Electrochemical multiplex biosensors

Abstract

It is provided an electrochemical biosensor for determining an analyte concentration comprising a sensing element comprising a first redox reporter such as an oxazine ATTO 700 and a co-functionalized electrochemical biosensor for determining analytes concentrations in a sample comprising a first sensing element comprising a first redox reporter attached to a first sensing receptor that interacts to a first analyte, at least one other sensing element comprising another redox reporter attached to at least one other sensing receptor that interacts to a another analyte, and a solid surface where the first and at least one other sensing elements are attached thereto, wherein upon interaction of the first or another analyte onto the first or at least one other sensing receptor, the first redox reporter and / or the other redox reporters undergoes a change in electron transfer, and wherein the first redox reporter and the other redox reporters have a distinct electrochemical signature.

Description

ELECTROCHEMICAL MULTIPLEX BIOSENSORSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is claiming priority from U.S. Provisional Application No. 63 / 570,952 filed March 28, 2024, the content of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] It is provided an electrochemical biosensor for determining an analyte in a fluid comprising a sensing element modified with at least two different redox reporters.BACKGROUND

[0003] When envisioning the future, one could see tools able to provide a detailed molecular profile conveniently and rapidly at the point-of-need. The simultaneous measurement of insulin and glucose in a closed-loop fashion, for example, could completely remove the incidence of low-glycemic events. Bedside monitoring of narrow therapeutic drugs like vancomycin, methotrexate, and associated biomarkers of renal clearance such as creatinine can likewise allow for efficient intervention and death prevention. These examples illustrate how valuable having access to a platform technology capable of rapidly achieving measurements of several molecules at once, a process commonly referred as “multiplexing”, could be transformative.

[0004] Several analytical approaches can achieve multiplex measurements. While chromatography can do so, it remains a time-consuming laboratory-bound technique. Sensing platforms relying on optical and plasmonic transduction schemes can also provide multiplexed measurements of different targets via, for example, fluidics to analyze undiluted complex matrices. Electrochemical sensors, in contrast, can readily deploy in undiluted complex matrices, such as the body for continuous real-time measurements. Multiplexing these, however, has largely relied on the rule “measure one molecule per electrode”. Using multielectrode devices, can be detrimental to individuals and can complicate the design of devices requiring multi-channel capabilities that increases their size and cost.

[0005] Thus, there is a need to have access to a platform technology that achieves convenient, minimally invasive, multiplex molecular measurements via, for example, the use of a single electrode device.SUMMARY

[0006] It is provided an electrochemical biosensor for determining an analyte concentration comprising a sensing element comprising a redox reporter attached to at least one sensing receptor that binds or interacts to the analyte, and a solid surface where the sensing element is attached thereto, wherein upon interacting or binding of the analyte onto the sensing receptor, the redox reporter undergoes a change in electron transfer.

[0007] It is also provided a co-functionalized electrochemical biosensor for determining analytes concentrations in a fluid comprising a first sensing element comprising a first redox reporter, attached to a first sensing receptor that binds to a first analyte, at least one other sensing element comprising another redox reporter attached to at least one other sensing receptor that binds to another analyte, and a solid surface where the first and at least one other sensing elements are attached thereto, wherein upon presence of the first or another analyte onto the first or the at least one other sensing receptor, the first redox reporter and / or the other redox reporters undergoes a change in electron transfer, and wherein the first redox reporter and the other redox reporters have a distinct electrochemical signature.

[0008] In an embodiment, the co-functionalized electrochemical biosensor comprises more than two aptamers.

[0009] In an embodiment, the surface is an electrode.

[0010] In a particular embodiment, the electrode is made of gold, carbon or silicon.

[0011] In a further embodiment, the analyte is in an environmental sample, a fluid, a tissue or an organ.

[0012] In another embodiment, the fluid is a body fluid, and said body fluid is an interstitial fluid, blood, plasma, saliva or urine.

