and optionally comprises or consists of whole blood, serum, plasma, urine, saliva, sweat, spinal fluid, sputum, a cell lysate, and mixtures thereof.

[0060] In other examples of the porous substrate according to the present disclosure for detecting nucleic acid analytes associated with allergens or contaminants, the sample can be a biological or organic sample selected from, but not limited to, the following: a soil sample, a water sample, a plant material, a food or beverage sample, an agricultural product, a surface debris, a surface swab, or any combination thereof. In some examples disclosed herein, the biological or organic sample can be obtained in a liquid form, or treated into a liquid form, for use with the porous substrate.

[0061] According to any of the examples disclosed herein, the porous substrate is capable of detecting at least one nucleic acid analyte in a sample at a concentration of about 1 pM or less. When used to detect at least one nucleic acid analyte in a sample, the porous substrate, electrode, electrochemical cell, and / or device as described herein should be capable of completing detection within 20, 30, 40, 50, or 60 minutes of contact with the sample. In one example, the porous substrate, electrode, electrochemical cell, and / or device as described herein is capable of detecting at least one nucleic acid analyte in a sample within 20 minutes of contact with the sample. In one example, the porous substrate, electrode, electrochemical cell, and / or device as described herein is capable of detecting at least one nucleic acid analyte in a sample within 30 minutes of contact with the sample. In one example, the porous substrate, electrode, electrochemical cell, and / or device as described herein is capable of detecting at least one nucleic acid analyte in a sample within 40 minutes of contact with the sample. In one example, the porous substrate, electrode, electrochemical cell, and / or device as described herein is capable of detecting at least one nucleic acid analyte in a sample within 50 minutes of contact with the sample. In one example, the porous substrate, electrode, electrochemical cell, and / or device as described herein is capable of detecting at least one nucleic acid analyte in a sample within 60 minutes of contact with the sample. BRIEF DESCRIPTION OF DRAWINGS

[0062] While it should be understood that various embodiments disclosed herein can be utilized, a number of examples will be described herein in connection with the following drawings:

[0063] Figure 1 Top surface and cross-sectional SEM images of various thermal hydrocarbonated porous silicon (THCpSi) samples prepared under different conditions in Example 1 are depicted.

[0064] Figure 2 Water contact angle measurements of pSi and THCpSi samples prepared in Example 1 are depicted.

[0065] Figure 3 FTIR spectra of freshly etched pSi, THCpSi, and thermal hydrosilation of THCpSi prepared in Example 2 are depicted.

[0066] Figure 4 (a) Cyclic voltammograms of freshly etched pSi, THCpSi, COOH-terminated THCpSi, and ssDNA-modified THCpSi electrodes prepared according to Example 3, and (b) Electrochemical impedance spectra of THCpSi, COOH-terminated THCpSi, and ssDNA-modified THCpSi electrodes are depicted.

[0067] Figure 5Differential pulse voltammograms (DPVs) of control and working electrodes made of THCpSi with different thicknesses according to Example 4 were plotted, as well as dose response curves of the biosensor made of THCpSi.

[0068] Figure 6 Differential pulse voltammograms (DPVs) of control and working electrodes made of THCpSi with different channel sizes according to Example 4 are depicted, as well as dose response curves of the biosensor made of THCpSi.

[0069] Figure 7 The sensing of target miRNA (miR-142-3p) using an optimized THCpSi-based nanochannel blocking biosensor according to Example 5 is described.

[0070] Figure 8 Differential pulse voltammograms (DPVs) of the biosensor according to Example 6 after incubation in clinical samples were depicted.

[0071] Figure 9 The calculated concentrations of the miR-142-3p biomarker were depicted in three different clinical human serum samples.

[0072] Figure 10 The assembly steps of the screen-printed porous silicon electrode according to Example 7 are described.

[0073] Figure 11 The insulating ink screen printing performed on a silicon wafer according to Example 7 is depicted.

[0074] Figure 12 Different templates for printing different electrode designs according to Example 7 are depicted.

[0075] Figure 13 A novel electrode design utilizing a pSi working electrode, an Ag / AgCl reference electrode, and a carbon-based counter electrode, according to Example 7, is described. Detailed Implementation

[0076] General Definitions

[0077] Unless otherwise expressly defined, all technical and scientific terms used herein should be assumed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., chemistry, biochemistry, engineering, physics, etc.).

[0078] As used herein, the term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be considered to provide explicit support for both meanings or either meaning, such as A and / or B including options i) A, ii) B or iii) A and B.

[0079] As used herein, the term about means ±20%, or ±10%, or ±5% of the indicated value, unless otherwise indicated.

[0080] As used herein, the terms "a," "an," and "the" include both the singular and plural aspects, unless the context clearly dictates otherwise.

[0081] It will be appreciated that, for clarity, purposes, certain features have been described herein with respect to individual embodiments separately. Conversely, for brevity, features described with respect to a single embodiment can also be provided separately or in any subcombination.

[0082] Throughout this specification, various aspects and components of the application can be presented in a range format. The range format is included to provide flexibility and to allow for a broadening of the range when specific values within the range are explicitly recited. Unless otherwise stated, the description of a range format is meant to include all possible sub-ranges within the range. For example, describing a range as 1 to 5 is intended to convey that the range includes all possible sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 5, 3 to 5, etc., as well as individual values and non-integer values within the indicated range, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc. This same applies to ranges reciting integers and fractions, such as 1 to 5.5, 2.5 to 4.5, etc. Unless otherwise indicated, the description of a range format is meant to include all possible sub-ranges within the range.

[0083] Throughout this specification, the word "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0084] Throughout this specification, the term "consisting essentially of" or variations such as "consists essentially of" or "consisting essentially thereof" will be understood to permit the inclusion of additional an element, integer or step of matter not specifically-recited in the range format, provided that such additional element, integer or step of matter does not materially affect the characteristics of the composition claimed.

[0085] It will be clearly understood that, although a number of prior art publications can be referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country or jurisdiction.

[0086] In the present text, the term "channel" or "channels" can be used interchangeably with "pore" or "pores".

[0087] In the present text, "reference electrode" refers to an electrode with a precisely maintained potential, used as a reference for measurements of other electrodes.

[0088] In the present text, "counter electrode" (which can also be referred to as "auxiliary electrode") refers to an electrode used only to carry the current through the electrochemical cell.

[0089] In the present text, "working electrode" refers to an electrode comprising or consisting of a porous substrate as described herein, in which one or more reactions can take place.

[0090] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described below. In case of conflict, the

[0091] As used herein, the term "treating" (or "treat", "treatment", etc.) includes detecting, identifying, alleviating, relieving, and / or eliminating one or more symptoms associated with a particular disorder or condition.

[0092] As used herein, the term "subject" can be used interchangeably with the terms "patient", "recipient" and "individual". In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The subject can be a human, male or female.

[0093] Traumatic Brain Injury

[0094] In an embodiment, the “traumatic brain injury” or “TBI” can be a mild, moderate, or severe traumatic brain injury. In an embodiment, the TBI is a mild TBI, such as a concussion. In an embodiment, the mild TBI can involve one or more symptoms selected from the group consisting of: headache, dizziness, insomnia, decreased attention and concentration span, depression, anxiety, mood swings, impaired balance, reduced information processing speed, decreased ability to learn new things and memory, and combinations thereof. In an embodiment, the TBI is a moderate TBI. The moderate TBI can include hemorrhage. In an embodiment, the moderate TBI can include brain hemorrhage and / or loss of consciousness. In an embodiment, the TBI is a severe TBI. The severe TBI can include a prolonged unconscious state or coma lasting days, weeks, or months. Symptoms of a severe TBI can include one or more of the following: loss of consciousness, headache, nausea, vomiting, lack of coordination, dizziness, balance disorder, dilated pupil on one or both sides, slurred speech, behavioral or emotional changes, loss of coordination, restlessness, and agitation, and combinations thereof.

[0095] In this document, the TBI can be the result of an “injury to the head” or “head injury.” In this document, “injury to the head” or “head injury” are used interchangeably to refer to any trauma to the scalp, skull, or brain. Such an injury can include only a minor bump on the head, or it can be a severe brain injury. Such an injury can include primary brain injury and / or secondary brain injury. Primary brain injury occurs during the initial injury and results from the displacement of the physical structure of the brain. More specifically, primary brain injury is a physical damage to the parenchyma (tissue, blood vessels) that occurs during the traumatic event, resulting in shearing and compression of surrounding brain tissue. Secondary brain injury occurs after the primary injury and can involve a series of cellular processes. More specifically, secondary brain injury refers to changes that develop over a period of time (from hours to days) after the primary brain injury. It includes a complete cascade of cellular, chemical, tissue, or vascular changes in the brain that further damage brain tissue.

[0096] A head injury can be a closed injury or an open injury (penetrating injury). A closed head injury refers to trauma to the scalp, skull, or brain where the striking object did not penetrate the skull. An open head injury refers to trauma to the scalp, skull, or brain where the striking object penetrated the skull. A head injury can be caused by physical jolts to the body, mechanical external forces or other external forces that result in closed or open head trauma (e.g., vehicle accidents such as cars, planes, trains, etc.; blunt force trauma such as hitting the head with a baseball bat, or injuries caused by a firearm), cerebrovascular accidents (e.g., stroke), one or more falls (e.g., falls during sports or other activities), explosions or blasts (collectively referred to as “explosive injuries”), and other types of blunt force trauma. Alternatively, a head injury can be caused by ingestion and / or exposure to a chemical, a toxin, or a combination of chemicals and toxins. Examples of such chemicals and / or toxins include fire, mold, asbestos, pesticides and insecticides, organic solvents, paint, glue, gases such as carbon monoxide, hydrogen sulfide, and cyanide, organic metals such as methylmercury, tetraethyl lead, and organotin, and / or one or more drugs of abuse. Alternatively, a head injury can be caused by the subject having an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof. In some cases, it can not be possible to determine whether any such event or injury has occurred or happened. For example, the patient or subject can not have a history, the subject can not be able to speak, the subject can know which events they have been exposed to, etc. Such cases can be described as the subject “can have suffered a head injury” or as a “suspected injury.” In certain embodiments herein, a closed head injury can not include and expressly exclude a cerebrovascular accident such as a stroke.

[0097] In this document, a TBI can comprise or result in one or more signs and / or symptoms, optionally selected from, but not limited to: headache, convulsions or seizures, blurred or double vision, unequal or dilated pupils, clear fluid draining from the nose or ears, nausea and vomiting, new neurologic deficits such as slurred speech, weakness in arms, legs, or face; loss of balance, loss or change of consciousness ranging from seconds to hours, decreased level of consciousness (e.g., difficulty waking up), mild to severe confusion or disorientation, impairment of memory, attention, or decision-making abilities, changes in sleep patterns (e.g., sleeping more, difficulty falling asleep, or difficulty staying asleep); inability to wake up from sleep, frustration, irritability, sensory / perceptual abnormalities, dizziness, vertigo, or loss of balance or coordination, blurred vision, hearing abnormalities such as tinnitus, bad taste in mouth, sensitivity to light or sound, emotional changes or fluctuations, agitation, aggression, or other abnormal behaviors, feelings of anxiety or depression, fatigue or lethargy; lack of energy or motivation, and combinations thereof.

[0098] Porous Substrate

[0099] Disclosed herein is a porous substrate for detecting nucleic acid analytes (e.g., nucleic acid biomarkers) in complex biological samples. The porous substrate for use according to the present application comprises a plurality of channels, wherein:

[0100] - at least a portion of the plurality of channels has an average depth of about 1.0 pm to about 6.0 pm; and / or

[0101] - at least a portion of the plurality of channels has an average diameter or cross-section in the range of about 15 nm to about 40 nm, wherein optionally one or more of: ■ one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more functional groups capable of reacting with amine groups; and / or ■ one or more functional groups capable of forming a covalent bond with complementary functional groups on one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more carboxylic acid groups, is disposed on at least one surface of at least a portion of the plurality of channels.

[0102] The porous substrate can comprise one or more “layers”, wherein each layer comprises one or more different chemical functional groups and / or modifications.

[0103] In one embodiment, the porous substrate comprises silicon, wherein the silicon comprises at least one layer that has been modified by at least one of heat and / or chemical treatment.

[0104] In another embodiment, the porous substrate optionally comprising, consisting essentially of, or consisting of silicon comprises a single layer, consists essentially of, or consists of a single layer, which single layer comprises one or more of: ■ one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more functional groups capable of reacting with amine groups; and / or ■ one or more functional groups capable of forming a covalent bond with complementary functional groups on one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more carboxylic acid groups.

[0105] In another embodiment, the porous substrate optionally comprising silicon, consisting essentially of silicon, or consisting of silicon comprises a layer comprising one or more of: ■one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more functional groups capable of reacting with amine groups; and / or ■one or more functional groups capable of forming a covalent bond with complementary functional groups on one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more carboxylic acid groups.

[0106] In another embodiment, the porous substrate optionally comprising silicon, consisting essentially of silicon, or consisting of silicon comprises at least one layer comprising one or more of: ■one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more functional groups capable of reacting with amine groups; and / or ■one or more functional groups capable of forming a covalent bond with complementary functional groups on one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more carboxylic acid groups.

