The present disclosure relates to an electrochemical immuno-biosensorfor the detection of blood circulating protein biomarkers indicative of various dis eases or conditions. More particularly the present method and sensor system relates to the detection of otolin-1 and prestin proteins, which are circulating bi- omarkers of the inner ear.

BACKGROUND

In the past two decades, rapid point-of-care diagnostic approaches based on the detection of biomarkers have been penetrating in many areas of medical diagnostics including infectious diseases ¹ · ² , cancers ³ , and neurologi cal disorders ⁴ _’ ⁵ , but not yet inner ear diseases. In fact, current methods to measure inner ear function utilize a set of physical and neurological examina tions such as audiograms ⁶ (for hearing thresholds), vestibular evoked myo genic potentials ⁷ (for vestibular function), or posturography ⁸ (for balance evalu- ations), which do not indicate the specific sites of degeneration within the inner ear ⁹ .

Permanent damage to the cellular sites of the inner ear - sensory hair cells, neurons, synapses, or stria vascularis - due to noise exposure, aging, and side effect of antibiotics (aminoglycosides) or chemotherapeutic drugs (cis- platin) can result in hearing loss or vestibular disorders ¹⁰ _’ ⁿ , which can only be identified post mortem. This leads clinicians to adopt a one-size-fits-all ap proach to the treatment, as they are not equipped with the information required to apply targeted treatment or rehabilitation for the specific injury. As new strat egies based on gene and stem cell therapies are being developed ^{12 16} , there is an unmet need for the development of novel diagnostic approaches based on the detection of inner ear effective biomarkers circulating in the blood, to be able to identify the sites of cellular damage resulting in more precise diagnos tics to ultimately guide therapy.

The lack of knowledge about inner ear specific biomarkers has been a main challenge toward the development of such advancements. However, re cent studies have shown the alteration of a number of serum proteins in human and animal models as indicators of inner ear disorders ¹⁷ - ²⁰ . Otolin-1 is a scaf folding protein expressed in the utricle and saccule - the otolith organs which are part of the vestibular component of the inner ear ²¹ , and prestin is a motor protein uniquely expressed in the cochlea, particularly in outer hair cells ²² _’ ²³ . Changes in serum otolin-1 levels are detectable in patients with balance/vestib ular end organ problems such as benign paroxysmal positional vertigo ²⁴ . Fur thermore, in an animal-ototoxicity model, changes in prestin blood levels were detectable before any shifts in audiometric thresholds could be traced ²⁵ , indi cating the ability of the two proteins to perform as potential biomarkers for inner ear blood-based diagnosis.

Recently, approaches for rapid point-of-care detection of macromole cules, particularly proteins, for disease diagnostics have been developed with the aim of reducing the time and limit of detection compared to currently availa ble multiple-step detection processes (e.g., Enzyme-Linked Immunosorbent As say (ELISA ²⁶ ) and Western blots ²⁷ ) ²⁸ - ³⁰ . Among platforms based on optical or mass detection, electrochemical biosensors, in principle, provide selectivity for capturing target molecules while delivering a specific measurable signal ³¹ _’ ³² . However, achieving high levels of sensitivity and selectivity in whole blood re mains challenging. In this case, various recognition strategies are proposed uti lizing antibodies, proteins, synthetic deoxyribonucleic acids (DNAs), and small- molecules interactions with the target of interest ³³ - ³⁷ . Furthermore, the sensor’s surface, when combined with nanostructured electrodes, offers a large surface area in small sample volumes ³⁸ , and enhancement in the molecular capturing mechanism on the limited geometry of the surface ³⁹ , resulting in the improve ment of the sensitivity and detection limit of electrochemical biosensors ⁴⁰ .

SUMMARY

The present disclosure provides a biosensor platform utilizing a DNA- based immunoassay immobilized on nanostructured electrodes for the detec tion of otolin-1 and prestin proteins, which are potential biomarkers of balance and hearing disorders, respectively. Taking advantage of the steric hindrance mechanism ⁴¹ on the nanostructured electrodes ⁴² , the present inventors have designed a recognition strategy adapting the conjugated antibodies that can be extended further to a variety of different target proteins by incorporating their specific antibodies. The electrochemical biosensor can potentially overcome the challenges toward one-step rapid detection at the point-of-care.

The present inventors have developed the first biosensor for inner ear biomarkers. The electrochemical biosensor is analogous to commercially avail able glucose-meter (for measuring blood glucose in diabetic patients) for the di rect non-invasive detection of otolin-1 and prestin, two blood-circulating protein biomarkers specifically expressed in the balance organs (utricle and saccule) and cochlea of the inner ear, respectively. The platform is designed based on two advances in nanobiotechnology to improve the functionality of the sensor toward one-step protein detection in complex media (e.g. whole blood). (1) A DNA-based immunoassay employs, first, interactions of oligonucleotide se quences to specify the sensors electrochemical signal when deployed in com plex media; second, conjugated antibodies for target protein recognition, which when bound alters the DNA-DNA hybridization on the surface with the steric ef fects resulting in the generation of the concentration-dependent electrochemical signal output. (2) The high-curvature nanostructured electrode is used for signal amplification to enable sensitive sample analysis at low picomolar concentra tions with a three-fold quantitative range, as well as to acquire the analysis in a low 10 mM (micromolar) sample volume in under 30 minutes (min).