[0013] In an embodiment, the sensing receptor is an aptamer, an antibody, a nucleic acid molecule, a peptide, a biopolymer or a combination thereof.

[0014] In a further embodiment, the nucleic acid molecule is a DNA molecule, a RNA molecule, a XNA oligonucleotide molecule, a spiegelmer, a peptide aptamer, or an affimer.

[0015] In another embodiment, the analyte is a hormone, a cytokine, creatinine, glucose, adenosine triphosphate (ATP), carnitine, lactate, lactic acid, malate, malic acid, maltose, adenosine monophosphate (AMP), or a drug.

[0016] In an embodiment, the drug is cocaine, vancomycin, methotrexate, or dopamine.

[0017] In a further embodiment, the hormone is epinephrine, norepinephrine, testosterone, or thyroid hormone.

[0018] In an embodiment, one of the redox reporter comprises ferrocene, anthraquinone, methylene blue, an oxazine (like ATTO700) a molecule bearing a thiazine core, a thiafulvalene core, or a phenylenediamine core.

[0019] In a particular embodiment, the redox reporter is ATTO 700.

[0020] In a further embodiment, it is provided a kit comprising the electrochemical biosensor or the co-functionalized electrochemical biosensor as described herein and instruction for use.

[0021] In another embodiment, the kit further comprises a monitoring device.

[0022] In an embodiment, the monitoring device monitors the change in electron transfer.

[0023] In a further embodiment, the electron transfer is monitored via a change in the peak height of square-wave voltammograms.

[0024] In another embodiment, the monitoring device monitors the change in electron transfer voltametrically, chronoamperometrically or impedimetrically.

[0025] In an embodiment, it is also provided a method of measuring at least one analyte level in a fluid sample comprising the steps of contacting the fluid sample with the electrochemical biosensor or the co-functionalized electrochemical biosensor as described herein; and monitoring the change in electron transfer indicating the presence of the at least one molecule in the fluid sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Reference will now be made to the accompanying drawings.

[0027] Fig. 1 illustrates an electrochemical aptamer-based (E-AB) biosensors comprising a redox-reporter-modified aptamer attached on an alkanethiol-coated gold electrode surface. As seen in Fig. 1A to multiplex E-AB biosensors, it is provided, in an embodiment, two co-functionalized aptamers specific for different molecules (cocaine and dopamine) on a single gold electrode which were modified with different redox reporters (methylene blue and ATTO 700). In Fig. 1 B upon electrochemical interrogation, two distinct faradaic signatures were observed in square-wave voltammetry corresponding to each of the aptamer (dopamine with methylene blue and cocaine with ATTO 700, respectively). The addition of the aptamers’ corresponding targets initiates independent electrochemical response of each redox reporter in a quantitative fashion enabling “true” multiplex measurements.

[0028] Fig. 2 illustrates that ATTO 700 exhibits a pH sensitive electrochemical response. It is showed by interrogating the MN19 ATTO labeled E-AB biosensors in PBS 1X adjusted at pHs ranging from 5.68 to 9.82. While the ATTO modified E-AB biosensors appeared to exhibit reversible electrochemistry at all pHs, its reduction potential shifted cathodically when moving in more alkaline solutions.

[0029] Fig. 3 illustrates the comparison of the electrochemical properties and sensor performances of the same cocaine-binding aptamer labelled with ATTO or methylene blue. As seen in Fig. 3A firstly sensors were interrogated over a broad range of square-wave frequencies. In absence of cocaine, the ATTO labelled E-AB biosensors showed an electron transfer rate of « 12 s1. When in presence of 200 M of cocaine, an increase was measured in charge transfer rate to « 22 s1. This difference resulted in sensor signal change of 47%. In Fig. 3B, when using methylene blue as the redox reporter, in contrast, lower electron transfer rates was obtained of « 2 s1and of « 4 s '1when in the absence or presence of the same concentration of cocaine which equally corresponded to a sensor signal change of 49%. In Fig. 3C, it was then verified that both sensors produced comparable responses when undergoing calibration. For this, the concentration of cocaine was incrementally increased and interrogated using square wave voltammetry at a frequency of 100 Hz. The resulting response trace was fitted using a Hill equation to extract dissociation constants of 1.5 mM and 1.3 mM and maximum gain of 79% and 146% for the ATTO labelled aptamer and methylene blue sensors, respectively. Error bars represent standard deviation from at least 3 independently made sensors.