[0107] In another embodiment, the porous substrate optionally comprising silicon, consisting essentially of silicon, or consisting of silicon does not comprise, consists essentially of, or consists of a plurality of layers comprising one or more of: ■one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more functional groups capable of reacting with amine groups; and / or ■one or more functional groups capable of forming a covalent bond with complementary functional groups on one or more capture probes capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more carboxylic acid groups.

[0108] As used herein, the term "probe" or "capture probe" can refer to a capture molecule having sufficient binding properties to specifically bind to a target analyte. For example, a probe can include a polynucleotide having sufficient complementarity to specifically hybridize to a target nucleic acid. In another example, a probe includes an antibody or protein tag. A capture probe can function as an affinity binding molecule for separating a target nucleic acid from other nucleic acids and / or components in a mixture. A target nucleic acid can also be specifically bound by a capture probe through an intervening molecule, such as a linker, adaptor, and other bridging nucleic acids having sufficient complementarity to specifically hybridize to both the target sequence and the capture probe. The term "capture probe" can also refer to a small organic molecule or polymer having sufficient binding properties to specifically bind to a target nucleic acid analyte.

[0109] In one embodiment, a capture probe is capable of specifically binding to at least one biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex, wherein the presence of the probe-analyte complex within a channel results in at least partial steric and / or charge blockage of at least a portion of the plurality of channels as compared to the plurality of channels in the absence of the probe-analyte complex. In some embodiments herein, the term "nucleic acid analyte" can be replaced with "biomarker."

[0110] The selection and number of capture probes can be tailored to the target-specific analyte. For example, a plurality of identical capture probes can be employed to increase selectivity for a single nucleic acid analyte. In other examples, a plurality of different capture probes can be employed, each capable of specifically binding to a different nucleic acid analyte, to enable simultaneous detection of multiple nucleic acid analytes. In the latter case, this can be advantageous in the case of diagnosing a single injury or medical condition using a plurality of biomarkers (e.g., a panel of biomarkers). Alternatively, a plurality of different capture probes can be used to detect a plurality of nucleic acid analytes, each associated with a different injury or medical condition, thereby allowing for simultaneous screening of multiple injuries or conditions. In some examples disclosed herein, the multi-well substrate and / or electrodes as described herein can be produced or designed to be replaceable in an article or device as described herein.

[0111] The plurality of channels can have various shapes, including circular, substantially circular, oval, substantially oval, regular, and / or irregular. The shape of the plurality of channels can be substantially the same across the multi-well substrate, or the shape can vary across different portions of the multi-well substrate. For at least a portion of the plurality of channels, the shape of the cross-section can be uniform, substantially uniform, or varying, e.g., along a particular dimension, such as depth, in one or more channels.

[0112] In one embodiment, at least a portion of the plurality of channels has an average depth of about 1.0 μm to about 6.0 μm. For example, the average depth is in a range of about 1.0 μm to about 2.0 μm or about 1.0 μm to about 4.0 μm. In another embodiment, at least a portion of the plurality of channels has an average diameter or cross-section in a range of about 15 nm to about 40 nm. In yet another embodiment, at least a portion of the plurality of channels has an average depth of about 1.0 μm to about 6.0 μm and at least a portion of the plurality of channels has an average diameter or cross-section in a range of about 15 nm to about 40 nm. For example, at least a portion of the plurality of channels has an average depth of about 1.0 μm to about 6.0 μm and an average diameter or cross-section in a range of about 15 nm to about 40 nm.

[0113] In one embodiment, the average depth of at least a portion of the plurality of channels is about or at least about: 1.00 pm, 1.10 pm, 1.20 pm, 1.30 pm, 1.40 pm, 1.50 pm, 1.60 pm, 1.70 pm, 1.80 pm, 1.90 pm, 2.00 pm, 2.10 pm, 2.20 pm, 2.30 pm, 2.40 pm, 2.50 pm, 2.60 pm, 2.70 pm, 2.80 pm, 2.90 pm, 3.00 pm, 3.10 pm, 3.20 pm, 3.30 pm, 3.40 pm, 3.50 pm, 3.60 pm, 3.70 pm, 3.80 pm, 3.90 pm, 4.00 pm, 4.10 pm, 4.20 pm, 4.30 pm, 4.40 pm, 4.50 pm, 4.60 pm, 4.70 pm, 4.80 pm, 4.90 pm, 5.00 pm, 5.10 pm, 5.20 pm, 5.30 pm, 5.40 pm, 5.50 pm, 5.60 pm, 5.70 pm, 5.80 pm, 5.90 pm, or 6.00 pm. In another embodiment, the average depth of at least a portion of the plurality of channels is less than about: 6.00 pm, 5.90 pm, 5.80 pm, 5.70 pm, 5.60 pm, 5.50 pm, 5.40 pm, 5.30 pm, 5.20 pm, 5.10 pm, 5.00 pm, 4.90 pm, 4.80 pm, 4.70 pm, 4.60 pm, 4.50 pm, 4.40 pm, 4.30 pm, 4.20 pm, 4.10 pm, 4.00 pm, 3.90 pm, 3.80 pm, 3.70 pm, 3.60 pm, 3.50 pm, 3.40 pm, 3.30 pm, 3.20 pm, 3.10 pm, 3.00 pm, 2.90 pm, 2.80 pm, 2.70 pm, 2.60 pm, 2.50 pm, 2.40 pm, 2.30 pm, 2.20 pm, 2.10 pm, 2.00 pm, 1.90 pm, 1.80 pm, 1.70 pm, 1.60 pm, 1.50 pm, 1.40 pm, 1.30 pm, 1.20 pm, or 1.10 pm. In yet another embodiment, the average depth of at least a portion of the plurality of channels can range from any of the values described above.

[0114] In one embodiment, at least about 10% of the plurality of channels have an average depth ranging from about 1.0 μιη to about 6.0 μιη. For example, about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the plurality of channels have an average depth ranging from about 1.0 μιη to about 6.0 μιη. This can be measured by any appropriate technique known in the art, such as scanning electron microscopy (SEM).

[0115] Alternatively, or in addition to at least a portion of the plurality of channels having an average depth in the range of about 1.0 μιη to about 6.0 μιη as described above, at least a portion of the plurality of channels can have an average diameter or cross-section in the range of about 15 nm to about 40 nm. For example, in the range of about 15 nm to about 30 nm or about 20 nm to about 30 nm. In one embodiment, at least a portion of the plurality of channels has an average diameter or cross-section of about or at least about: 15.00 nm, 15.50 nm, 16.00 nm, 16.50 nm, 17.00 nm, 17.50 nm, 18.00 nm, 18.50 nm, 19.00 nm, 19.50 nm, 20.00 nm, 20.50 nm, 21.00 nm, 21.50 nm, 22.00 nm, 22.50 nm, 23.00 nm, 23.50 nm, 24.00 nm, 24.50 nm, 25.00 nm, 25.50 nm, 26.00 nm, 26.50 nm, 27.00 nm, 27.50 nm, 28.00 nm, 28.50 nm, 29.00 nm, 29.50 nm, 30.00 nm, 30.50 nm, 31.00 nm, 31.50 nm, 32.00 nm, 32.50 nm, 33.00 nm, 33.50 nm, 34.00 nm, 34.50 nm, 35.00 nm, 35.50 nm, 36.00 nm, 36.50 nm, 37.00 nm, 37.50 nm, 38.00 nm, 38.50 nm, 39.00 nm, or 39.50 nm.In another embodiment, at least a portion of the plurality of channels has an average diameter or cross-section of less than about: 40.00 nm, 39.50 nm, 39.00 nm, 38.50 nm, 38.00 nm, 37.50 nm, 37.00 nm, 36.50 nm, 36.00 nm, 35.50 nm, 35.00 nm, 34.50 nm, 34.00 nm, 33.50 nm, 33.00 nm, 32.50 nm, 32.00 nm, 31.50 nm, 31.00 nm, 30.50 nm, 30.00 nm, 29.50 nm, 29.00 nm, 28.50 nm, 28.00 nm, 27.50 nm, 27.00 nm, 26.50 nm, 26.00 nm, 25.50 nm, 25.00 nm, 24.50 nm, 24.00 nm, 23.50 nm, 23.00 nm, 22.50 nm, 22.00 nm, 21.50 nm, 21.00 nm, 20.50 nm, 20.00 nm, 19.50 nm, 19.00 nm, 18.50 nm, 18.00 nm, 17.50 nm, 17.00 nm, 16.50 nm, 16.00 nm, or 15.50 nm. In yet another embodiment, at least a portion of the plurality of channels can have an average diameter or cross-section in the range of any of the above values.

[0116] In one embodiment, at least about 10% of the plurality of channels has an average diameter or cross-section in the range of about 15 nm to about 40 nm. For example, about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the plurality of channels has an average diameter or cross-section in the range of about 15 nm to about 40 nm. This can be measured by any appropriate technique known in the art, such as SEM.

[0117] The overall dimensions of the porous substrate used in accordance with the present application can vary and can be dictated by the end application or use of the porous substrate, for example based on any sample used in conjunction with the porous substrate, and / or based on the device in which the porous substrate is present as a component, for example as part of an electrode, electrochemical sensor, and / or device.

[0118] The porous substrate can comprise, consist essentially of, or consist of any suitable electrically conductive or semiconductive material in any suitable form. Examples of suitable forms include, but are not limited to, a wire, a rod, a disc, a foil, a wafer, or a chip. The electrically conductive or semiconductive material can be in the form of a coating deposited on another substrate, such as a non-conductive substrate, by any known deposition process. Suitable materials include, but are not limited to, platinum, gold, silver, cobalt, gallium, indium, tin, vanadium, zirconium, copper, aluminum, iridium, palladium, rhodium, silicon, zinc, iron, steel, brass, carbon (e.g., graphite), alloys, oxides, or metal compounds of these elements, etc., and mixtures thereof. Examples of semiconductors include, but are not limited to, silicon semiconductors, germanium semiconductors, and indium tin oxide semiconductors, and mixtures thereof, such as silicon-germanium hybrid semiconductors. The semiconductors can be n-type or p-type doped semiconductors (e.g., silicon semiconductors). If present, the dopants can be conventional n-type dopants, such as phosphorus, boron, or antimony.

[0119] In one embodiment, the porous substrate comprises a wafer. In another embodiment, the porous substrate comprises silicon, optionally a silicon wafer. In yet another embodiment, at least a portion of the silicon (e.g., silicon wafer) can be modified via chemical and / or physical treatment.

[0120] In one embodiment, the porous substrate is not in the form of a thin film.

[0121] In one embodiment, the porous substrate does not comprise, or consist of, nanoparticles.

[0122] In one embodiment, the porous substrate does not comprise one or more of: aluminum, platinum, gold, and / or silver. In another embodiment, the porous substrate does not comprise an alloy comprising gold and / or silver.

[0123] In one embodiment, the porous substrate comprises, consists essentially of, or consists of a material selected from silicon. The silicon can be doped and can be n-type or p-type silicon.

[0124] The surface of the porous substrate can be modified to introduce and / or immobilize a compound (optionally one or more capture probes) and / or a functional group. For example, linkers, functional groups, and / or capture probes can be attached to the porous substrate, either directly or indirectly, by any means known in the art, including covalent and non-covalent interactions, or any combination thereof (see, e.g., Chan et al., 2007, PLoS One 2:ell64; Cazalis et al., Bioconj. Chem. 15: 1005-1009; Soellner et al., 2003, J. Am. Chem. Soc. 125: 11790-11791; Sun et al., 2006, Bioconjug. Chem. 17-52-57; Decreau et al., 2007, J. Org. Chem. 72:2794-2802; Camarero et al., 2004, J. Am. Chem. Soc. 126: 14730-14731; Girish et al., 2005, Bioorg. Med. Chem. Lett. 15:2447-2451; Kalia et al., 2007, Bioconjug. Chem. 18: 1064-1069; Watzke et al., 2006, Angew. Chem. Int. Ed. Engl. 45: 1408-1412; Parthasarathy et al., 2007, Bioconjugate Chem. 18:469-476; and Bioconjugate Techniques, G. T. Hermanson, Academic Press (2013), each of which is incorporated herein by reference in its entirety). Any suitable molecule or material can be used for this purpose, including proteins, nucleic acids, carbohydrates, and small molecules. For example, in one embodiment, the capture probe or surface of the substrate comprises an azido group, which can react with an alkyne on another entity to facilitate association or binding.

[0125] The surface of the porous substrate can be modified using methods known as "click chemistry." Exemplary reactions include copper-catalyzed azide and alkyne reaction to form triazoles (Huisgen 1,3-dipolar cycloaddition), strain-promoted azide alkyne cycloaddition (SPAAC), diene and dienophile reaction (Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, strain alkenes and azides, tetrazines or tetrazoles, alkenes and azides [3+2] cycloaddition, alkenes and tetrazine reverse electron demand Diels-Alder (IEDDA) reaction (e.g., m-tetrazine (mTet) or phenyltetrazine (pTet) with trans-cyclooctene (TCO); or pTet with alkenes), alkenes and tetrazole photoreaction, Staudinger ligation of azides and phosphines, and various displacement reactions, such as a nucleophile attacking an electrophilic atom to displace a leaving group (e.g., Horisawa, Front Physiol (2014). 5: 457; Knall, Hollauf et al., Tetrahedron Lett (2014) 55(34): 4763-4766). Exemplary displacement reactions include amines reacting with activated esters; N-hydroxysuccinimidyl esters; isocyanates; isothiocyanates, aldehydes, epoxides, and the like.