The synthetic assay utilizes a high population of short single-strand cap turing DNA probes immobilized on the surface of gold nanostructure electrodes deposited on glass chips with addressable electrodes. The signaling DNA probes, which are mixed with the target sample before being added on the sen sor surface, are designed to carry and place the redox moiety, methylene blue (MB), on the sensing surface and generate an electrochemical current signal upon hybridization. On the other extremity, the signaling DNA probes are at tached to the antibody recognition element utilizing a streptavidin-biotin interac tion. The recognition strategy disclosed herein is unique as it offers a universal detection mechanism knowing the high-affinity-interaction of streptavidin-biotin (Ko =40 fM) and the ability to incorporate different antibodies specific to various targets. The steric effects of such a recognition molecule on the surface hybridi zation can be extensively diminished within the curvatures of surface nanostructuring. When the target protein is bound to the recognition element on the signaling DNA probe, the steric hindrance of the target protein limits the more significant number of successful hybridizations to the surface resulting in an elevated reduction of the current signal.

There is provided an electrochemical immuno-biosensor-based method for detecting blood circulating target protein biomarker, comprising: selecting a target protein biomarker to be detected for; identifying an antibody complimentary to the target protein biomarker; preparing a recognition complex of antibody with streptavidin (1:1) thereby preparing a streptavidin-conjugated-antibody recognition complex; mixing the recognition complex with signaling DNA probes to produce a final recognition complex comprising signaling probe plus streptavidin-conju- gated-antibody complex, the signaling DNA probes being complexed with a re dox moiety; preparing a mixture of the final recognition complex with a sample being tested for the presence of the target protein biomarker such that any target pro teins present in the sample bind with the antibody of the final recognition com plex; preparing high curvature gold nanostructure working electrode and im mobilizing capturing DNA probes onto a surface of the gold nanostructure elec trode and adding the mixture of final recognition complex with a sample to the surface of the working electrode to the mixture of the sample and final recogni tion complex; and performing square wave voltammetry (SWV) on the sample and plotting the current versus voltage and comparing the sample current versus voltage plots to current versus voltage plots obtained using a calibration solution not containing any target protein biomarker and based on differences between the sample and calibration current versus voltage plots determining the presence or absence of the target protein biomarker. The step of mixing the recognition complex with signaling DNA probes to produce a final recognition complex may comprise the signaling DNA probe be ing added to the mixture (5:1) and (10:1) to make a final recognition solution of 25 nM signaling probe + 5nM (nanomolar) streptavidin-conjugated-antibody and 10 nM signaling probe + 100 pM (picomolar) streptavidin-conjugated-antibody, respectively.

The signaling DNA probes are bound to the final recognition complex uti lizing a streptavidin-biotin interaction.

The signaling DNA probes are shorter and complementary to the captur ing DNA probes, which upon hybridization, bring the redox moiety, to the sur- face and generate the current signal.

The redox moiety may be methylene blue (MB), or any other organic or inorganic molecules that can be attached to the probes and generate redox ac tivity upon applying proper voltage.

The target protein being detected may be otolin-1 , so that the antibody is anti-otolin-1 antibody.

The target protein being detected may be otolin-1 in a blood sample, and wherein the antibody can be replaced with the antibody Fab fragment or a pep- tide-derivate of otolin-1 protein, or replace with the otolin-1 protein or otolin-1 protein antigen for indirect detection of target otolin-1 , in a competition assay. The target protein being detected may be prestin, and wherein the anti body is anti-prestin antibody.

The target protein being detected may be prestin in a blood sample, and wherein the antibody can be replaced with a peptide-derivate of prestin protein or antibody Fab fragment, or replaced with the prestin protein or prestin protein antigen for indirect detection of target prestin, in a competition assay.

The target protein being detected may be prestin in a blood sample, and wherein the antibody may be prestin protein or a peptide-derivate of prestin pro tein for indirect detection of target prestin, in a competition assay.

The sample may be human blood.

The sample may be human biofluid, including serum, plasma, saliva, na sopharyngeal, urine, perilymph, and any other liquid-based biofluid.

The sample may be animal biofluid including blood.

A further understanding of the functional and advantageous aspects of the present disclosure can be realized by reference to the following detailed de scription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will now be described, by way of example only, with refer ence to the accompanying drawings, in which:

FIGS. 1 A to 1C shows a schematic illustration and proof of principle for the immunosensor developed on nanostructures, in which:

FIG. 1A shows a 20-lead chip having 200 pm nanostructures electrode- posited (top) on the 10 pm-apertures (bottom) of addressable electrodes. The immunoassay is having a layer of capturing probes immobilized on the surface of nanostructures.

FIG. 1B shows when no attachment to target proteins (panel B’) and when attached, (panel B”). When not attached to the target proteins (panel B’, top), the current signal from hybridization of signaling probes carrying the recognition-antibodies is relatively high (without target). Upon binding to the tar get protein (B”, bottom), the steric hindrance of target proteins will reduce the number of successful hybridizations to the surface and consequently suppress the current signal (with target) to give the lower current signal compared to the higher current plot obtained without the target proteins present.