[0030] Fig. 4 illustrates the comparison of the electrochemical properties of ATTO or methylene blue labeled cocaine-binding aptamer and dopamine-binding aptamer when singly or co-functionalized. As seen in Fig. 4A, a cyclic voltammogram was performed to measure the redox potentials of ATTO and methylene blue. In doing so, the co-functionalized electrode showed similar redox potentials than when singly functionalized. In Fig. 4B when comparing the electron transfer rates of ATTO when singly or co-functionalized via square-wave voltametric interrogations over a broad range of frequencies, no significant change was observed when in absence (« 12 s1vs « 12 s'1) or presence of cocaine (« 22 s1vs « 22 s1) thus yielding comparable sensor signal gain (34 % vs 47%). In Figs. 40 and D these conclusions also hold for the methylene blue modified dopamine aptamer (absence (< 4 s1vs < 4 s1) or presence of dopamine (« 18 S'1vs « 18 S'1) with comparable sensor signal gain (100% vs 80%)). Error bars represent standard deviation from at least 3 independently made sensors.

[0031] Fig. 5 illustrates E-AB biosensor that have similar analytical performances when singly or co-functionalized. In Fig. 5A, the ATTO labelled cocaine-binding aptamer was exposed when either singly or co-functionalized to increasing amounts of cocaine in PBS 1X while monitoring the change in signal at an interrogating frequency of 100 Hz in square wave voltammetry. Using a Hill equation, similar dissociation constants were obtained and signal change (84% vs 79%) in the two sensor configurations. In Fig. 5B, the experiment was repeated, this time for the methylene blue labelled dopamine-binding aptamer when challenged with increasing amounts of dopamine. In doing so, dissociation constants were measured in the same order of magnitude and similar signal response for both electrode configurations. Error bars represent standard deviation from at least 3 independently made sensors.

[0032] Fig. 6 illustrates E-AB biosensors support multiplex measurements of a pair of molecular targets. When aptamers for cocaine and dopamine are co-functionalized on the same electrode and when challenged with increasing amounts of cocaine in Fig. 6A or dopamine in Fig. 6B, only the redox reporter associated with the corresponding aptamer produced a measurable response. A sensor saturation was not obtained as reaching high target concentrations can cause changes in pH or non-specific interaction with the aptamer ultimately resulting in confounding effects of E-AB sensors.

[0033] Fig. 7 illustrates that E-AB biosensors support continuous and real-time measurements of a pair of molecular targets directly in buffer. When deploying and electrochemically interrogating the co-immobilized E-AB biosensors in PBS 1X, onlythe corresponding electrochemical signal (methylene blue or ATTO 700) associated with either aptamer increased upon addition of cocaine or dopamine. Error bars represent standard deviation from at least 3 independently made sensors.

[0034] Fig. 8 illustrates that the E-AB biosensors support continuous and real-time measurements of a pair of molecular targets directly in an undiluted complex matrix. When deploying and electrochemically interrogating the co-functionalized E-AB biosensors in bovine whole blood, only the corresponding electrochemical signal (methylene blue or ATTO 700) associated with either aptamer increased upon addition of cocaine or dopamine.

[0035] Fig. 9 illustrates that E-AB biosensors support continuous and real-time measurements of a pair of molecular targets directly upon implantation of the biosensor in a jugular vein of animal placed under anesthesia. When electrochemically interrogating the co-functionalized E-AB biosensors, only the corresponding electrochemical signal (methylene blue or ATTO 700) associated with either aptamer increased upon addition of creatinine or vancomycin, respectively.DETAILED DESCRIPTION

[0036] In accordance with the present description, it is provided an electrochemical biosensor for determining an analyte concentration comprising a sensing element comprising a redox molecule for example, but not limited to, ATTO 700, attached to at least one sensing receptor that interacts with the analyte, and a solid surface where the sensing element is attached thereto, wherein upon interacting with the analyte onto the sensing receptor, the redox reporter undergoes a change in electron transfer.