[0126] The porous substrate used in accordance with the present application can comprise one or more functional groups capable of reacting with one or more complementary functional groups on the capture probe, and / or a linking compound (or "tether") that can be subsequently modified or used to immobilize at least one capture probe to the substrate. Examples of functional groups can be independently selected from, but are not limited to, an a,b-unsaturated carbonyl, an acid, an acyl halide, an acyl azide, a halogenated imide, an alcohol, an aldehyde, an alkene, an alkyne, an amide, an amine, an azide, an aziridine, a biotin or thiirane functional group containing a complementary reactive group, a carbocyclic acid, a disulfide, an epoxide, an ester, an activated ester (e.g., N-hydroxysuccinimidyl ester, pentynoic acid STP ester), a halide, a heteroaryl, a heterocycle or heteroaryl, a hydrazide, a hydrazine, a hydrazone, an imidate, an imine, an isocyanate, an isothiocyanate, a ketone, a maleimide, a nitrile, a nitrone, an oxime, a phosphine, a sulfhydryl, a sulfinic acid, a sulfonic acid, a sulfonyl halide, a tetrazine, a tetrazole, a thioester, a triazole, or mixtures thereof.

[0127] For example, in one embodiment, one or more carboxylic acid groups are disposed on at least one surface of at least a portion of the plurality of channels. The carboxylic acid groups can result from the attachment of one or more compounds comprising at least one carboxylic acid group and at least one other functional group that can react with a complementary functional group on at least one surface of at least a portion of the plurality of channels. In another embodiment, one or more carboxylic acid groups of Formula (I) are disposed on at least one surface of at least a portion of the plurality of channels: Formula I

[0128] wherein n is an integer from 1 to 15, and * indicates attachment to at least one surface of at least a portion of the plurality of channels. For example, n can be: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In one embodiment, a plurality of carboxylic acid groups of Formula (I) can be disposed on at least one surface of at least a portion of the plurality of channels, wherein n is the same or different.

[0129] In one embodiment, one or more functional groups capable of reacting with an amine group are disposed on at least one surface of at least a portion of the plurality of channels. The reaction can include the formation of one or more covalent bonds. Examples of functional groups capable of reacting with an amine group include, but are not limited to: aldehyde groups, esters (optionally succinimidyl esters), anhydrides, alkyl halides, acyl halides, ketones, epoxies, carboxylic acids, isocyanates, isothiocyanates, fluorophenyl esters, and mixtures thereof.

[0130] In one embodiment, at least a portion, e.g., a layer, of the porous substrate is modified. The modification can be performed to protect underlying segments / portions of the porous substrate from degradation, e.g., to minimize or reduce degradation when the porous substrate or a portion thereof is exposed to an aqueous medium; and / or to increase the electrical conductivity of the porous substrate or a portion thereof. Optionally in the presence of a gas, e.g., nitrogen, the porous substrate can be exposed to heat and / or a carbon source, e.g., acetylene, for a period of time. In one embodiment, the porous substrate is modified such that it comprises 1, 2, 3, 4, or 5 physically and / or chemically distinct layers.

[0131] In one embodiment, one or more capture probes are disposed on at least one surface of at least a portion of the plurality of channels. In another embodiment, the one or more capture probes are capable of binding at least one nucleic acid analyte. Those skilled in the art will appreciate that the selection of the capture probe will depend on the type of nucleic acid analyte to be detected. Examples of nucleic acid analytes include, but are not limited to: DNA analytes, messenger RNA (mRNA) analytes, ribosomal RNA (rRNA) analytes, transfer RNA (tRNA) analytes, microRNA (miRNA) analytes, short interfering RNA (siRNA) analytes, plasmid DNA (pDNA), and mixtures thereof. In another embodiment, at least one capture probe is a DNA capture probe or a peptide nucleic acid probe (PNA) probe.

[0132] According to the example where the capture probe is a DNA capture probe, the probe can comprise a polynucleotide sequence of about 15 to about 70 nucleotides in length that is sufficiently complementary to a sequence of corresponding length in a nucleic acid analyte (e.g., DNA or RNA) to be detected such that the capture probe hybridizes to the sequence in the nucleic acid analyte. For example, the DNA capture probe can be 15, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides in length. As used herein, the term "sufficiently complementary" as used in the context of a capture probe and a nucleic acid analyte is intended to indicate a sufficient degree of complementarity or exact pairing so that stable and specific binding occurs between the nucleic acid sequences, e.g., between a capture probe and a sequence within a nucleic acid analyte (e.g., DNA or RNA). It will be appreciated that the sequence of the probe need not be 100% complementary to the sequence of its target nucleic acid analyte. The term encompasses sequences that are complementary to another sequence, but excludes mismatches that prevent hybridization and subsequent duplex formation. In some cases, the probe is complementary to a sequence within the target nucleic acid analyte except for 1-2 mismatches. In some cases, the sequence is complementary except for 1 mismatch. In some cases, the sequence is complementary except for 2 mismatches. In other cases, the sequence is complementary except for 3 mismatches. In other cases, the sequence is complementary except for 4 mismatches. Preferably, there is sufficient complementarity between the capture probe and the nucleic acid analyte so that hybridization can occur at a temperature that is not significantly different from the temperature of the multi-well substrate.

[0133] As used in the context of nucleic acid binding, the term "hybridise", "hybridising" or a similar term refers to the process of two complementary or substantially complementary nucleic acid sequences annealing to each other according to Watson-Crick base pairing rules.

[0134] In this context, the partial steric and / or charge occlusion of the probe-analyte complex to at least a portion of the plurality of channels can reduce the diffusion of the redox species through the channel. The redox species can be a transition metal species, where the transition metal can adopt two valence states (e.g., a metal ion (M) that can adopt M(II) and M(III) states). In some embodiments, the redox probe contains a metal ion, where the metal of the metal ion can be selected from, but not limited to, iron, ruthenium, iridium, osmium, cobalt, tungsten, and molybdenum. Examples of redox species include, but are not limited to: Fe(CN)6 3- / 4-Fe(NH3)6 3+ / 2+ Fe(phen)3 3+ / 2+ Fe(bipy)2 3+ / 2+ Fe(bipy)3 3+ / 2+ Ru3 3+ / 2+ RuO4 3- / 2- Ru(CN)6 3- / 4- Ru(NH3)6 3+ / 2+ Ru(en)3 3+ / 2+ Ru(NH3)5(Py) 3+ / 2+ Ir 4+ / 3+ Ir(Cl)6 2- / 3- Ir(Br)6 2- / 3- Os(bipy)2 3+ / 2+ Os(bipy)3 3+ / 2+ OxC16 2- / 3- Co(NH3)6 3+ / 2+ W(CN)6 3- / 4- Mo(CN)6 3- / 4- optionally substituted ferrocene, polyferrocene, quinones such as p-benzoquinone and hydroquinone, and phenol. In particular embodiments, the redox species is a ferrous species, where iron is in the Fe(II) and / or Fe(III) state. The redox species can be present in the biological sample in an amount of 0.1 mM to 100 mM, optionally 0.5 mM to 10 mM, optionally 0.5 mM to 2 mM, optionally 0.5 mM to 1.5 mM, optionally about 1 mM.

[0135] In one embodiment, the porous substrate is used or can be used to detect at least one nucleic acid analyte in a complex biological sample, optionally wherein the nucleic acid analyte is selected from, but not limited to: a messenger RNA (mRNA) analyte, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a microRNA (miRNA), a short interfering RNA (siRNA), and mixtures thereof. The at least one nucleic acid analyte can be associated with a disease state or injury. One exemplary injury for which the porous substrate can be used is a brain injury, optionally a traumatic brain injury (TBI). Exemplary nucleic acid analytes for detecting TBI include microRNAs described in PCT / US2015 / 051518, the entire contents of which are incorporated herein by reference in their entirety. For example, the miRNAs associated with TBI can be selected from miR-142-3p, miR-196b-5p, let-7f-5p, miR-150-5p, miR-196b-5p, and combinations thereof. Exemplary capture probes for detecting the above miRNAs are described in Table 1 of the Examples herein. However, one skilled in the art will appreciate that the porous substrate can be used to detect nucleic acid analytes associated with any injury, disorder, or disease, including but not limited to: cancer, kidney disease (including acute kidney injury), cardiovascular disease, autoimmune disease, infectious disease, metabolic disease, liver disease, and blood disease. In addition, the porous substrate can be used to detect nucleic acid analytes associated with a target pathogen and / or contaminant. Suitable pathogens include, but are not limited to, a virus, a bacterium, an algae, a fungus, a prion, or a protozoan. The pathogen can cause a disease or disorder in a human or non-human animal. In other examples, the pathogen can be a plant pathogen.

[0136] In one embodiment, the porous substrate can be used to detect at least one nucleic acid analyte in a liquid sample. The liquid sample can comprise or consist of a biological sample or medium, such as a complex biological sample or medium. In one example, the liquid sample is a bodily fluid. Targeted bodily fluids include, but are not limited to, one or more of: whole blood, fractionated blood, serum, plasma, urine, saliva, sweat, spinal fluid, mucus (including nasal discharge and sputum), amniotic fluid, aqueous humor, vitreous humor, breast milk, cerebrospinal fluid (CSF), pericardial fluid, peritoneal fluid, pleural fluid, pus, thin mucus, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine, and exhaled breath condensate. In other examples, the liquid sample is a cell lysate, a cell culture medium, and mixtures thereof. In certain examples, the porous substrate can be used to directly detect at least one nucleic acid analyte in a liquid sample. In other examples, the liquid sample can be subjected to one or more treatments (e.g., dilution, lysis, fractionation) prior to detection with the porous substrate or a device comprising the porous substrate. In some examples disclosed herein that describe biological liquid samples, the sample can be obtained from a mammalian subject, such as a human.

[0137] Other examples of porous substrates according to the present application for detecting nucleic acid analytes associated with allergens or contaminants, the sample can be a biological or organic sample selected from, but not limited to, soil samples, water samples, plant material, food or beverage samples, agricultural products, surface debris, surface swabs, or any combination thereof. In some examples disclosed herein, the biological or organic sample can be obtained in liquid form, or processed into liquid form, for use with the porous substrate.

[0138] Liquid samples described herein can be used and / or tested directly, for example using a substrate, electrode, electrochemical cell, and / or device as described herein, or can be diluted prior to use and / or testing. For example, a sample can be diluted to about, at least about, or less than about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the original concentration (e.g., w / w, w / v, or v / v). According to examples in which a sample is diluted to about 2% concentration, the diluted sample can comprise about 2% of the original sample and 98% of a diluent, such as a buffer (e.g., w / w, w / v, or v / v). Likewise, in examples in which a sample is diluted to about 5% concentration, the diluted sample can comprise about 2% of the original sample and 95% of a diluent, such as a buffer (e.g., w / w, w / v, or v / v). Other dilutions can be determined based on this approach.

[0139] In this context, a liquid sample can also refer to a liquid comprising or consisting essentially of water (optionally seawater, wastewater, or freshwater), for example for water quality testing. Alternatively, a liquid sample can also be an environmental sample, for example as part of a bioremediation test. The sample can comprise or consist of wastewater, which can form part of any water quality test. Alternatively, a liquid sample comprises or consists essentially of plant and / or animal products, which can be applied in fields such as biosafety.

[0140] In one embodiment, the porous substrate used according to the present application is capable of detecting at least one nucleic acid analyte in a sample. In another embodiment, the porous substrate is capable of detecting at least one nucleic acid analyte in a sample associated with a disease state or injury, such as a brain injury, optionally a TBI. In another embodiment, the porous substrate is capable of detecting a plurality of nucleic acid analytes in a sample, wherein the plurality of nucleic acid analytes are optionally associated with a disease state or injury, such as a brain injury, optionally a TBI. In yet another embodiment, the porous substrate is capable of detecting at least one nucleic acid analyte in a sample at a concentration of about 1 pM or less.

[0141] In one embodiment, the porous substrate is capable of detecting and / or quantifying at least one nucleic acid analyte in a sample within about 60 minutes, 30 minutes, or 20 minutes of contacting the sample. For example, detection of the at least one nucleic acid analyte can be performed within about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or about 60 minutes.

[0142] Electrode

[0143] Disclosed herein is an electrode comprising a porous substrate as defined herein for detecting a nucleic acid analyte in a complex biological sample.

[0144] In one embodiment, the electrode comprises: - a working electrode comprising a porous substrate as defined herein; - a reference electrode; and - a counter electrode.