FIG. 1C shows sensor performance in PBS buffer (panel C’) and in whole blood (panel C”). The sensor’s performance was tested and confirmed the same in PBS buffer (panel C’) and in whole blood (panel C”) by comparing the signal gain reduction at 10 nM (34% vs 33%) and 20nM (64% vs 67%) of otolin-1 (OTOL1) and 20 nM (77% vs 78%) of prestin (PRES). Sample media: PBS 1X 10mM MgCh buffer, and 100% human whole blood; recognition com pound: 5 nM of streptavidin-conjugated antibody previously bound to 25 nM of signaling probes; chips were divided into two zones with a hydrophobic line, each zone loaded with 10 pL of target solution for 10 min-incubation, kept in hu mid chamber; ^**** P < 0.0001 , ^** P < 0.01 significance versus concentrations.

FIG. 2 shows steric-based assay validation for the detection of otolin-1 and prestin. Assay illustration, square wave voltammetry responses, and the re sulting current signal of the immobilized nanostructured electrodes reported, (1, 1’, 1”) in buffer, then in signaling probes (2, 2’, 2”) alone, (3, 3’, 3”) with otolin- 1 (OTOL1) or prestin (PRES), (4, 4’, 4”) with otolin-1 antibody (anti-OTOL1) or prestin antibody (anti-PRES), (5, 5’, 5”) with streptavidin (SA), and signaling probes with recognition elements of SA-antiOTOL1 and SA-antiPRES (6, 6’, 6”) in the absence of target, and (7, 7’, 7”) in the presence of target. Ctrl.: con trol/signaling probes; 10nM signaling probes, 100pM recognition element (1 :1 SA:antibody), 500 pM otolin-1 and 500 pM prestin protein; buffer media: PBS (Phosphate-buffered saline) 1X 10mM (millimolar) MgCh. ^**** P < 0.0001 , ^*** P < 0.001 , ^* P < 0.05, and ns: non-significant versus different concentrations.

FIG. 3 show assay response-time using kinetics and gain reduction in which panel A shows a plot of current in microamps (mA) versus time (minutes) which shows hybridization kinetics of the signaling probes in the absence and presence of target otolin-1 , and panel B is a plot of gain reduction % versus time (minutes) which shows the slight changes in the gain reduction versus time, indicating the maximum performance of the assay was within the first 10 min.

FIG. 4 Optimization of the assay through dose-response curves. Com parison of dose-response curves on NE1 , NE2, NE3 when applied in signaling probes (A, A) without recognition element have C5O%,A,NEI = 16± 3 nM,

C5O%,A,NE2 = 10±3 nM, and CSO%,A\NE3 = 85±13 nM, and (B, B’) with recognition element in the absence of protein have CSO%,B,NEI = 750 nM and CSO%,B\NE3 = 85 nM, and (C, C’) with recognition element in the presence of protein have C5O%,C,NEI = 280 pM and CSO%,C\NE3 = 2 pM. NEs1 to 3 are introduced in Table 3; ^**** p < 0.0001 , ^*** P < 0.001 , ^* P < 0.05, and ns: non-significant versus different concentrations. ^† Although the ANOVA model for panel 4C was not significant, at the highest level of the Otolinl antigen (500 pM), the reduction in the re sponse was at the start of the dose-response curve and was significantly re duced ( ^* P<0.05). DETAILED DESCRIPTION

Various embodiments and aspects of the deep orbital access retractor device disclosed herein will be described with reference to details discussed below. The following description and drawings are illustrative of the disclosure and are not to be construed as limiting the disclosure. The figures are not to scale. Numerous specific details are described to provide a thorough under standing of various embodiments of the present disclosure. However, in certain instances, well-known or conventional details are not described in order to pro vide a concise discussion of embodiments of the present disclosure.

As used herein, the terms, “comprises” and “comprising” are to be con strued as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and claims, the terms “comprises” and “comprising” and variations thereof mean the specified features, steps or components are in cluded. These terms are not to be interpreted to exclude the presence of other features, steps or components.

As used herein, the term “exemplary” means “serving as an example, in stance, or illustration,” and should not be construed as preferred or advanta geous over other configurations disclosed herein.

As used herein, the terms “about” and “approximately” are meant to cover variations that may exist in the upper and lower limits of the ranges of val ues, such as variations in properties, parameters, and dimensions. In one non limiting example, the terms “about” and “approximately” mean plus or minus 10 percent or less.

As used herein, the terms “generally” and “essentially” are meant to refer to the general overall physical and geometric appearance of a feature and should not be construed as preferred or advantageous over other configura tions disclosed herein.

It is to be understood that unless otherwise specified, any specified range or group is as a shorthand way of referring to each and every member of a range or group individually, as well as each and every possible sub-range or sub-group encompassed therein and similarly with respect to any sub-ranges or sub-groups therein. Unless otherwise specified, the present disclosure relates to and explicitly incorporates each and every specific member and combination of sub-ranges or sub-groups.

As used herein, the term "on the order of", when used in conjunction with a quantity or parameter, refers to a range spanning approximately one tenth to ten times the stated quantity or parameter.