[0037] It is further provided co-functionalized electrochemical biosensor for determining analytes concentrations in a fluid comprising a first sensing element comprising a first redox reporter, like an oxazine, attached to a first sensing receptor that interacts with a first analyte, at least one other sensing element comprising another redox reporter attached to at least one other sensing receptor that interacts with a another analyte, and a solid surface where the first and at least one other sensing elements are attached thereto, wherein upon interaction of the first or another analyte onto the first or at least one other sensing receptor, the redox reporter and / or the other redox reporters undergoes a change in electron transfer, and wherein the first redoxreporter and the other redox reporters have a distinct electrochemical signature. In an embodiment, the biosensor can comprise more that two redox reporters.

[0038] As provided herewith, an electrochemical aptamer-based biosensors were adapted to afford at least the measurement of two targets using a single electrode. It is described the co-functionalization of two aptamers (one for cocaine and another for dopamine) bearing different redox reporters to transduce the response of both aptamers. It is encompassed that the biosensor is not limited to two aptamers and can comprise more than two aptamers. Accordingly, it is demonstrated the multiplexing capabilities of electrochemical aptamer-based biosensors by overcoming a lasting challenge in the field: the lack of redox reporter. It is also provided the first introduction of the oxazine ATTO 700 in electrochemical aptamer-based biosensors. As encompassed herein, given the generalizability of aptamer-based sensors that this strategy can be further adapted allowing for the multiplexed measurements of various targets of interest at the point-of-need.

[0039] Accordingly, it is provided in an embodiment a new redox reporter, ATTO 700, in electrochemical aptamer-based biosensors to achieve “true” multiplex measurements with the commonly used methylene blue. In view of the ease with which one aptamer can be substituted by another in E-AB sensors, it is now possible to measure the concentrations of most pairs of molecules directly in undiluted complex matrices.

[0040] In an embodiment, it is provided an electrochemical biosensor for determining an analyte concentration comprising a sensing element comprising a redox reporter, like ATTO700, attached to at least one sensing receptor that interacts with the analyte and a solid surface where the sensing element is attached thereto, wherein upon interacting with the analyte onto the sensing receptor, the redox reporter undergoes a binding-induced change in electron transfer.

[0041] In an embodiment, the solid surface is an electrode, e.g. a gold electrode, a carbon electrode or a silicon electrode.

[0042] It is encompassed that E-AB sensors provided herewith allow measuring targets molecules in a fluid, tissue, organ, such as e.g. and not limited to, interstitial fluid, blood, plasma, saliva or urine.

[0043] As described herein, aptamers can be used as a sensing receptor.

[0044] As used herein, the term “aptamer” means a molecule that interacts with an analyte. Such molecules include, e.g., natural or modified DNA, RNA, or XNA oligonucleotide sequences, spiegelmers, peptide aptamers, and affimers. Modifications may include substituting unnatural nucleic acid bases for natural bases within the aptamer sequence, replacing natural sequences with unnatural sequences, or other suitable modifications that improve sensor function.

[0045] It is encompassed that a sensing receptor can be an aptamer, an antibody, a nucleic acid molecule, peptides, or other biopolymers and / or combinations or modifications thereof.

[0046] In an embodiment, the analyte detected by the biosensor provided herewith can be for example, but not limited to, a hormone such as epinephrine, norepinephrine, testosterone, or thyroid hormone, or a biomarker like cytokines, serotonin, creatinine, glucose, adenosine triphosphate (ATP), carnitine, lactate, lactic acid, malate, malic acid, maltose, or adenosine monophosphate (AMP). The analyte can also be a drug as demonstrated herewith (e.g. cocaine or dopamine), vancomycin or methotrexate.