[0145] In one embodiment, the reference electrode comprises, consists essentially of, or consists of at least one material selected from, but not limited to, silver, a silver salt (optionally silver halide), calomel, and mixtures thereof. In another embodiment, the reference electrode can be a hydrogen electrode.

[0146] In one embodiment, the counter electrode comprises, consists essentially of, or consists of at least one material selected from, but not limited to, carbon, graphite, platinum, and mixtures thereof.

[0147] The electrode can be integrated into an article or device using suitable techniques in the art, such as screen printing, spray coating, inkjet printing, and mixtures thereof.

[0148] The electrode can be tailored to target a particular analyte, such as a nucleic acid analyte. The electrode can be in the form of a disposable article, where it can be integrated into / used in a device as described herein, and then potentially replaced with an electrode of the same type (e.g., for testing a new sample), or a different electrode comprising different capture probes (e.g., for testing a sample or different sample for the presence of one or more different nucleic acid analytes).

[0149] In another embodiment, an insulating material is disposed on at least a portion of the electrode. For example, the insulating material can be disposed on about, at least about, or less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the electrode. Examples of insulating materials include, but are not limited to: silicon dioxide; polymers, glass, ceramic, and the like. For example, the insulating material can be polyvinyl chloride, polycarbonate, epoxy, polyester, paper, paperboard, ceramic, ceramic-coated metal, polydimethylsiloxane, and blends of these materials (e.g., a blend of polycarbonate and polyester).

[0150] In one embodiment, the electrode is used in conjunction with an apparatus, such as an electrochemical analysis apparatus capable of scanning a potential over a range and measuring a current, such as a potentiostat.

[0151] In one embodiment, the electrode further comprises a coating. The coating can be a polymer, such as a polydimethylsiloxane or an epoxy-based polymer, and mixtures thereof. The coating can be applied by any method known in the art.

[0152] In one embodiment, the electrode comprises a region adapted for deposition of a liquid, such as a complex biological sample or a liquid sample as described herein, wherein the region adapted for deposition of a liquid is optionally in the form of a reservoir, such as a reservoir formed in the coating or in the porous substrate itself. In yet another embodiment, the region for deposition of a liquid is in contact with one or more of: the working electrode; the reference electrode; and / or the counter electrode.

[0153] In one embodiment, the electrode is capable of detecting at least one nucleic acid analyte in a sample. The at least one nucleic acid analyte can be associated with a condition, disease state, or injury, such as a brain injury, optionally a TBI. However, other conditions, diseases, or injuries are also contemplated and described herein in the context of the porous substrate. In another embodiment, the electrode is capable of detecting a plurality of nucleic acid analytes in a sample, wherein the plurality of nucleic acid analytes are optionally associated with a disease state or injury, such as a brain injury, optionally a TBI. In yet another embodiment, the electrode is capable of detecting at least one nucleic acid analyte in a sample at a concentration of about 1 pM or less.

[0154] In one embodiment, the electrode is capable of detecting at least one nucleic acid analyte in a sample within about 60 minutes, 30 minutes, or 20 minutes of contacting the sample. For example, detection of the at least one analyte can be performed within about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or about 60 minutes.

[0155] In one embodiment, the electrode is used to detect at least one nucleic acid analyte in a sample, wherein the nucleic acid analyte is optionally associated with a disease state or injury, such as a brain injury, optionally a TBI. In another embodiment, the nucleic acid analyte is selected from, but not limited to, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), short interfering RNA (siRNA), and mixtures thereof.

[0156] Electrochemical Cell

[0157] Disclosed herein is an electrochemical cell comprising an electrode as defined herein for detecting a nucleic acid analyte in a complex biological sample.

[0158] The electrochemical cell can be an electrochemical sensor. In one embodiment, the electrochemical cell is capable of detecting at least one nucleic acid analyte in a sample. In another embodiment, the electrochemical cell is capable of detecting at least one nucleic acid analyte in a sample that is associated with a disease state or injury, such as a brain injury, optionally a TBI. However, other conditions, diseases, or injuries are also contemplated and described herein in the context of the porous substrate. In another embodiment, the electrochemical cell is capable of detecting a plurality of nucleic acid analytes in a sample, wherein the plurality of nucleic acid analytes is optionally associated with a disease state or injury, such as a brain injury, optionally a TBI. In yet another embodiment, the electrochemical cell is capable of detecting at least one nucleic acid analyte in a sample at a concentration of at least about 1 pM.

[0159] In one embodiment, the electrochemical electrolytic cell is capable of detecting at least one nucleic acid analyte in a sample within about 60 minutes, 30 minutes, or 20 minutes of contacting the sample. For example, detection of the at least one analyte can be performed within about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or about 60 minutes.

[0160] In one embodiment, the electrochemical electrolytic cell is used to detect at least one nucleic acid analyte in a sample, wherein the nucleic acid analyte is optionally associated with a disease state or injury, such as a brain injury, optionally a TBI. In another embodiment, the nucleic acid analyte is selected from, but not limited to, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and short interfering RNA (siRNA).

[0161] Device

[0162] Disclosed herein is a device for detecting a nucleic acid analyte in a complex biological sample comprising a porous substrate as described herein, an electrode as described herein, and / or an electrochemical electrolytic cell as described herein.

[0163] In one embodiment, the device is an electrochemical biosensor capable of detecting at least one nucleic acid analyte in a sample. For example, the device can be a diagnostic device, e.g., a point-of-care diagnostic device. In this context, the phrase “electrochemical biosensor” can be used interchangeably with “electrochemical sensor,” and vice versa.

[0164] In one embodiment, the device is capable of detecting at least one nucleic acid analyte in a sample. In another embodiment, the device is capable of detecting at least one nucleic acid analyte in a sample that is associated with a disease state or injury, such as a brain injury, optionally a TBI. However, other conditions, diseases, or injuries are also contemplated and described herein in the context of the porous substrate. In another embodiment, the device is capable of detecting a plurality of nucleic acid analytes in a sample, wherein the plurality of nucleic acid analytes are optionally associated with a disease state or injury, such as a brain injury, optionally a TBI. In yet another embodiment, the device is capable of detecting at least one nucleic acid analyte in a sample at a concentration of at least about 1 pM.

[0165] In one embodiment, the device is capable of detecting at least one nucleic acid analyte in a sample within about 60 minutes, 30 minutes, or 20 minutes of contacting the sample. For example, detection of the at least one analyte can be performed within about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or about 60 minutes.

[0166] In one embodiment, the device is used to detect at least one nucleic acid analyte in a sample, wherein the nucleic acid analyte is optionally associated with a disease state or injury, such as a brain injury, optionally a TBI. In another embodiment, the nucleic acid analyte is selected from, but not limited to, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and short interfering RNA (siRNA).

[0167] The device can be in a form suitable for deployment within a clinical setting or outside of a clinical setting, for example, within an urban hospital or a field hospital, or within a rural setting, and / or in other settings, such as a clinical setting. The device can provide one or more of the following: assist in making clinical and / or operational decisions, such as decisions related to diagnosis and / or treatment of a subject; facilitate improvements in short-term and long-term outcomes based on results obtained from the device; achieve analysis in a short period of time (e.g., about or less than about 60, 40, 30, or 20 minutes); determine the concentration of one or more analytes in an accessible physiological medium (e.g., a complex biological sample or a fluid sample as described herein); be easy to operate without requiring extensive or substantial training.

[0168] Disclosed herein is the use of a porous substrate as described herein, an electrode as described herein, an electrochemical cell as described herein, and / or a device as described herein for detecting a nucleic acid analyte in a sample. In some examples, the nucleic acid analyte is associated with a disease state or injury, such as a brain injury, optionally a TBI.

[0169] Applications and Uses

[0170] The porous substrate, electrode, electrochemical cell, and / or device can be used to detect a nucleic acid analyte or biomarker in a complex biological sample or medium. Accordingly, the present invention provides a method of detecting at least one biomarker (e.g., at least one nucleic acid analyte) in a complex biological sample, the method comprising: (i) introducing a complex biological sample into an electrochemical biosensor comprising a porous substrate comprising a plurality of channels, wherein: - at least a portion of the plurality of channels has an average depth of about 1.0 pm to about 6.0 pm; and / or - at least a portion of the plurality of channels has an average diameter or cross-section ranging from about 15 nm to about 40 nm, wherein one or more capture probes capable of specifically binding to at least a biomarker, e.g., at least one nucleic acid analyte, to form a probe-analyte complex are disposed on at least one surface of at least a portion of the plurality of channels; and (ii) optionally determining whether at least one biomarker, e.g., at least one nucleic acid analyte, is present in the sample based on measurements of peak current obtained from differential pulse voltammograms before and after the complex biological sample is introduced into the electrochemical biosensor, wherein a change in peak current detected after the complex biological sample is introduced into the electrochemical biosensor indicates that at least one biomarker, e.g., at least one nucleic acid analyte, is present in the complex biological sample, and no change in peak current detected after the complex biological sample is introduced into the electrochemical biosensor indicates that at least one biomarker, e.g., at least one nucleic acid analyte, is not present in the complex biological sample above a threshold level.

[0171] As used herein, the term "complex biological sample," "complex biological medium," or a similar term is intended to mean a heterogeneous biological sample or medium containing nucleic acids, proteins, and / or cellular material of different molecular weights and / or origins. In some examples, the complex biological sample or medium upon which the detection methods of the present application can be performed is a clinically relevant biological sample. Exemplary complex biological samples that are clinically relevant are described herein, including but not limited to whole blood, fractionated blood, serum, plasma, urine, saliva, sweat, spinal fluid, mucus (including nasal discharge and sputum), amniotic fluid, aqueous humor, vitreous humor, breast milk, cerebrospinal fluid (CSF), pericardial fluid, peritoneal fluid, pleural fluid, pus, thin mucus, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine, exhaled breath condensate, cell lysate, cell culture media, and mixtures thereof. In other examples, the complex biological sample or medium upon which the detection methods of the present application can be performed is an environmentally relevant biological sample. Exemplary complex biological samples that are environmentally relevant are described herein, including but not limited to water samples, sewage, soil samples, and mixtures thereof. In yet other examples, the complex biological sample or medium upon which the detection methods of the present application can be performed is an agriculturally relevant biological sample. Exemplary complex biological samples that are agriculturally relevant are described herein, including but not limited to plant and / or animal products or tissues, including products and tissues relevant to the field of biosecurity. In another example, the complex biological sample or medium upon which the detection methods of the present application can be performed is a food or beverage sample. In some examples disclosed herein, the complex biological sample or medium can undergo minimal sample preparation procedures or no sample preparation procedures. An example of a complex biological sample or medium that undergoes minimal sample preparation is simple dilution, dissolution, or stabilization of the complex biological sample in an appropriate liquid for testing, such as water, saline, or a buffer. In other examples, fractionation is performed on the complex biological sample or medium that undergoes minimal sample preparation. In some examples, the processing steps performed on the complex biological sample can reduce the complexity of the sample. However, in other examples, the complex biological sample can be untreated such that the detection methods of the present application are performed directly on the sample.

[0172] Detection of nucleic acid analytes or biomarkers can constitute part of a diagnosis of a condition, disorder, or injury in a subject, e.g., a brain injury, such as a TBI. Accordingly, at least one nucleic acid analyte can be associated with a condition, disease state, or injury. In one particular example, at least one nucleic acid analyte is associated with a brain injury, optionally a traumatic brain injury (TBI), and the method is used to diagnose a brain injury (e.g., a TBI). Other medical indications are described and contemplated herein. However, as described herein, the porous substrate, electrode, electrochemical cell, and / or device are not limited to detecting only nucleic acid analytes associated with brain injuries, or even to detecting nucleic acid analytes associated with medical conditions. The porous substrate, electrode, electrochemical cell, and / or device can likewise be used to detect nucleic acids associated with a biological source (e.g., an injury, a disease state, a pathogen, a contaminant, etc.) in other complex biological samples. This can occur via detection of one or more nucleic acid analytes associated with a condition, injury, disease state, pathogen, contaminant, etc., which can take various forms as described herein. As described herein, at least one nucleic acid analyte can take various forms. Exemplary nucleic acids that can be detected using the methods of the present application include DNA, messenger RNA, ribosomal RNA, transfer RNA, microRNA, short interfering RNA, and plasmid DNA. In one example, the nucleic acid analyte detected in the present methods is a miRNA. In one particular example, the methods of the present application comprise detecting the presence or absence of one or more miRNAs associated with a TBI in a complex biological sample (e.g., blood, a blood fraction, or a diluted blood solution). For example, the one or more miRNAs can be selected from the group consisting of miR-142-3p, miR-150-5p, miR-196b-5p, let-7f-5p, miR-196b-5p, and combinations thereof.

[0173] In some examples, the method comprises determining the severity of the condition, disease state, or injury by determining the concentration of at least one biomarker (e.g., at least one nucleic acid analyte) in the sample. In the case of a disease or injury, this can allow the user to determine the severity of a particular injury or disease, and possibly distinguish between mild and severe cases. In the case of a pathogen or contaminant, it can enable the user to determine whether the amount of analyte present is above or below a threshold level relevant to decision making. The analysis and result generation can be completed in a short timeframe, e.g., within 60, 40, 30, 20, 15, or 10 minutes.