As used herein, the phrase “high-curvature nanostructured electrode(s)” refers to a structure that consists of thousands of nano-needles with sharp tips that are located in close proximity representing a tree-like /spiky-like/shrub-like structure in microscales (e.g., about 1 to about 300 pm). They can be compared with low curvature nanostructures where the structure contains round-shape tips such as nanoparticles and/or nanorods. The bigger the size of the structure (within about 1 to about 300 micrometers (pm)) is, the more the number of branches and nano-needles are generated, as well as more sites for probe im mobilization. It has been shown that immobilized probes are displayed at a high deflection angle on these branches resulting in suppression of the probe aggre gation among adjacent probes allowing greater accessibility and more efficient attachment of the target molecules to the surface, which can tremendously im prove the sensitivity of the sensor. As used herein the phrase “target protein” refers to the protein that is in dicative of the disease being tested for which is known to circulate in blood.

As used herein, the term “antibody” refers to a blood protein produced in response to and counteracting the target protein (antigen). Antibodies combine chemically with substances which the antibody recognizes as alien, such as bacteria, viruses, and foreign substances in the blood.

As used herein, the phrase “recognition complex” refers to the molecular complex which “recognizes” and forms a complex with the target protein. In this disclosure the recognition complex comprises streptavidin conjugated with the antibody being used to detect the selected protein, thus giving a streptavidin- conjugated-antibody complex.

As used herein, the phrases “capturing DNA probes” or “capturing probes” refers to single-strand capturing DNA probes or DNA-analog probes (e.g., peptide nucleic acid (PNA) strands) immobilized on the surface of the gold nanostructure electrodes.

As used herein the phrase “signaling DNA probes” refers to DNA strands or DNA-analog strands (e.g., PNA strands) designed to carry and place a redox moiety on the sensing surface and generate an electrochemical current signal upon hybridization. The signaling DNA probes are attached to the antibody recognition element utilizing a streptavidin-biotin interaction. The signaling DNA probes are shorter and complementary to the capturing DNA probes (or strands), which upon hybridization, bring the redox moiety, to the surface and generate the current signal. The redox moiety (or redox label, or redox indica tor) may be methylene blue (MB), or any other organic or inorganic molecules that can be attached to the probes and generate redox activity upon applying proper voltage.

Several DNA probe immobilization techniques have been employed in electrochemical DNA sensing for immobilized or captured DNA probes on the electrode surface, such as adsorption methods, covalent bonding and avidin- biotin interaction.

The adsorption methods include physical adsorption and electrochemical adsorption. In physical adsorption or physisorption, forces of attraction are due to Van der Waals’ forces between the solid surface and the bio-molecule and the quantity binding to the surface depends on surface properties including tem perature, pressure and the surface roughness. In electrochemical adsorption, forces of attraction between the surface and DNA are due to ion-to-ion interac tions between the negatively charged DNA and the positively charged surface. By applying a constant positive potential (e.g., 0.8 Volts (V)), the phosphate group of the DNA molecule binds to the positively charged surface due to elec trostatic attraction. In both techniques, the immobilized electrodes can be washed with distilled water to remove loosely adsorbed DNA and dried under nitrogen gas. However, the DNA adsorption immobilization method results in orientation of the molecules parallel to, rather than perpendicular to the surface where the DNA backbone is attached to the surface and base pairing sites ex posed to the liquid. This configuration is not ideal for the present design of as say on the surface which relies on the spatial orientation of DNA probes stand ing on the surface to hybridize to the target molecule ⁴³ _’ ⁴⁴ .

The covalent immobilization of DNA on the surface has some ad vantages when compared to adsorption method mainly because the DNA probes are bound to the electrode surface by one end only, which provides more structural flexibility and increases the accessibility for more efficient hy bridization. The covalent binding of DNA probes to the surface is based on a modification on the surface to provide some active groups in the electrode ma terial such as carboxylic or amino groups. The active group on the electrode surface is in charge of interacting with the DNA probe through either the gua nine or one of the ends (5 or 3) of DNA. Covalent immobilization provides a sta ble detection layer preventing the desorption of DNA probe from the electrode unlike the adsorption technique ⁴⁵ .

The immobilization method based on interactions between biomolecules such as avidin-biotin complex formation is more secured by the affinity strength of the interactions. In this case, the surface is modified to carry avidin mole cules as binding points for the biotinylated-DNA probes (the DNA probes can be modified with biotin on either 5’ or 3’ ends) to be immobilized on the surface

46

Herein, the method and system rely upon a property of gold electrodes that can generate a strong binding to the thiol (-SH) to form the self-assembly monolayer of DNA probe on the surface. In this case, the DNA probe is modi fied with a thiol on the 5’ end, which upon activation with TCEP (Tris(2-carboxy- ethyl) phosphine hydrochloride) can directly bind to the surface of gold elec trode without further modification on the surface. The density of the probes on the surface can be easily adjusted by the concentration of DNA probes during immobilization. Furthermore, the probes spatial orientation and structural flexi bility are advantageous for more efficient signal response in the proposed as say. The present method and system will now be illustrated using the non-lim iting and exemplary example of the detection of otolin-1 and prestin proteins in blood. EXAMPLES

In the example of detection of otolin-1 , the recognition molecules brought to the surface of the nanostructured electrode include anti-otolin-1 antibody, which is conjugated to streptavidin (ratio 1 :1), and further bound to the biotin on the signaling DNA probes through streptavidin-biotin conjugation. However, it will be appreciated that the anti-otolin-1 antibody may be replaced by otolin-1 protein or otolin-1 protein antigen or a peptide-derivate of otolin-1 protein for in direct detection of target otolin-1 , for example in a competition assay.