[0047] Typically, as encompassed herein, the sensing receptor, e.g. an aptamer, used in electrochemical sensors are tagged with a redox molecule such as methylene blue.

[0048] It is further encompassed a redox molecule constructed from ferrocene and anthraquinone units that are alternatives to methylene blue. It is further encompassed a redox molecule which comprises a thiazine core, such as methylene blue or ones comprising a thiafulvalene core e.g. tetrathiafulvalene (TTF) or a phenylenediamine core such as dimethyl-p-phenylenediamine (DMPD)

[0049] It is further provided a co-functionalized electrochemical biosensor for determining analytes concentrations in a fluid comprising a first sensing element comprising a first redox reporter like, but not limited to, an oxazine attached to at least one sensing receptor that binds to a first analyte, and at least one other sensing element comprising another redox reporter attached to at least one other sensing receptor that binds to a another analyte, and a solid surface where the first and at least one other sensing elements are attached thereto, wherein upon interaction of the first or another analyte onto the first or at least one other sensing receptor, the redox reporter and / or the other redox reporters undergoes a change in electron transfer, andwherein the first redox reporter and other redox reporters have a distinct electrochemical signature.

[0050] It is encompassed a co-functionalized electrochemical biosensor comprising more than two different sensing receptors.

[0051] Electrochemical aptamer-based (E-AB) biosensors rely on a redox-reporter- modified aptamer attached on a gold electrode substrate using thiol chemistry. Upon target binding, the redox reporter undergoes a change in electron transfer that quantitatively report on target concentration as measured using various electroanalytical techniques like square-wave voltammetry. E-AB biosensors have the potential to achieve multiplex measurements at the point-of-care as the platform has proven generalizable (aptamers can be seamlessly prepared for most molecular targets) and able to readily deploy directly in undiluted complex matrices.

[0052] While an increasing number of E-AB biosensors are reported, only two examples of reagentless and multiplexed measurements have been done using a single electrode (Liu et al., 2015, Biosensors and Bioelectronics, 64: 43-50; Shen et al., 2021 , Sensors and Actuators B: Chemical, 336: 129747). E-AB biosensors have been relying on a single redox reporter molecule, methylene blue, to support the change in electron transfer. Methylene blue strikingly provides for stable measurements in unprocessed complex and buffered matrices like whole blood. Although other redox reporters (mainly iron-based (like ferrocene) and quinone-based (like anthraquinone)) have been investigated, they experience lower stabilities ultimately questioning their usability. Nonetheless, two to three different cytokine-binding aptamers have been functionalized with, for example, methylene blue, ferrocene and anthraquinone to allow multiplexed measurements of inflammation markers in E-AB sensors. Measuring the concentrations of these molecules can prove transformative for precision health particularly in a simultaneous and real-time fashion to report on cytokine interactions. These past examples, however, did not achieve this feat and the resulting sensors only produced signal off responses making it difficult to distinguish from sensor degradation or electrode fouling.

[0053] In response to the lack of redox reporters, several groups have explored new ones. This is the case of oxazines, a class of fluorescent dyes which has shown increased stability to photobleaching in comparison to others. Smiljanic and co-workers reported the use of the oxazine ATTO 655 as redox reporter for electrochemicalmeasurements of DNA concentrations. The authors showed that ATTO 655 had an electron transfer rate approaching methylene blue (of « 40 s_1vs « 24 s_1) and with stabilities surpassing ferrocene and anthraquinone (Smiljanic et al., 2024, Bioelectrochemistry, 155: 108582). ATTO 655 also presented a potential of -0.36 V vs Ag|AgCI that is cathodic with respect to methylene blue (-0.28 V vs Ag|AgCI).