[0174] The porous substrates, electrodes, electrochemical electrolytic cells, and / or devices or methods of the present application can also be used in personalized medicine, for example, to determine the likelihood that a subject will develop a certain disease or condition based on the presence, absence, or concentration of one or more nucleic acid biomarkers; to determine the likelihood that a subject with a disease or condition will respond to a treatment based on the presence, absence, or concentration of one or more nucleic acid biomarkers; to determine the prognosis (or likely progression or regression of a disease) of a subject with a disease or condition based on the presence, absence, or concentration of one or more nucleic acid biomarkers; and / or to determine the effectiveness of a treatment on a subject with a disease or condition based on the presence, absence, or concentration of one or more nucleic acid biomarkers. In some examples disclosed herein, the methods of the present application are capable of detecting at least one nucleic acid analyte at a concentration of about 1 pM or less in a complex biological sample.

[0175] The use of the porous substrates, electrodes, electrochemical electrolytic cells, and / or devices can allow for the manufacture of equipment that can be used at a benchtop, in a handheld form, for use in a clinical setting or in the field, allowing for rapid diagnosis and the potential for in situ results (which can guide medical decisions). Training for the equipment can also allow for more individuals to be trained, requiring simpler training relative to other technologies, such as PCR-based detection.

[0176] Also disclosed herein is the use of a porous substrate as described herein, an electrode as described herein, an electrochemical electrolytic cell as described herein, and / or a device as described herein for determining the concentration of at least one nucleic acid analyte in a complex biological sample. The nucleic acid analyte can be selected from, but is not limited to, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), short interfering RNA (siRNA), and mixtures thereof. In one particular example, the nucleic acid analyte is associated with a brain injury, such as a TBI. However, the skilled person will appreciate that the nucleic acid analyte can be used as a biomarker for a range of injuries, conditions, or diseases, including but not limited to cancer, kidney disease (including acute kidney injury), cardiovascular disease, autoimmune disease, infectious disease, metabolic disease, liver disease, and blood disease. Thus, the porous substrate, electrode, electrochemical electrolytic cell, and / or device as described herein can equally be used to determine the concentration of at least one nucleic acid analyte in a sample that is associated with any of the aforementioned conditions. Furthermore, the porous substrate, electrode, electrochemical electrolytic cell, and / or device as described herein can equally be used to determine the concentration of at least one nucleic acid analyte that is associated with a target pathogen and / or contaminant. Suitable pathogens are described above, which should be applicable to this example mutatis mutandis. In some of the foregoing examples, the concentration of the nucleic acid analyte can be used to make a diagnostic decision, for example, to determine the disease state or severity of a condition in a subject, or to determine the level of a contaminant, etc. In one example, the concentration of one or more nucleic acid analytes associated with a brain injury (e.g., a TBI) is used to determine the severity of the brain injury (e.g., TBI).

[0177] In one embodiment, the porous substrate, electrode, electrochemical electrolytic cell, and / or device as described herein is used to detect at least one nucleic acid analyte in a liquid complex biological sample. Exemplary complex biological samples are described herein. In one example, the liquid sample is a bodily fluid. In one particular example, the complex biological sample is blood or a blood fraction (e.g., serum).

[0178] Also disclosed herein is a method of detecting at least one nucleic acid analyte in a sample, the method comprising introducing the sample (e.g., a liquid sample) into an electrochemical biosensor comprising: a porous substrate as described herein, an electrode as described herein, an electrochemical electrolytic cell as described herein, and / or a device as described herein, and determining whether the nucleic acid analyte is present in the sample.

[0179] Also disclosed herein is a method of detecting at least one nucleic acid analyte in a sample, the method comprising introducing a sample (e.g., a liquid sample) into a porous substrate as described herein, an electrode as described herein, an electrochemical cell as described herein, and / or a device as described herein, and determining whether at least one nucleic acid analyte is present in the sample. In one embodiment, this is based on measurements of peak current obtained from differential pulse voltammograms before and after introducing the sample into the electrochemical biosensor, wherein a change in peak current detected after introducing the sample into the electrochemical biosensor indicates that a nucleic acid analyte is present in the sample, and no change in peak current detected after introducing the sample into the electrochemical biosensor indicates that a nucleic acid analyte above a threshold level is not present in the sample. An advantage of using a porous substrate, an electrochemical biosensor, an electrochemical cell, and / or a device as described herein to detect a nucleic acid analyte is that it enables detection of a nucleic acid in a complex biological sample or medium as described herein. In this regard, controlling and selecting the dimensions of the plurality of channels in the porous substrate can eliminate or reduce problems associated with biological contamination. In this regard, for a sensor deployed and / or exposed to a biological sample, signals from any channel blockage can be affected by biological contamination mechanisms, for example, in the case of a contaminant adhering to a portion of an electrode (e.g., an electrode derived from or based on a porous substrate as described herein) via non-specific adsorption. The contaminant can potentially reduce the area of the electrode surface available for one or more analytes, resulting in, for example, a reduction in the differential pulse voltammetry signal. In the case of the present invention, controlling and selecting the dimensions of the plurality of channels can eliminate or reduce problems associated with biological contamination. This can be due, at least in part, to the dimensions of the channels being at a scale such that proteins and / or other potential biological molecules that cause contamination, such as those that can be present in a complex biological sample containing a target analyte, are unable to enter or progress within the channels, whereas the target analytes are at a scale meaning that they are small enough and mobile enough to reach the capture probes disposed within the plurality of channels. Thus, a potential advantage of the method of the present invention is that it enables rapid detection of a nucleic acid analyte in a complex biological sample.

[0180] The method of determining a change in peak current can be performed using any suitable method in the art. For example, the change in peak current can be normalized via the relationship of Equation 1 : (1)

[0181] wherein is the normalized current change, is the peak current value measured after incubation in buffer for a first period of time, and is the peak current value measured after incubation in a target solution at a given concentration for a second period of time compared to a control sample. In this context, the first period of time and / or the second period of time can independently be or less than about 60 minutes, 50 minutes, 40 minutes, 30 minutes, or 20 minutes of contact with the sample. For example, detection of the at least one analyte can be performed in about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or about 60 minutes.

[0182] The method can comprise determining a concentration of the detected nucleic acid analyte in the sample based on the level of reduction in peak current intensity. The method can further comprise comparing the concentration of the at least one nucleic acid analyte to a calibration curve.

[0183] In some examples, the one or more nucleic acid analytes are detected, and optionally quantified for concentration, within about 60 minutes or less, 30 minutes or less, or 20 minutes or less of contact with the sample. For example, detection of the at least one analyte can be performed in about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or about 60 minutes.

[0184] Example embodiments

[0185] The following are example embodiments according to various aspects described herein:

[0186] 1. A method of detecting at least one nucleic acid analyte in a complex biological sample, the method comprising: (i) introducing the sample into an electrochemical biosensor comprising a porous substrate comprising a plurality of channels, wherein: - at least a portion of the plurality of channels has an average depth of about 1.0 pm to about 6.0 pm; and - at least a portion of the plurality of channels has an average diameter or cross-section ranging from about 15 nm to about 40 nm, wherein one or more capture probes capable of specifically binding to at least the biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex are disposed on at least one surface of at least a portion of the plurality of channels; and (ii) determining whether at least one nucleic acid analyte is present in the sample based on measurements of peak current obtained from the differential pulse voltammogram before and after the sample is introduced into the electrochemical biosensor, wherein a change in peak current detected after the sample is introduced into the electrochemical biosensor indicates that at least one nucleic acid analyte is present in the sample, and no change in peak current detected after the sample is introduced into the electrochemical biosensor indicates that at least one nucleic acid analyte above a threshold level is not present in the sample.

[0187] 2. The method according to example embodiment 1, wherein the capture probe is capable of specifically binding to at least one nucleic acid analyte to form a probe-analyte complex, wherein the presence of the probe-analyte complex within the channel causes at least partial steric and / or charge blockage of the channel compared to a channel in which the probe-analyte complex is not present.

[0188] 3. The method according to any one of example embodiments 1 to 3, wherein: - at least 10% of the plurality of channels in the porous substrate have an average depth in the range of about 1.0 pm to about 2.0 pm, or about 1.0 pm to about 4.0 pm; and / or - at least 10% of the plurality of channels in the porous substrate have an average diameter or cross-section in the range of about 15 nm to about 30 nm, or about 20 nm to about 30 nm.

[0189] 4. The method according to any one of example embodiments 1 to 3, wherein: - the porous substrate comprises, consists essentially of, or consists of an electrically conductive or semiconductive material; and / or - the porous substrate comprises, consists essentially of, or consists of a material selected from the group consisting of platinum, gold, silver, cobalt, gallium, indium, tin, vanadium, zirconium, copper, aluminum, iridium, palladium, rhodium, silicon, zinc, iron, steel, brass, carbon, graphite, alloys, oxides, or metal compounds of these elements, and mixtures thereof; and / or - the porous substrate is not in the form of a thin film; and / or - the porous substrate does not comprise and / or consist of nanoparticles; and / or - the porous substrate does not comprise one or more of aluminum, platinum, gold, and / or silver; and / or - the porous substrate comprises, consists essentially of, or consists of a material selected from silicon, silicon optionally heat treated and / or chemically modified; and / or - a layer comprising, consisting essentially of, or consisting of carbon is disposed on at least a portion of the porous substrate.

[0190] 5. The method according to any one of example embodiments 1 to 4, wherein: - the porous substrate optionally comprising silicon comprises, consists essentially of, or consists of a monolayer comprising one or more of: ■ one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more functional groups capable of reacting with an amine group; and / or ■ one or more functional groups capable of forming a covalent bond with a complementary functional group on one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more carboxylic acid groups; or - the porous substrate optionally comprising silicon comprises, consists essentially of, or consists of at least one layer comprising one or more of: ■ one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more functional groups capable of reacting with an amine group; and / or ■ one or more functional groups capable of forming a covalent bond with a complementary functional group on one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more carboxylic acid groups.

[0191] 6. The method according to any one of example embodiments 1 to 5, wherein: - the one or more capture probes are capable of binding to a plurality of different nucleic acid analytes; and / or - the at least one nucleic acid analyte is selected from the group consisting of: a DNA analyte, a messenger RNA (mRNA) analyte, a ribosomal RNA (rRNA) analyte, a transfer RNA (tRNA) analyte, a microRNA (miRNA) analyte, a short interfering RNA (siRNA) analyte, a plasmid DNA (pDNA), and mixtures thereof; and / or - one or more of the at least one capture probe is a DNA capture probe or a Peptide Nucleic Acid (PNA) probe.

[0192] 7. The method according to any one of example embodiments 1 to 6, wherein: - the at least one nucleic acid analyte is associated with a condition, disease state, or injury; and / or - the at least one nucleic acid analyte is associated with a brain injury, optionally a traumatic brain injury.

[0193] 8. The method according to any one of example embodiments 1 to 7, wherein the at least one nucleic acid analyte is a miRNA associated with a brain injury, optionally a traumatic brain injury.

[0194] 9. The method according to example embodiment 6 or example embodiment 8, wherein the one or more miRNAs are selected from miR-142-3p, miR-150-5p, miR-196b-5p, let-7f-5p, or any combination thereof.

[0195] 10. The method according to any one of example embodiments 1 to 9, wherein the at least one nucleic acid analyte is detected in the sample at a concentration of about 1 pM or less.

[0196] 11. The method according to any one of example embodiments 1 to 10, wherein the complex biological sample is or comprises a bodily fluid, a cell culture medium, a water sample, a soil sample, a food or beverage sample, an agricultural product, a surface swab, or any combination thereof.

[0197] 12. The method according to any one of example embodiments 1 to 11, wherein the complex biological sample is: - from a human; and / or - a liquid sample; and / or - selected from the group consisting of: whole blood, serum, plasma, urine, saliva, sweat, spinal fluid, sputum, cell lysate, cell culture medium, and mixtures thereof; and / or - obtained from a subject suspected of having a brain injury, optionally a traumatic brain injury.

[0198] 13. The method according to any one of example embodiments 1 to 12, wherein the electrochemical biosensor comprises one or more electrodes.

[0199] 14. The method according to example embodiment 13, wherein the electrochemical biosensor comprises: - a working electrode, the working electrode comprising a porous substrate; - a reference electrode; and - a counter electrode.

[0200] 15. The method according to either Example Embodiment 13 or Example Embodiment 14, wherein an insulating material is disposed on at least a portion of the electrode.

[0201] 16. The method according to any one of Example Embodiments 1 to 15, wherein the electrochemical biosensor is used in conjunction with (i) a device capable of scanning a range of potentials and measuring current or (ii) a potentiostat.

[0202] 17. The method according to any one of Example Embodiments 1 to 16, wherein the change in peak current is normalized via a relationship of Equation 1 : (1)

[0203] wherein is the normalized current change, is the peak current value measured after incubation in the buffer for a first time period, and is the peak current value measured after incubation in a target solution at a given concentration for a second time period compared to a control sample.

[0204] 18. The method according to Example Embodiment 17, wherein the first time period and / or the second time period is independently about 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, or 20 minutes or less.