For the detection of prestin proteins in blood, the recognition molecules bound to the surface of the nanostructured electrode include anti-prestin anti- body, which is conjugates to streptavidin (ratio 1 :1), and further bound to the bi otin on the signaling DNA probes through streptavidin-biotin conjugation. The recognition antibody, which is used for direct capturing of target prestin can be replaced with a peptide-derivate of prestin antibody or antibody Fab fragment as long as the affinity of binding is still maintained. On the other hand, the recognition molecule can also be the prestin protein or a peptide-derivate of prestin protein for indirect detection of target prestin, for example in a competi tion assay.

Materials and Methods Reagents Glass chips (Telic; Valenica, CA), HAuCI4 solution (Sigma Aldrich), 6N- hydrochloric acid (HCI; VWR), Tris(2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma-Aldrich), 6-Mercapto-1-hexanol (MCH; Sigma-Aldrich), Phos phate-buffered saline (PBS, pH 7.4, 1X; Invitrogen), Magnesium chloride (>=98%; Sigma-Aldrich), were all used as received. The DNA constructs (Table 2) synthesized and HPLC purified (Biosearch Technologies Inc., Novato, CA), were aliquoted and stored at -20 °C (degrees Celsius).

Table 2. Sequences of capturing and signaling DNA probes.

Capture strands

5’-HS-(CH2)6- AAGG AAA GGG AAG AAG TTTA CTC CAC GTG CTC Signaling strands 5 - CTT CTT CCC TTT CCTT-MB

Biotin-conjugated rabbit polyclonal antibody to human otolin-1 and to hu man SLC26A5 (prestin) (anti-OTOL1 and anti-PRES; LifeSpan Biosciences Inc.); streptavidin (Sigma Aldrich), otolin-1 protein antigen (26kDa (kiloDal- tons)); Novus Biologicals) were all aliquoted and stored at -20°C for long-term storage and at 4°C for short-term use. SLC26A5 or prestin protein (81 4kDa; Novus Biologicals) was aliquoted and stored at -80°C. Single-donor human whole blood from Innovative Research, that contains heparin as an anticoagu lant, was aliquoted and frozen at -20°C prior to use.

Instrumentation

Direct current (DC) potential amperometry and square wave voltammetry (SWV) was carried out by PalmSens4 potentiostat/galvanostat/impedance ana lyzer combined with MUS08R2 multiplexer. A conventional three-electrode cell was used with a platinum wire counter electrode (CE; Sigma-Aldrich), an Ag/AgCI reference electrode (RE; CH Instruments), and the chip substrate as the working electrode.

On-chip Electrode Preparation

Glass chips were patterned with the leads and the electrodes at their ter minals by first precoating with a 5 nm-Cr, coating with a 50 nm-Au, coating with a layer of AZ 1600 positive photoresist, selective exposure to 900 W UV for 12 s, developing in MF 312 for 40 s, and wet etching of Au and Cr on the unpro tected areas. The 10 pm-apertures were then formed on the electrodes by spin- cast of the negative photoresist (SU-82002) at 4500 revolutions per minute (rpm) for 40 s on the patterned chips, exposing for 12 seconds (s), and then de veloping for 1 min. Chip substrates with twenty addressable 10-pm-apertures were rinsed with acetone, isopropyl alcohol, and Dl water, then dried with the flow of nitrogen.

Chips were immersed in a 3 milliliter (ml) electrolyte solution containing 50 mM HAuCU and 0.5 M HCI. Using DC potential amperometry at 0 mV for 200 s (for 200 pm NE1 and NE2) and 100 s (for 100 pm NE3), the gold nanostructured electrodes (NEs) are electrodeposited on the apertures. All the experiments were done at room temperature. The chips were then rinsed with Dl water and dried with air blow to become ready for capturing probe-immobili zation.

Surface immobilization was done with 100 nM and 200 nM capturing probes in 1X PBS + 10 mM MgCh. Prior to immobilization, 1 pi of 0.1 mM cap turing probes were incubated with 2 mI of 10 mM TCEP for 1 h for reduction of disulfide bonds, then diluted in 1X PBS + 10mM MgCh to the desired concen trations; 100 mI_ of capturing probe solutions of 100 nM (on NE1 and NE3) and 200 nM (on NE2) were applied on the individual chips, to cover all over the area of the electrodes and kept overnight. After washing with 1X PBS, 100 mI_ of 3mM MCH was put on the electrodes for 3 hr and then washed with 1X PBS. The surface density of the capturing probes was calculated between 1 *10 ¹² - 5x10 ¹² cm ² depending on the size of the electrode ⁴¹ .

Electrochemical Measurement

SWV was used to collect the experimental data from -0.45 to 0.05V in in crements of 0.001V vs. Ag/AgCI, with an amplitude of 50 mV and a frequency of 60 Hz. Peak currents were fitted using the PSTrace software.

In Buffer Media

The recognition complex was prepared by pre-incubation of antibody with streptavidin (1 :1) overnight. Then the signaling probe was added to the mixture (5:1) and (10:1) overnight to make a final recognition solution of 25 nM signaling probe + 5nM streptavidin-conjugated-antibody and 10 nM signaling probe + 100 pM streptavidin-conjugated-antibody, respectively.