[0054] ATTO 700, an oxazine dye structurally resembling ATTO 655 that is commercially available as a phosphoramidite, has electrochemical properties resembling methylene blue when interrogated and attached on electrode-bound aptamers. As showed herein, a cocaine-binding aptamer variant (MN19; Neves et al., 2010, Biochemistry, 49: 8478-8487) with ATTO 700 or methylene blue. The aptamers were attached on gold electrodes, which was then passivated using the hydroxy alkanethiol, mercaptohexanol (Fig. 1A). When voltametrically interrogated in phosphate-buffered saline, a reversible electrochemical behaviour was observed with a potential of -0.39 V vs Ag|AgCI for the ATTO 700 functionalized aptamer, a more cathodic value when compared to the same aptamer modified with methylene blue (- 0.28 V vs Ag|AgCI) (Fig. 1 B). The ATTO 700 potential is thus sufficiently anodic with respect to the competing oxygen reduction reaction (< -0.5 V vs Ag|AgCI) and cathodic to methylene blue to support E-AB biosensors multiplexing measurements. As is also the case of methylene blue, the potential of ATTO 700 appears to be dependent on pH (Fig. 2), which should allow for measurements in buffered matrices, like blood or interstitial fluid in contrast to urine or sweat.

[0055] ATTO 700 has an electron transfer rate that approaches methylene blue. It was determined that the MN19 aptamer when modified with ATTO 700 had an electron transfer rate of = 13 s1(as determined using Lovric’s formalism with kmax—0.88). This value approaches the electron transfer rate of the same methylene blue-modified aptamer 2 S’1) in comparison to when ferrocene-modified1190 S’1with kmax = 1.19) (Fig. 3A). The ATTO 700 electron transfer properties measured should support binding- induced change in electron transfer in E-AB biosensor as it is slower than electrical double layer formation and faster than oxygen reduction on alkanethiol-coated gold. Thus, changes in electron transfer rate of ATTO 700 could be measured using different electroanalytical techniques like the commonly used square-wave voltammetry.

[0056] Similar to methylene blue, ATTO 700 supports signaling in E-AB biosensors. It is demonstrated by measuring the electron transfer rate of the MN19 E- AB biosensor when in presence of 200 M of cocaine. An electron transfer of = 22 s1was measured (Fig. 3A). In taking the difference of the charge transferred in absence and presence of target, a maximal E-AB sensor response of 47 % was measured, a value approaching ones obtained with the methylene-blue-modified aptamer (48 %) (Fig. 3B). These results support that ATTO 700 could afford measurements of target concentrations in E-AB biosensors with signal-OFF or signal-ON responses and thus dual-frequency drift correction strategies.

[0057] Again, similar to methylene blue, ATTO 700 supports quantitative target concentration measurements in E-AB biosensors. Using the charge transferred difference between the two conditions, a signal-ON square-wave frequency was selected to maximize the response of the E-AB biosensor for cocaine (100 Hz) while increasing target concentration. A maximal signal change of 79 % was measured with an estimated dissociation constant of 1.5 mM (Fig. 3C). Compared to the same methylene blue labelled aptamer, signal change obtained was = 2-fold lower (79 % vs 146 %). The difference in signal change was attributed because of the faster electron transfer rate of ATTO 700 when E-AB biosensors are in absence of target. As expected, when looking at dissociation constants, both ATTO 700 and methylene blue- modified aptamers provided similar values (1.5 mM vs 1.3 mM) supporting the argument that they undergo a similar signaling mechanism.

[0058] ATTO-labelled and methylene-blue-labeled aptamers can be cofunctionalized on the same electrode while retaining their electrochemical properties. To achieve this, electrodes bearing an ATTO-labelled cocaine binding aptamer (Neves et al., 2010, Biophysical Chemistry, 153: 9-16) and a methylene blue-labelled dopamine binding aptamer (Nakatsuka et al., 2018, Science, 362: 319-324) were fabricated through co-functionalization on the same bare electrodes and concentrations. Using this protocol, a similar surface coverage was obtained (1.7-1011molecule-c r2vs 2.5-1011molecule-cmr2) of both aptamers and no significant change in electrochemical potentials or electron transfer rate arising from co-functionalization were noticed (Fig. 4). Given the similarities in the electrochemical performances of cofunctionalized aptamers, they should support real-time measurements in a similar fashion as when functionalized alone.