[0205] 19. The method according to any one of Example Embodiments 1 to 18, comprising: - determining a concentration of the detected nucleic acid analyte in the sample based on the level of decrease in peak current intensity; - optionally comparing the concentration of the at least one nucleic acid analyte to a calibration curve; and - optionally determining a disease state or severity of an injury, optionally a brain injury, of the subject based on the concentration of the at least one nucleic acid analyte.

[0206] 20. The method according to any one of Example Embodiments 1 to 19, wherein the at least one nucleic acid analyte is detected and optionally quantified for its concentration within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 15 minutes, or about 10 minutes of introducing the sample to the electrochemical biosensor.

[0207] 21. A porous substrate for use in the method according to any one of Example Embodiments 1 to 20, the porous substrate comprising a plurality of channels, wherein: - at least a portion of the plurality of channels has an average depth of about 1.0 pm to about 6.0 pm; and / or - at least a portion of the plurality of channels has an average diameter or cross-section ranging from about 15 nm to about 40 nm, wherein one or more capture probes capable of specifically binding to at least one nucleic acid analyte to form a probe-analyte complex are disposed on at least one surface of at least a portion of the plurality of channels.

[0208] 22. The porous substrate in use according to example embodiment 21, wherein the capture probes are capable of specifically binding to at least one nucleic acid analyte to form a probe-analyte complex, wherein the presence of the probe-analyte complex within the channel causes at least partial steric and / or charge blockage of the channel compared to a channel in which no probe-analyte complex is present.

[0209] 23. The porous substrate in use according to example embodiment 21 or example embodiment 22, wherein: - at least a portion of the plurality of channels has an average depth in the range of about 1.0 pm to about 6.0 pm; and - at least a portion of the plurality of channels has an average diameter or cross-section in the range of about 15 nm to about 40 nm.

[0210] 24. The porous substrate in use according to any one of example embodiments 21 to 23, wherein: - at least 10% of the plurality of channels has an average depth in the range of about 1.0 pm to about 6.0 pm; and / or - at least 10% of the plurality of channels has an average diameter or cross-section in the range of about 15 nm to about 40 nm.

[0211] 25. The porous substrate in use according to any one of example embodiments 21 to 24, wherein: - the porous substrate comprises, consists essentially of, or consists of an electrically conductive or semiconductive material; and / or - the porous substrate comprises, consists essentially of, or consists of a material selected from the group consisting of platinum, gold, silver, cobalt, gallium, indium, tin, vanadium, zirconium, copper, aluminium, iridium, palladium, rhodium, silicon, zinc, iron, steel, brass, carbon, graphite, alloys, oxides or metal compounds of these elements, and mixtures thereof; and / or - the porous substrate comprises, consists essentially of, or consists of a material selected from silicon; and / or - a layer comprising, consisting essentially of, or consisting of carbon is disposed on at least a portion of the porous substrate.

[0212] 26. The porous substrate in use according to any one of example embodiments 21 to 25, wherein: - the porous substrate, optionally comprising silicon, comprises, consists essentially of, or consists of a monolayer, the monolayer comprising one or more of: ■one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more functional groups capable of reacting with an amine group; and / or ■one or more functional groups capable of forming a covalent bond with a complementary functional group on one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more carboxylic acid groups; or - the porous substrate optionally comprising silicon comprises, consists essentially of, or consists of at least one layer comprising one or more of: ■one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more functional groups capable of reacting with an amine group; and / or ■one or more functional groups capable of forming a covalent bond with a complementary functional group on one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more carboxylic acid groups.

[0213] 27. The porous substrate in use according to any one of Example Embodiments 21 to 26, wherein: - the one or more capture probes are capable of binding to a plurality of different nucleic acid analytes; and / or - the at least one nucleic acid analyte is selected from the group consisting of: a DNA analyte, a messenger RNA (mRNA) analyte, a ribosomal RNA (rRNA) analyte, a transfer RNA (tRNA) analyte, a microRNA (miRNA) analyte, a short interfering RNA (siRNA) analyte, a plasmid DNA (pDNA), and mixtures thereof; and / or - one or more of the at least one capture probe is a DNA capture probe or a peptide nucleic acid probe (PNA) probe.

[0214] 28. The porous substrate in use according to any one of Example Embodiments 21 to 27, wherein: - the at least one nucleic acid analyte is associated with a condition, a disease state, or an injury; and / or - the at least one nucleic acid analyte is associated with a brain injury, optionally a traumatic brain injury.

[0215] 29. The porous substrate in use according to any one of example embodiments 21 to 28, wherein the at least one nucleic acid analyte is a miRNA associated with brain injury, optionally traumatic brain injury.

[0216] 30. The porous substrate in use according to example embodiment 29, wherein the one or more capture probes are capable of binding one or more miRNAs selected from the group consisting of miR-142-3p, miR-196b-5p, let-7f-5p, miR-150-5p, and combinations thereof.

[0217] EXAMPLE

[0218] MATERIALS

[0219] P-type Si wafers with resistivity of 0.00055-0.001 Ω cm, (100) orientation were purchased from Siltronix (France). Hydrofluoric acid (HF) (48%, AR grade) was purchased from Scharlau (Australia). Potassium ferrocyanide (K4[Fe(CN)6]), potassium ferricyanide (K3[Fe(CN)6]), undecylenic acid, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC), phosphate buffered saline (PBS) tablets, 2-(N- morpholino)-ethanesulfonic acid (MES), sodium chloride (NaCl), sodium hydroxide, ethanolamine and tris(hydroxymethyl)aminomethane were purchased from Sigma-Aldrich (Australia). Acetylene gas cylinder was purchased from BOC (Australia). All DNA and miRNA strands were purchased from Integrated DNA Technologies, Inc. Sequences of the capture ssDNA, target ssDNA and target miRNA are listed in Table 1.

[0220] Table 1. Sequences of ssDNA capture probes, ssDNA targets and miRNA targets.

[0221] Custom Oligomer Channel Depth (pm) miR-142-ssDNA-capture 5'- / 5AmMC6 / TC CAT AAA GTA GGA AAC ACT ACA-3' miR-142-ssDNA-target 3'-TGT AGT GTT TCC TAC TTT ATG GA-5' miR-142-RNA-target 3' rUrGrT rArGrU rGrUrU rUrCrC rUrArC rUrUrU rArUrG rGrA-5' miR-423-ssDNA-capture 5'- / 5AmMC6 / AC TGA GGG GCC TCA GAC CGA GCT- 3'

[0222] Fabrication of porous silicon

[0223] An entire 6-inch p-type Si wafer was anodically etched in an electrolyte solution containing 1 : 1 (v:v) HF and absolute ethanol using an MPSB wet etching system (AMMT GmbH) to produce a first pSi layer. First, a sacrificial layer was produced at an anodic current of 1 A for 90 s. This was removed with 1 M sodium hydroxide. Then, the etching cell was rinsed with water, absolute ethanol and dried with N2 gas. This step helps to ideally suppress the formation of parasitic layers during further etching of a suitable pSi sensing layer. Next, pSi samples with different channel sizes and thicknesses / diameters were fabricated in 1 : 1 etchant by varying the current density and time to investigate the influence of morphological features on their electrochemical performance (Table 2). The freshly etched pSi was rinsed with ethanol and then immediately used for carbon stabilization.

[0224] Table 2. Etching conditions for the fabrication of pSi with different parameters.

[0225] Channel Size (nm) Channel Depth (pm) Current (A) Etching Time (s) 27 1.0 1 272 27 1.7 1 463 27 5.7 1 1300 15 1.7 0.55 633 40 1.7 5 100

[0226] Thermal hydrocarbonization treatment of freshly etched porous silicon

[0227] To improve the stability and conductivity of the pSi single-layer nanostructure, the freshly etched pSi was placed in a quartz tube under a stream of N2 at 2 L min -1 for 45 min at room temperature. After the purging step, a stream of 1 : 1 N2-ethyne mixture was introduced into the tube at room temperature for 15 min, and then the quartz tube was placed in a preheated tube furnace at 525 °C for 15 min under a continuous mixture stream. Finally, the tube was cooled back to room temperature under a stream of N2 to obtain a THCpSi sample with a THCpSi layer.

[0228] Surface functionalization and immobilization of capture probes

[0229] First, to functionalize the THCpSi layer with -COOH groups, the sample was immersed in pure undecylenic acid at 150 °C for 10 h under an inert atmosphere (N2). After cooling to room temperature, the sample was rinsed with absolute ethanol. The -COOH groups were activated by immersion in a solution containing 10 mg mL -1 1 -ethyl-3 -(3 -dimethylaminopropyl)carbodiimide (EDC) and 15 mg mL -1The -COOH groups of the THCpSi layer were activated by incubation of the functionalized substrate in 0.1 M NHS in MES buffer (pH 5.5) for 30 min at room temperature to yield succinimidyl ester groups. Subsequently, 200 μL of 10 μM NH2-SSDNA capture probe or non-specific NH2-SSDNA capture probe prepared in 10 mM PBS were incubated on the activated surface overnight at 4°C. The samples were then washed extensively with 10 mM PBS and 200 μL of 0.1 M ethanolamine in 10 mM PBS was added to the electrochemical cell and incubated for 45 min at room temperature to quench unreacted succinimidyl ester groups. Finally, the samples were washed again with 10 mM PBS and were ready for sensing experiments.

[0230] Scanning electron microscopy (SEM)

[0231] For the characterization of the morphology of the biosensor surface, the samples were mounted on an aluminum sample holder using double-sided conductive tape. SEM images were obtained with a FEI NovaNano SEM 430 at an acceleration voltage ranging from 10 kV to 19 kV.

[0232] Fourier-transform infrared spectroscopy (FTIR)

[0233] IR spectra were recorded on a Thermo Scientific Nicolet 6700 FT-IR spectrometer using reflection mode. (100)-oriented boron-doped (p-type) Si with resistivities of 1.1-1.8 Ω cm and 0.00055-0.001 Ω cm, respectively, were used for IR transmission and reflection measurements. All spectra were recorded as an average of 64 scans in the range of 650 to 4000 cm -1 with a resolution of 8 cm -1 -1. A clean flat Si substrate was used as background.

[0234] Water contact angle measurements

[0235] Water contact measurements were performed on the pSi structures using a custom-made goniometer with a Panasonic CCTV camera (WV-BP550 / G). After placing a 1 μL drop of ultrapure water on the surface using a 10 μL syringe, a picture was taken immediately. The contact angle of the water drop with the surface was determined using ImageJ software (drop analysis plug-in).

[0236] Electrochemical characterization

[0237] All electrochemical measurements were performed using a three-electrode electrochemical cell on an electrochemical analyzer (Metrohm Autolab potentiostat). A custom designed Teflon cell was assembled to house the modified THCpSi glass slide as the working electrode, silver / silver chloride was used as the reference electrode, and platinum wire was used as the counter electrode. Data acquisition and analysis were done using Metrohm Nova 2.1 software. All potentials are provided with respect to Ag / AgCl. Cyclic voltammetry (CV) measurements were performed by scanning the potential in the potential range depending on the redox species used at a scan rate of 0.1 V s -1 3- / 4- Electrochemical Impedance Spectroscopy (EIS) measurements were performed at open circuit potential with a scan frequency from 100 kHz to 0.1 Hz (log scale) and an AC amplitude of 5 mV. Differential Pulse Voltammetry (DPV) measurements were done by scanning the potential from -0.2 to 0.6 V.

[0238] Detection of DNA and miRNA using THCpSi-based sensors

[0239] To evaluate the performance of the developed miRNA sensor, ssDNA and miRNA target solutions were prepared in 10 mM Tris buffer containing 75 mM NaCl, pH 7.5 at various concentrations (0.1 to 1000 pM) and incubated on the sensor surface for 15 min, respectively. After each incubation step, the biosensor surface was thoroughly washed with PBS and transferred to 10 mM PBS containing 2 mM [Fe(CN)6] 3- / 4- solution. To detect miRNA biomarkers in diluted serum, fetal bovine serum (FBS) was added to the above Tris buffer solution to obtain a final concentration of 2% FBS, and miRNA target solutions were prepared in 2% FBS / Tris buffer at various concentrations (0.1 to 1000 pM). To verify that the current change is only caused by specific hybridization between the ssDNA capture probe immobilized on the carbon-stabilized pSi surface and the incubated target ssDNA, the same measurement was repeated using a control biosensor prepared under the same conditions, with the ssDNA capture probe using a random sequence. Human serum samples were collected from human healthy donors and mTBI patients at Alfred Hospital (Melbourne, Australia). The collected serum samples were then diluted to a concentration of 2% with the same Tris buffer solution.

[0240] Example 1 : Fabrication and thermal hydrocarbonation of pSi

[0241] Following the aforementioned procedure, five representative pSi samples were fabricated by anodic etching of p-type Si wafers in a wet etching system. The channel size and depth were easily controlled and adjusted by regulating the anodic oxidation conditions, including current density and etching time. To stabilize the pSi surface and improve the semiconductor's conductivity, the newly etched pSi samples were heat-treated at 525°C.