In human whole blood (>71%): 10 mI_ of the pre-(overnight) incubated recognition complex solution of 20 nM (described above) is mixed with 1 mI_ of the 1 mM signaling probe overnight and reached to 40 mI_ volume by adding whole blood. Proteins were first spiked in whole blood (0.2 mI_ of protein to 10 mI_ of whole blood) prior to mixing with the recognition compound.

All measurements were taken immediately after 15 min incubation of protein with the mixture of recognition bound to signaling probe solution. For the incubation, the chips were divided into two zones using a hydrophobic pen, each having ten electrodes that were loaded with 10 pL-solution. One of the zones on the chip was always assigned for the control test. After 10 min of ac- quisition (extracted from FIG. 3), the chips were unloaded and reloaded with 1X PBS or with whole blood (100%) for the signal measurement in buffer media and whole blood, respectively. Results are presented in terms of current (know ing that the geometric area of the electrode is 0.03 mm ² for NE1 and NE2 and 0.008 mm ² for NE3). Control samples (Ctrl.) are representing the response to 10 nM of signaling probes after 10 min.

Gain Reduction

This value is calculated as the difference in peak current of the samples with and without the target protein divided by the initial peak current (without target protein). Binding Curves

Binding curves or dose-response curves were obtained by testing vari ous concentrations of protein on the platform. Individual curves were fitted to a single-site binding mechanism (Co= background current; Cso% is the concentra tion of target proteins at when the sensor reaches 50% of the signal amplitude:

[Target] (current amplitude)

Probe Density Calculations

Capturing probe surface density is defined by the number of moles of capturing probes per unit area of the NE (Nt) that is equivalent to the number of methylene blue (MB) molecules being placed on the surface through hybridiza- tion: n = 2: number of electrons transferred per MB label

F: Faraday constant

R: universal gas constant

T: temperature

Eac: amplitude f: frequency of the applied voltage perturbation.

We estimated the capturing probes surface density on three different NEs (based on the size and immobilization concentration) after 10 min of hybridizing to 100 nM signaling probes as presented in Table 3.

Table 3. The estimated density of capturing probes. area, [probe], density, 1/cm ² cm ² nM

NE1 0.0003 100 (1.26±

0.38)x10 ^{12 ~} NE2 0.0003 200 (4.57±

0.84)x10 ¹²

NE3 0.00008 Ϊ00 (3.64±

0.56)x10 ¹²

Statistics

Data for bar charts and binding curves are reported as mean values ± standard errors of the means and were analyzed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA). Data for FIGS. 1C-OTOL1 , 2, and 4 were analyzed by one-way analysis of variance (ANOVA) with omnibus statis tics presented in Table 4.

Table 4. Statistics measured by one-way analysis of variance for otolin-1 and prestin electrochemical immuno-biosensor. Owing to the fact that we performed multiple ANOVA tests (N = 10) for detection of otolin-1 and prestin, we used Bonferroni correction to adjust our alpha criterion to 0.005. ^† = not significant.

Figure AN OVA table

1C’, F (2, 20) = 23.78; P<0.0001

OTOL1

1C”, F (2, 35) = 35.21; P<0.0001

OTOL1

2 F (9, 136) = 52.90; P<0.0001

4A, NE1 F (4, 95) = 38.75; P<0.0001

4A, NE2 F (4, 92) = 70.62; P<0.0001

4B F (3, 68) = 5.253; P=0.0026

4C F (5, 48) = 3.801; P=0.0056 ^†

4A’ F (4, 91) = 299.3; P<0.0001

4B’ F (3, 36) = 20.48; P<0.0001

4C’ F (4, 35) = 125.9; P<0.0001

Post hoc comparisons were done by Tukey’s Honest Significant Differ- ence (HSD) test. P-values below the alpha criterion (probability of Type 1 error) of 0.05 were considered statistically significant in all post hoc tests, whereas the alpha criterion for one-way ANOVA tests (described in Table 4) were Bonfer- roni-corrected due to multiple testing. For the data in FIG. 1C-PRES, the inde pendent f-tests were done for the analysis with statistics presented in Table 5. Table 5. Statistics measured by independent t-tests for prestin electrochemical immuno-biosensor.

Figure target prestin

1C PRES t(15) = 7.235; P<0.0001

1C”, PRES t(32) = 45.70; P<0.0001 Disclosed herein is a single-step assay taking advantage of the specific ity of interactions of oligonucleotide sequences in the sensor’s signal, even when deployed in complex media ⁴⁷⁴⁹ (see FIGS. 1A to 1C). Nanostructures are electrodeposited with high curvature, as working electrodes (NEs) on the 10 pm-apertures of a glass chip with twenty addressable leads (see FIG. 1A). The nanostructuring of the surface provides tunable sensitivities and linear detection ranges for the electrochemical detection platform. Short single-strand capturing DNA probes (longer strands) are immobilized on the gold nanostructures (see panel 1 B of FIG. 1 B). The signaling DNA probes (shorter strands) are shorter and complementary to the capturing strands, which upon hybridization, can bring the redox moiety, methylene blue (MB), to the surface and generate a cur rent signal (see panel 1B’ of FIG. 1B, top row). On the other extremity, the sig naling probes carry the recognition element utilizing a streptavidin-biotin inter action. This can apply steric effects on surface hybridization, which can be di minished within the curvatures of the nanostructured surface. When the target protein is bound to the recognition element on the signaling DNA probe (see panel 1B” of FIG. 1B, bottom row), steric hindrance is increased, limiting the number of successful hybridizations to the surface resulting in a reduction in the current signal (with-target curve vs. without-target curve).