[0059] The ATTO labelled cocaine-binding aptamer and the methylene blue labelled dopamine-binding aptamer retain their analytical response upon co- functionalization to the same electrode. Biosensors were interrogated using optimal square wave frequencies to maximize signal-ON response for each aptamer uponexposure to increasing amounts of cocaine and dopamine (Fig. 5A and B). No noticeable difference was observed between the maximum response and calculated dissociation constants of singly or co-functionalized electrodes.

[0060] Co-functionalized ATTO labelled cocaine-binding aptamer and methylene blue labelled dopamine-binding aptamer E-AB biosensors can afford simultaneous quantification of cocaine and dopamine using a single electrode. To achieve dual quantification, the co-functionalized E-AB biosensor as described herein was exposed with increasing amounts of cocaine and monitored the peak current of each redox reporters (ATTO and methylene blue) using square wave voltammetry at an interrogation frequency of 100 Hz (Fig. 6A). During the experiment, a signal increase was observed of the ATTO 700-labelled MN19 at concentrations > 10 M while the dopamine methylene blue-labelled aptamer produced negligible response. Similarly, when interrogating sensors with increasing concentrations of dopamine, only the dopamine methylene blue-labelled aptamer produced a measurable response (Fig. 6B). These experiments illustrate the exquisite specificity of the signal produced by each redox reporter provided by the aptamers and demonstrate “true” multiplex capabilities.

[0061] The multiplexed E-AB biosensor as described herein can conveniently and quickly measure concentrations of dopamine and cocaine in real-time in buffer or undiluted bovine whole-blood. To demonstrate these benchmarks of point-of-care sensing, the sensor was sequentially exposed to increasing amounts of dopamine and cocaine and measured the corresponding response over time while performing continuous square wave voltammetry interrogations (1 scan every 20 seconds) (Fig. 7 and Fig. 8). In doing so, rapid (< 15 s) and stable sensor response was obtained when each specific aptamers are exposed to their designated targets over the interrogation period while the other was unaltered by this change. Upon sensor rinsing into target- free blood, both ATTO 700 and methylene blue aptamers responses were regenerated. These results showcase the ability of the provided co-functionalized E-AB biosensors to afford “true” multiplex, continuous and real-time measurements in complex matrices, feats that could prove useful in monitoring pharmacokinetics of different molecules directly in the body.

[0062] The multiplexed E-AB biosensor as described herein can conveniently and quickly measure concentrations of creatinine and vancomycin in real-time directly in the body. To demonstrate this, a creatinine-binding aptamer and a vancomycin-bindingaptamer were co-functionalized on a gold wire. This sensor was implanted in the jugular vein of a rat placed under anesthesia while performing bolus injection of each target while performing continuous square wave voltammetry interrogations (1 scan every 15 seconds) (Fig. 9). In doing so, rapid (< 15 s) sensor response was obtained when each specific aptamers are exposed to their respective target over the interrogation period while the other was unaltered by this change. These results showcase the ability of the provided co-functionalized E-AB biosensors to afford “true” multiplex, continuous and real-time measurements in the body, feats that could prove useful in monitoring pharmacokinetics of different molecules.EXAMPLE I Biosensors electrodes production and characterization

[0063] Phosphate-buffered saline (PBS) tablets, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 6-mercapto-1 -hexanol, dopamine hydrochloride and cocaine hydrochloride were obtained from Sigma Aldrich (Ontario, Canada).