[0242] Figure 1 The SEM images in the image show the tops of five THCpSi samples with various channel sizes and thicknesses. Figure 1 a, c, e, g, and i) and cross-section ( Figure 1 Features b, d, f, h, j). Under different etching conditions, the average channel diameter can be modified from 15 nm to 50 nm, while the channel depth ranges from 1.0 μm to 5.7 μm.

[0243] Furthermore, water contact angle measurements demonstrate that the surface chemical properties of pSi changed after thermal hydrocarbonation. Figure 2 Under the same dimensions, compared with pSi substrate ( Figure 2 a) Compared to THCpSi substrate ( Figure 2 b) has a significantly larger contact angle, which clearly demonstrates the hydrophobic behavior of THCpSi and the hydrophilic behavior of pSi, which is expected from thermally hydrocarbonated coatings and saturated hydrocarbon chains.

[0244] Example 2: Surface functionality on THCpSi

[0245] The surface of the THCpSi sample was modified according to the above procedure. The surface modification steps included the introduction of carboxyl groups and the covalent connection of biological receptors.

[0246] The functionalization of THCpSi with carboxyl groups was characterized using FTIR. Figure 3 The FTIR spectra collected from the newly etched pSi, THCpSi, and COOH-terminated THCpSi are shown.

[0247] As expected, the newly etched pSi values ​​are at 2087 and 2114 cm⁻¹. -1 The characteristic spectral bands of Si-H and Si-H2 stretching vibrations are displayed at 905 cm⁻¹. -1 There is also a band at this location attributed to Si-H deformation (spectral line A). After the formation of a hydrocarbon layer across the entire surface, the characteristic bands of Si-H in THCpSi disappear, but remain at 1250-1260 cm⁻¹. -1 A new peak appears at 2852 cm⁻¹, which is related to the CH₃ symmetry deformation of Si-CH₃ (spectral line B). Additionally, peaks appear at 2852 and 2920 cm⁻¹. -1At two distinct absorption bands become apparent, which can be assigned to the symmetric and asymmetric CH2stretching modes, respectively. After thermal treatment, the surface of THCpSi was thermosilylated with COOH-terminated alkenes (10-undecenoic acid), which in turn was used to link NH2-ssDNA capture probes as biological receptors inside the nanochannels. Here, a biosensor was fabricated using miR-142-ssDNA capture probes. Prior to the attachment of the biological receptors, a peak at 1720 cm -1 in the FTIR spectrum was observed, which is attributed to the carbonyl stretching vibration (line C). These results reveal the successful change in chemical composition during surface functionalization.

[0248] Example 3: Electrochemical characterization of THCpSi electrodes

[0249] The electrochemical properties of THCpSi nanostructures were first investigated by cyclic voltammetry (CV) in the presence of [Fe(CN)6] 3- / 4- in 10 mM phosphate buffered saline solution (PBS, pH 7.4). The evolution of the oxidation and reduction reactions was monitored by observing the changes in current intensity as well as the changes in the redox potential when a cyclic potential was applied between the working and reference electrodes.

[0250] Figure 4 a depicts the electrochemical characterization of THCpSi, which are cyclic voltammograms (CV) of freshly etched pSi (line 1), THCpSi (line 2), COOH-terminated THCpSi (line 3), and ssDNA-modified THCpSi (line 4) electrodes. Figure 4 b depicts the electrochemical impedance spectra (EIS) of THCpSi (line 1), COOH-terminated THCpSi (line 2), and ssDNA-modified THCpSi (line 3) electrodes. The measurements were performed in 10 mM PBS (pH 7.4) containing 2 mM [Fe(CN)6] 3- / 4- solution.

[0251] As shown in Figure 4 a, the low oxidation and reduction peak currents indicate poor performance of freshly etched pSi as an electrochemical transducer (I Figure 4 a, line 1). THCpSi (I Figure 4 a, line 2) shows a significant increase in the peak current values compared to freshly etched pSi (I 3- / 4-A rapid and reversible electrochemical response was observed, representing a fully reversible single-electron transfer reaction. The results indicate that THCpSi possesses the surface structure and electronic properties required to achieve rapid electron transfer. The COOH-capped THCpSi surface was activated via an EDC / NHS reaction prior to immobilizing the ssDNA capture probe to generate succinimide ester groups, which are known to react with available primary amino groups on ssDNA. Both COOH-capped THCpSi and ssDNA-modified THCpSi were electrochemically characterized by CV measurements. Compared to unmodified THCpSi, COOH-capped THCpSi exhibited a larger peak spacing and a lower peak current (…). Figure 4 a, spectral line 3), and these features are more pronounced in ssDNA-modified THCpSi ( Figure 4 a, spectral line 4).

[0252] Electrochemical impedance spectroscopy (EIS) was used to further investigate the relationship between the THCpSi electrode and [Fe(CN)6]. 3- / 4- Electron transfer processes at the interface were investigated. Impedance measurements were performed at an open-circuit potential at a range of frequencies (100 kHz–0.1 Hz) and an amplitude of 5 mV. The impedance spectrum was fitted to a Randle circuit, which is an equivalent circuit consisting of solution resistance (Rs), charge transfer resistance (Ret), Warburg impedance (W), and double-layer capacitance (Cdl). Notably, unmodified THCpSi exhibits a much smaller Ret value, such as... Figure 4 The straight line in b (spectral line 1) demonstrates the high conductivity of the carbon-stabilized pSi nanostructure as an electrochemical transducer. After surface functionalization, the impedance spectrum reveals COOH-terminated THCpSi (… Figure 4 b, spectral line 2) and ssDNA-modified THCpSi ( Figure 4 The Ret values ​​for both b and 3) were significantly increased. Therefore, these results confirm that the ssDNA capture probe exists on the surface after surface conjugation, demonstrating the feasibility of using these carbon-stabilized pSi nanostructures as promising electrochemical transducers. Furthermore, the reproducibility and reproducibility of the THCpSi platform were evaluated by analyzing CV and EIS data, showing excellent reproducibility.

[0253] Example 4: Optimizing the parameters of THCpSi

[0254] Compared to conventional carbon-based electrodes such as glassy carbon electrodes and carbon screen-printed electrodes, the electrode surface area of ​​THCpSi can be easily tuned by changing the etching conditions. As a result, one of the main advantages of this carbon-stabilized pSi over conventional carbon electrodes is the ability to improve the sensitivity of the sensing platform by tailoring the channel size to the desired application. To demonstrate the suitability of THCpSi nanostructures for electrochemical biosensing, a label-free voltammetric miRNA biosensor was constructed.

[0255] The sensing mechanism of the label-free voltammetric miRNA sensor developed here relies on the assumption that ssDNA-miRNA hybridization will cause partial steric hindrance or charge-based blockage of the nanochannels. When the target miRNA hybridizes with an ssDNA capture probe immobilized on a THCpSi layer, partial blockage of the porous nanochannels can be expected. This partial nanochannel blockage will hinder the sensing of probes such as [Fe(CN)6]. 3- / 4- As redox substances diffuse into the electrochemically active layer (electrochemical transducer), the peak current intensity monitored by DPV measurements decreases. The change in peak current obtained from differential pulse voltammograms acquired before and after hybridization is normalized using Equation 1: (1)

[0256] in It is the normalized current change, I o The peak current value was measured after incubation for 20 minutes in a buffer blank control, and I n The peak current value was measured after incubation for 20 minutes in a miRNA target solution of a given concentration. All control samples were prepared using a random sequence of ssDNA as a capture probe (miR-423-ssDNA-capture).

[0257] To develop a rapid, reliable, and highly sensitive electrochemical biosensor for miRNA biomarker detection, it is necessary to optimize the fundamental parameters of the THCpSi platform, including channel size and depth. Initially, the average channel size was maintained at 27 nm, while the pore thickness was adjusted from 1.0 μm to 5.7 μm. Figure 5 As shown in af. Because ssDNA is significantly more stable in buffer than similar miRNAs, the miR-142-ssDNA target, with a sequence similar to the miR-142-miRNA biomarker, was selected during optimization. The biosensor was sequentially incubated with various concentrations of miR-142-ssDNA target titers diluted in Tris buffer (as disclosed earlier herein), from lowest to highest. All measurements were performed in triplicate to verify reproducibility.

[0258] Figure 5The performance of each DNA sensor was depicted as follows: differential pulse voltammetry (DPV) of the control electrode fabricated from (a) 1.0 μm thick and (d) 1.7 μm thick THCpSi after continuous incubation in target ssDNA solution; DPV of the working electrode fabricated from (b) 1.0 μm thick and (e) 1.7 μm thick THCpSi after continuous incubation in target ssDNA solution; and dose-response curves of the biosensor fabricated from (c) 1.0 μm thick and (f) 1.7 μm thick THCpSi after incubation with serially diluted target ssDNA.

[0259] like Figure 5 As shown, THCpSi control electrodes with average channel depths of 1.0 μm and 1.7 μm immobilized with mismatched ssDNA probes (miR-423-ssDNA-capture) did not show any significant signal changes in their differential pulse voltammograms when incorporating target ssDNA buffer at concentrations ranging from 0.1 pM to 1000 pM. Figure 5 (a) and (d) demonstrate the promising specificity of the biosensor. However, with increasing target ssDNA concentration, the current intensity of the two working electrodes immobilized by miR-142-ssDNA decreases, as... Figure 5 As shown in figures b and e, the observed current change is a result of the specific binding between the captured probe and the target ssDNA within the nanochannel. The sensor sensitivity was determined by calculating the slope of the linear curve fit, and the electrochemical sensing performance was compared. The biosensor with a 1.7 μm thick sensor can provide 0.05 pM compared to a 1.0 μm thick transducer. -1 Sensitivity.

[0260] After determining the appropriate thickness of THCpSi for a specific DNA sequence, biosensors with different average channel sizes of 15 nm and 40 nm were further evaluated in the same manner.

[0261] Figure 6 The performance of each DNA sensor was depicted, specifically: the DPV of the control electrode fabricated by THCpSi with an average pore diameter of (a) 15 nm and (d) 40 nm after continuous incubation in the target ssDNA solution; the DPV of the working electrode fabricated by THCpSi with an average pore diameter of (b) 15 nm and (e) 40 nm after continuous incubation in the target ssDNA solution; and the dose-response curves of the biosensor fabricated by THCpSi with an average pore diameter of (c) 15 nm and (f) 40 nm after incubation with serially diluted target ssDNA.

[0262] like Figure 6As shown, the control electrodes with these two average channel diameters exhibited nearly identical current intensities in their differential pulse voltammograms when incorporated with various concentrations of target ssDNA buffer. Figure 6 (a and d), and as the concentration of target ssDNA increases, the current intensity of both working electrodes decreases, such as Figure 6 As shown in b and e. Notably, when detecting the target ssDNA, both biosensors exhibited similar electrochemical sensitivities of 0.02 pM. -1 This is slightly lower than that of biosensors with a channel size of 27 nm and a depth of 1.7 μm. These results reveal that suitable nanochannel parameters for THCpSi-based biosensors have been optimized for the development of target miRNA sensing platforms.

[0263] Example 5: Electrochemical detection of target miRNA

[0264] TBI-associated miRNA (miR-142-3p) was selected as a biomarker to test the sensing performance of the optimized THCpSi-based nanochannel blocking biosensor in buffer and diluted serum. Figure 7 The results show the sensing effects of different concentrations of miR-142-miRNA-target in Tris buffer. Similar to the electrochemical detection of the analog ssDNA, the current intensity of the DPV on the working electrode showed a significant decrease as the target miRNA concentration increased from 0.1 pM to 1000 pM, while the current intensity on the control electrode remained almost constant. The associated maximum sensitivity is 0.04 pM. -1 This confirms that the platform has excellent sensing performance for TBI-related miRNA biomarkers.

[0265] Typically, the signal from channel-blocking electrochemical biosensors is also affected by contaminants that attach to the electrode surface via nonspecific adsorption. Contaminants can reduce the area of ​​the electrode surface available for electrolyte, leading to a decrease in the DPV signal. Before detecting miRNA biomarkers in clinical samples, the performance of the developed biosensor was first characterized in diluted serum. Selected miRNA biomarker solutions at concentrations of 0.1, 1, 10, 100, and 1000 pM were prepared in 2% fetal bovine serum (FBS) and used as contaminants. Notably, the control electrode exhibited excellent stability after incubation in 2% serum for 20 minutes. Figure 7 (d) demonstrates the excellent anti-fouling performance of the developed sensing platform. As expected, in 2% FBS solution, a continuous decrease in current was observed in the working electrode with increasing miRNA titer. Figure 7 e). Figure 7 The dose-response curve shown in f provides a value of 0.03 pM.-1 The sensitivity of the THCpSi biosensor was slightly lower than the value in pure buffer solution. The results showed that the THCpSi biosensor could selectively detect the miR-142-miRNA biomarker down to a concentration of 0.1 pM in 2% serum.