Detection of otolin-1 and prestin The mammalian inner ear contains proteins that circulate in the blood stream. Otolin-1 , a glycoprotein expressed within the vestibular supporting cells, provides a scaffold for otoconia on the sensory epithelia maintaining the body balance ²⁴ . Prestin is a motor protein in the outer hair cells and operates to elongate the cells in support of normal hearing sensitivity ⁵⁰ . As proof of prin ciple to our DNA-based immunoassay, we targeted the protein detection (here, a 26-kDa otolin-1 antigen and an 81-kDa prestin) in the buffer (see panel 1C’ of FIG. 1 C) as well as in whole blood (see panel 1 C” of FIG. 1 C). To do so, we first designed a recognition element by having their antibodies (anti-otolin-1 and anti-prestin) attached to the signaling probes via streptavidin-biotin conjugation (see FIG. 1B).

Then the sensor platform was developed to maintain the steric hindrance of the target protein despite the potential steric effects of other components of the assay (e.g., the antibody, streptavidin, or the DNA probes). The incorpora- tion of synthetic DNA probes, combined with the anti-protein specific antibody, regulates the capturing and detection of protein without any interference from the non-specific interactions when deployed in whole blood. This is evident by comparing the signal gain reductions of otolin-1 (OTOL1) in the buffer (34% and 64%) and whole blood (33% and 67%) at 10 nM and 20 nM protein concentra- tions. The detection of prestin (PRES) at 20 nM, further proved the similarity of the sensor’s response in the buffer (77%) and whole blood (78%). The increase in the signal gain of prestin compared to otolin-1 (78% vs. 67%) could be at tributed to the difference in the size of the target proteins (26 kDa vs. 81 kDa). Furthermore, increasing the concentration of protein (10 nM to 20 nM) resulted in more signal loss (33% vs. 67%), representing the quantitative ability of the sensor even in whole blood.

Immunoassay design and validation for otolin-1 and prestin.

Surface immobilization was done on nanostructures via sulfur-gold bonds, at a high surface density of the capturing probes, followed by back-filling with the 6-mercaptohexanol (MCH). We used a thiol-modified (on the 5’ end) 32-base DNA construct, as the capturing probe on the electrode’s surface. The current response of such a surface represents no peak (FIG. 2- (1, 1’, and 1”)). Signaling probes were designed to be 16-base long with a biotin on the 5’ end and a methylene blue (MB) on the 3’ end, complementary to the lower half of the capturing probe construct. Upon hybridization to the high-density of captur ing probes on the NEs, a relatively high peak current was measured as the MB is placed on the electrode surface resulting in a successful electron transfer (FIG. 2- (2, 2’, and 2”)). The response peak was not affected by the presence of otolin-1 (OTOL1) and prestin (PRES) proteins (3, 3’and 3”) or antibodies (4, 4’, and 4”).

However, once the signaling probes were introduced to the streptavidin (55 kDa) (FIG. 2- (5, 5’, and 5”)), or the streptavidin-conjugated antibodies (210kDa (kilodaltons)) (FIG. 2-(6, 6’, and 6”)), the current signal decreased by 25% and -60% (SA-anti-OTOL1 , 62% and SA-anti-PRES, 58%), respectively. This is due to the interactions between streptavidin and biotin on signaling probes, and consequently, the steric hindrance of attached molecules on the surface hybridization.

A further signal decrease occurred when the recognition molecule was attached to the target proteins (otolin-1 antigen: 26kDa and prestin: 81 kDa), re sulting in elevated steric hindrance effect of a bigger molecular compound (OTOL1 compound, 262kDa and PRES compound, 372kDa) on the surface hy bridization (FIGS. 2-(7, 7’, and 7”).

The signal suppression within this assay originated from, first, the smaller number of signaling probes reaching the surface due to the steric hin- drance of attached macromolecules, and second, their lower rate of hybridiza tion ⁵¹ . We studied the hybridization kinetics of signaling probes carrying the recognition element in the absence and presence of the target otolin-1 (FIG. 3A). The calculated rate and time of hybridization (Table 1) indicated that there was a slight delay in hybridization to the surface in the presence of the target. We then measured the increase in the signal gain reduction of the sensor ver sus time, which shows a gradual decrease after the first minutes. This implies that the maximum performance of the assay can be achieved within the first 10 min (FIG. 3B).

Table 1. The hybridization kinetics and the consequent rate and time of hybridi- zation. min no target 0.03 23 with tar- 0.02 37 get

Immunoassay Optimization

The maximum surface density of capturing probes combined with the op timum size of the electrode is required to differentiate the signal of the target protein from the background signal (steric hindrance of the conjugated antibody with and without target protein). Then, the minimum amount of signaling probes required to saturate the surface hybridization, and the minimum amount re quired to generate a measurable signal, can be deducted from the dose-re sponse curves on three NEs with variations in size and immobilized-capturing probe concentration - NE1 (200pm, 10OnM), NE2(200pm, 200nM) and

NE3(100pm, 100nM) (FIGS. 4A, 4A’). We used tree-like nanostructures that possess high-curvatures to ensure the high-surface density of capturing probes as well as the high density of hybridization to the surface ³⁹ . In this case, we showed that when the immobilization concentration is adjusted to 100 nM, and the concentration of the signaling probe to 10 nM, we could maintain the high current signal as well as we could minimize the background current induced by the large (210 kDa) recognition element.