[0266] Example 6: Detection of target miRNA biomarkers in clinical samples

[0267] The optimized THCpSi nanochannel-based biosensor was further tested for its ability to detect miRNA biomarkers in clinical samples. Human serum samples collected from healthy donors and mTBI patients were added to Tris buffer solution to obtain a final concentration of 2%. Figure 8 a and b show the electrochemical response of the biosensor with miR-142-3p complementary capture probe. The DPV current decreased after 20 minutes of incubation in the positive sample compared to the same signal in the negative sample, demonstrating that the nanochannel-based biosensor could effectively differentiate between human serum samples containing a specific miRNA biomarker and those not containing the biomarker. Another mTBI-related miRNA biomarker, miR-423-3p, was also measured in human serum samples by simply changing the complementary ssDNA capture probe immobilized on the electrode surface to miR-423-ssDNA-capture. As shown in Figure 8 c and d show the sensing results further demonstrating the ability of the developed miRNA biosensor to detect multiple miRNA biomarkers related to TBI, allowing accurate diagnosis of TBI within a 20-minute time window.

[0268] The actual concentration of miR-142-3p in human whole serum samples could be further determined by comparing the intensity ratio to the linear fitted curve according to the calibration curve obtained from 2% FBS buffer. Figure 9 The calculated concentrations of miR-142-3p in the three different clinical sample groups are clearly shown. These results were then compared to the clinical PCR test results, summarized in Table 3. It is worth noting that the detection results from the electrochemical detection system were very consistent with the PCR results obtained at Alfred Hospital and the medical history of the TBI patients, including falls from ladders, traffic accidents, and falls from scaffolding. Furthermore, since the gold standard PCR technique can only determine the relative concentration of miRNA biomarkers in human serum samples, the electrochemical detection system was also able to further determine the exact value of miRNA concentration in the picomolar range within a 20-minute window. Therefore, the accurate quantification of miRNA markers in human serum could allow for the immediate assessment of the severity of TBI.

[0269] Table 3. Comparison of miRNA detection results obtained by PCR and the electrochemical detection system disclosed herein.

[0270] Sample ID Clinical PCR (NER) Exemplary System Control 1 0.27 0 Control 2 0.359 0 Control 3 1.052 0 B20 (Fall from Ladder) 0.938 5.35 ± 0.65 pM B11 (Traffic Accident) 1.523 7.08 ± 0.71 pM B9 (Fall from Scaffolding) 4.060 15.46 ± 0.59 pM

[0271] Those skilled in the art will appreciate that various changes and / or modifications can be made to the application as shown in the specific embodiments without departing from the scope of the application as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

1. A method of detecting at least one nucleic acid analyte in a complex biological sample, the method comprising: (i) introducing the sample into an electrochemical biosensor comprising a porous substrate comprising a plurality of channels, wherein: - at least a portion of the plurality of channels has an average depth of about 1.0 pm to about 6.0 pm; and - at least a portion of the plurality of channels has an average diameter or cross-section in the range of about 15 nm to about 40 nm, wherein one or more capture probes capable of specifically binding at least a biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex are disposed on at least one surface of at least a portion of the plurality of channels; and (ii) determining whether the at least one nucleic acid analyte is present in the sample based on measurements of peak current obtained from differential pulse voltammograms before and after introducing the sample into the chemical biosensor, wherein a change in the peak current detected after introducing the sample into the electrochemical biosensor is indicative of the presence of the at least one nucleic acid analyte in the sample, and no change in the peak current detected after introducing the sample into the electrochemical biosensor is indicative of the absence of the at least one nucleic acid analyte above a threshold level in the sample.

2. The method of claim 1, wherein the capture probes are capable of specifically binding at least one nucleic acid analyte to form a probe-analyte complex, wherein the presence of the probe-analyte complex within a channel causes at least partial steric and / or charge blockage of the channel compared to a channel in which the probe-analyte complex is not present.

3. The method of any one of claims 1 to 3, wherein: - at least 10% of the plurality of channels in the porous substrate has an average depth in the range of about 1.0 pm to about 2.0 pm, or about 1.0 pm to about 4.0 pm; and / or - at least 10% of the plurality of channels in the porous substrate has an average diameter or cross-section in the range of about 15 nm to about 30 nm, or about 20 nm to about 30 nm.

4. The method of any one of claims 1 to 3, wherein: - the porous substrate comprises, consists essentially of, or consists of an electrically conductive or semiconductive material; and / or - the porous substrate comprises, consists essentially of, or consists of a material selected from the group consisting of platinum, gold, silver, cobalt, gallium, indium, tin, vanadium, zirconium, copper, aluminum, iridium, palladium, rhodium, silicon, zinc, iron, steel, brass, carbon, graphite, alloys, oxides, or metallic compounds of these elements, and mixtures thereof; and / or - the porous substrate is not in the form of a thin film; and / or - the porous substrate does not comprise and / or consist of nanoparticles; and / or - the porous substrate does not comprise one or more of aluminum, platinum, gold, and silver; and / or - the porous substrate comprises, consists essentially of, or consists of a material selected from silicon, optionally heat treated and / or chemically modified silicon; and / or - a layer comprising, consisting essentially of, or consisting of carbon is disposed on at least a portion of the porous substrate.

5. The method of any one of claims 1 to 4, wherein: - the porous substrate, optionally comprising silicon, comprises, consists essentially of, or consists of a monolayer comprising one or more of: ■ one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more functional groups capable of reacting with amine groups; and / or ■ one or more functional groups capable of forming a covalent bond with complementary functional groups on one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more carboxylic acid groups; or - the porous substrate, optionally comprising silicon, comprises, consists essentially of, or consists of at least one layer comprising one or more of: ■ one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more functional groups capable of reacting with amine groups; and / or ■ one or more functional groups capable of forming a covalent bond with complementary functional groups on one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more carboxylic acid groups.

6. The method of any one of claims 1 to 5, wherein: - the one or more capture probes are capable of binding to a plurality of different nucleic acid analytes; and / or - the at least one nucleic acid analyte is selected from the group consisting of: a DNA analyte, a messenger RNA (mRNA) analyte, a ribosomal RNA (rRNA) analyte, a transfer RNA (tRNA) analyte, a microRNA (miRNA) analyte, a short interfering RNA (siRNA) analyte, a plasmid DNA (pDNA), and mixtures thereof; and / or - one or more of the at least one capture probe is a DNA capture probe or a peptide nucleic acid probe (PNA) probe.

7. The method of any one of claims 1 to 6, wherein: - the at least one nucleic acid analyte is associated with a condition, a disease state, or an injury; and / or - the at least one nucleic acid analyte is associated with a brain injury, optionally a traumatic brain injury.

8. The method of any one of claims 1 to 7, wherein the at least one nucleic acid analyte is a miRNA associated with a brain injury, optionally a traumatic brain injury.

9. The method of claim 6 or claim 8, wherein the one or more miRNAs are selected from miR-142-3p, miR-196b-5p, let-7f-5p, miR-150-5p, or any combination thereof.

10. The method of any one of claims 1 to 9, wherein the at least one nucleic acid analyte is detected in the sample at a concentration of about 1 pM or less.

11. The method of any one of claims 1 to 10, wherein the complex biological sample is or comprises a bodily fluid, a cell culture medium, a water sample, a soil sample, a food or beverage sample, an agricultural product, a surface swab, or any combination thereof.

12. The method of any one of claims 1 to 11, wherein the complex biological sample is: - from a human; and / or - a liquid sample; and / or - selected from the group consisting of: whole blood, serum, plasma, urine, saliva, sweat, spinal fluid, sputum, cell lysate, cell culture medium, and mixtures thereof; and / or - obtained from a subject suspected of having a brain injury, optionally a traumatic brain injury.

13. The method of any one of claims 1 to 12, wherein the electrochemical biosensor comprises one or more electrodes.

14. The method of claim 13, wherein the electrochemical biosensor comprises: - a working electrode comprising the porous substrate; - a reference electrode; and - a counter electrode.

15. The method of claim 13 or claim 14, wherein an insulating material is disposed on at least a portion of the electrode.

16. The method of any one of claims 1 to 15, wherein the electrochemical biosensor is used in conjunction with (i) a device capable of scanning a range of potentials and measuring current or (ii) a potentiostat.

17. The method of any one of claims 1 to 16, wherein the change in peak current is normalized via the relationship of Equation 1: (1) wherein is the normalized current change, is the peak current value measured after incubation in the buffer for the first time period, and I n is the peak current value measured after incubation in the target solution at a given concentration for the second time period compared to the control sample.

18. The method of claim 17, wherein the first time period and / or the second time period is independently about 60 minutes, 50 minutes, 40 minutes, 30 minutes, or 20 minutes or less.

19. The method of any one of claims 1 to 18, comprising: - determining the concentration of the nucleic acid analyte detected in the sample based on the level of decrease in peak current intensity; - optionally comparing the concentration of the at least one nucleic acid analyte to a calibration curve; and - optionally determining the disease state or severity of injury, optionally brain injury, of a subject based on the concentration of the at least one nucleic acid analyte.

20. The method of any one of claims 1 to 19, wherein the at least one nucleic acid analyte is detected, and optionally quantified for its concentration, within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 15 minutes, or about 10 minutes of introducing the sample to the electrochemical biosensor.

21. A porous substrate when used in a method according to any one of claims 1 to 20, the porous substrate comprising a plurality of channels, wherein: - at least a portion of the plurality of channels has an average depth of about 1.0 pm to about 6.0 pm; and / or - at least a portion of the plurality of channels has an average diameter or cross-section in the range of about 15 nm to about 40 nm, wherein one or more capture probes capable of specifically binding at least one nucleic acid analyte to form a probe-analyte complex is disposed on at least one surface of at least a portion of the plurality of channels.

22. The porous substrate when used according to claim 21, wherein the capture probes are capable of specifically binding at least one nucleic acid analyte to form a probe-analyte complex, wherein the presence of the probe-analyte complex within a channel causes at least partial steric and / or charge blockage of the channel compared to a channel in which the probe-analyte complex is not present.

23. The porous substrate when used according to claim 21 or claim 22, wherein: - at least a portion of the plurality of channels has an average depth of about 1.0 pm to about 6.0 pm; and - at least a portion of the plurality of channels has an average diameter or cross-section in the range of about 15 nm to about 40 nm.

24. The porous substrate when used according to any one of claims 21 to 23, wherein: - at least 10% of the plurality of channels has an average depth in the range of about 1.0 pm to about 6.0 pm; and / or - at least 10% of the plurality of channels has an average diameter or cross-section in the range of about 15 nm to about 40 nm.

25. The porous substrate when used according to any one of claims 21 to 24, wherein: - the porous substrate comprises, consists essentially of, or consists of an electrically conductive or semiconductive material; and / or - the porous substrate comprises, consists essentially of, or consists of a material selected from the group consisting of platinum, gold, silver, cobalt, gallium, indium, tin, vanadium, zirconium, copper, aluminium, iridium, palladium, rhodium, silicon, zinc, iron, steel, brass, carbon, graphite, alloys, oxides or metal compounds of these elements, and mixtures thereof; and / or - the porous substrate comprises, consists essentially of, or consists of a material selected from silicon; and / or - a layer comprising, consisting essentially of, or consisting of carbon is disposed on at least a portion of the porous substrate.

26. The porous substrate when used according to any one of claims 21 to 25, wherein: - the porous substrate, optionally comprising silicon, comprises, consists essentially of, or consists of a monolayer, the monolayer comprising one or more of: ■ one or more capture probes capable of specifically binding at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■ one or more functional groups capable of reacting with amine groups; and / or and / or ■one or more functional groups capable of forming a covalent bond with a complementary functional group on one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more carboxylic acid groups; or - the porous substrate optionally comprising silicon comprises, consists essentially of, or consists of at least one layer comprising one or more of: ■one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more functional groups capable of reacting with an amine group; and / or ■one or more functional groups capable of forming a covalent bond with a complementary functional group on one or more capture probes capable of specifically binding to at least one biomarker, optionally at least one nucleic acid analyte, to form a probe-analyte complex; and / or ■one or more carboxylic acid groups.

27. The porous substrate for use according to any one of claims 21 to 26, wherein: - the one or more capture probes are capable of binding to a plurality of different nucleic acid analytes; and / or - the at least one nucleic acid analyte is selected from the group consisting of: a DNA analyte, a messenger RNA (mRNA) analyte, a ribosomal RNA (rRNA) analyte, a transfer RNA (tRNA) analyte, a microRNA (miRNA) analyte, a short interfering RNA (siRNA) analyte, a plasmid DNA (pDNA), and mixtures thereof; and / or - one or more of the at least one capture probe is a DNA capture probe or a peptide nucleic acid probe (PNA) probe.

28. The porous substrate for use according to any one of claims 21 to 27, wherein: - the at least one nucleic acid analyte is associated with a condition, a disease state, or an injury; and / or - the at least one nucleic acid analyte is associated with a brain injury, optionally a traumatic brain injury.

29. The porous substrate for use according to any one of claims 21 to 28, wherein the at least one nucleic acid analyte is a miRNA associated with a brain injury, optionally a traumatic brain injury.

30. The porous substrate for use according to claim 29, wherein the one or more capture probes are capable of binding to one or more miRNAs selected from the group consisting of miR-142-3p, miR-196b-5p, let-7f-5p, miR-150-5p, and combinations thereof.

Images