The inventors estimated the minimum amount of recognition molecule required to maintain the steric hindrance of the target molecule once it was at- tached to the signaling probe, as well as a high current signal in the absence of the target molecule. The dose-response curves of the recognition molecules were measured with 10nM of signaling probes (deducted from Cso% of FIGS. 4As) on the NEs (FIGS. 4B, 4B’; CSO%,NEI = 750 pM and CSO%,NE3 = 85 pM) im plying that the smaller NEs (NE3) were capable of complete suppression of the background current. With an optimum amount of recognition element (data is shown for 100 pM), we then conducted the binding curve of target protein (here, otolin-1 ; FIGS. 4C, 4C’). The resulting Cso% was measured at 280 pM on NE1 and 2 pM on NE3. We showed that with the NE3, we could achieve low picomo- lar concentrations of the protein within a three-fold quantitative range within less than 10 min, implying not only the impact of electrode’s dimension on the suc cessful reduction of the detection limit but also the capacity of this technique for rapid point-of-care detection of proteins.

Discussion

Using the idea of steric hindrance on nanostructured surfaces that could alter the hybridization of DNA probes to their complementary strands on the surface generating a measurable linear range, the present inventors have cre ated the first biosensor for electrochemical detection of otolin-1 and prestin, two circulating biomarkers of the mammalian inner ear. Compared with the compli cated multiple-step ELISA-based approaches that are used as the gold stand ard for the detection of inner ear proteins, the approach disclosed herein has the advantage of simplicity, rapidity together with the specificity of the signal, making it a strong candidate for the point-of-care diagnostic platforms for inner ear diseases. Although this disclosure has been illustrated with respect to the detection of two inner ear proteins, otolin-1 and prestin, it can be adapted for other inner ear biomarkers as well as well as for any other circulating protein bi omarkers.

The DNA-based detection platform disclosed herein incorporates a recognition strategy with an antibody conjugated to streptavidin for the detec tion of proteins at low concentrations. In this case, a combination of the high density of capturing DNA probes on the surface, and an optimal density of sig naling complementary DNA probes carrying the streptavidin-antibody recogni tion element to the surface was calculated to ensure the best performance of the assay on the nanostructured electrodes for quantitative detection of protein. The inventors have shown that by using small-scale nanostructured electrodes, they can significantly improve the sensitivity down to low picomolar concentra tions with a three-fold linear detection range. The inventors have also demon strated the assay detection time in less than ten minutes, indicating that the as say can be utilized for rapid diagnostic techniques. The physiological levels of otolin-1 and prestin in human blood are in the femtomolar ranges. Their variations start from around 100 pg/ml in healthy indi viduals by factors of 50 to 100 pg/ml up to about 1000 pg/ml depending on the age and level of damage ¹⁹ _’ ²⁴ _’ ⁵² . However, quantitative measurements within whole blood can be challenging, mainly when detecting low physiological levels, such as for the inner ear proteins ⁴⁰ . The inventors have provided a strategy to reduce the detection limit and yet maintain the linearity of the sensor by lower ing the electrode’s dimension while enabling the high curvatures of nanostruc turing. The combination of synthetic assays with nanostructured electrodes can also be improved to promote the rate of reactions and can be adapted within a microfluidic device to accelerate the rate of target delivery and eventually to de velop a rapid test platform in therapeutic ranges.

The present disclosure advantageously provides a new non-invasive di agnostic approach for inner ear diseases that is capable of rapid detection of blood-circulating biomarkers through a point-of-care biosensor platform. The present method and system has been illustrated with respect to a method and system for the single-step detection of otolin-1 and prestin protein but it will be appreciated that the present method and sensor platform made be applied to the detection of other unique and promising biomarkers of the inner ear, includ ing the circulating DNAs/RNAs (ribonucleic acids), proteins, metabolites, cells, exosomes, and small molecules, in order to develop a comprehensive multi plexing device for the point-of-care diagnosis of inner ear disorders.

Ultimately, more accurate diagnostics will help identify the sites of dam age in the inner ear or central auditory pathway and will provide the ability to monitor the occurrence and progression of a variety of inner ear disorders as well as the efficacy of treatment. Furthermore, while the initial design of the point-of-care biosensing platform is for the detection of inner ear protein bi omarkers, several clinically relevant circumstances, e.g., infectious or autoim mune diseases would benefit from such an approach for the rapid early-stage diagnosis.

It will be appreciated that while the present electrochemical immuno-bio- sensor-based method and system has been illustrated with respect to the de tection of otolin-1 and prestin proteins, it will be appreciated that this method and system can be adapted for all diseased characterized by circulating protein biomarkers, including for example cancers, infectious and autoimmune dis eases, as well as, any other acute and chronic illnesses. For each disease that the system is to be configured to detect, the method involves determining the circulating protein to be detected,

The foregoing description of the preferred embodiments of the invention has been presented to illustrate the principles of the invention and not to limit the invention to the particular embodiment illustrated. It is intended that the scope of the invention be defined by all of the embodiments encompassed within the following claims and their equivalents. References

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