[0064] Methylene blue-modified aptamers were obtained from Bio-Basic (Toronto, Canada) with the HPLC purification grade. ATTO 700-modified DNA was obtained from Integrated DNA Technologies Inc. (USA) as the HPLC purified. Sequences were used as received. The DNA was reconstituted in Milli-Q water to obtain a concentration of 100 pM, which was verified using a Nano-Drop spectrophotometer (Implen NP80, Germany) and the molar absorption coefficient at 260 nm. The DNA was aliquoted in 50 pL portions and kept at -20 °C prior to use.Electrochemical measurement

[0065] Electrochemical measurements were performed using an Ag|AgCI (saturated KCI) reference electrode, 2 mm diameter gold working electrodes and a platinum wire counter electrode in a three-electrode system interrogated with a CHI1040C potentiostat all acquired from CH Instruments (USA, Texas). The sensors were interrogated from -0.15 to -0.50 V using square-wave voltammetry with an amplitude of 25 mV, potential increment of 1 mV and a quiet time of 2 seconds with various frequencies. Peak potentials and peak currents were determined using an inhouse Python script relying on Pandas, Numpy and Peakutils for baseline subtraction.Electrode preparation

[0066] Electrodes were mechanically polished using a 0.1 pm diamond slurry solution on a polishing cloth (Buehler, USA) for 1 minute. The electrodes were rinsed and sonicated for 5 min in absolute ethanol. Then electrodes were polished using a 0.05 pm alumina slurry on a polishing cloth for 1 minute (Buehler, USA). The electrodes were sonicated for 5 min in Milli-Q water to remove adsorbed alumina. The polished electrodes were then electrochemically cleaned by cycling the potential from - 1 to -1.6 V for 300 scans at a scan rate of 1 V s1in 0.5 M NaOH. The electrodes were then moved and cycled in 0.5 M H2SO4from -0.35 to 1.5 V for 40 times at 4 V s1followed by cycling 8 times at a scan rate of 0.1 V s1in the same potential window. Finally, we determined the electroactive surface area of our electrodes with the gold oxide reduction peak obtained in 0.05 M FhSC when electrodes are cycled from -0.35 to 1.35 V at a scan rate of 0.1 V s1.Singly-functionalized sensor fabrication

[0067] Reconstituted DNA (2 pL, 120 pM) in Milli-Q water was reduced using TCEP (6 pL, 10 mM in Milli-Q water) for 1 h in the dark. The obtained DNA was diluted to 250 nM using PBS 1X buffer prepared by dissolving 2 PBS tablets in 400 ml_ yielding 137 mM NaCI, 2.7 mM KCI and 10 mM phosphate buffer at pH 7.4. The cleaned electrodes were immersed for 1 h in 100 pL of the 200 nM solution DNA strands. The resulting electrodes were rinsed and immersed in 5 mM 6-mercaptohexan-1-ol in PBS 1X for 3 h at room temperature or 16 h at 4 °C to protect the surface with a self-assembled monolayer.Co-functionalized sensor fabrication

[0068] Reconstituted ATTO-labelled DNA and methylene blue-labelled DNA (2 pL each, 120 pM) in Milli-Q water were reduced using TCEP (6 pL each, 10 mM in Milli-Q water) for 1 h in the dark. The obtained DNA solutions were diluted to 500 nM using PBS 1X and 50 pL of each DNA solution were combined. The cleaned electrodes were immersed for 1 h in 100 pL of the resulting DNA strands solution containing 250 nM ATTO labelled DNA and 250 nM methylene blue labelled DNA. The resulting electrodes were rinsed and immersed in 5 mM 6-mercaptohexan-1-ol in PBS 1X for 3 h at room temperature or 16 h at 4 °C to protect the surface with a self-assembled monolayer.Titration experiment

[0069] Titration experiments were performed in PBS 1X while interrogating sensors at a square-wave frequency maximizing signal response. Stock solutions of targets were prepared in degassed PBS 1X. Addition of target was performed, and the sensors were scanned once every 30 s. The solution was mixed and purged with argon with a BASI C3 Cell Stand in between interrogation to prevent dopamine oxidation.Real-time experiment

[0070] The real time experiment was performed at an interrogating square wave frequency of 100 Hz to maximize signal response of our sensors. The experiment was performed in argon flushed PBS 1X to prevent oxidation. Our sensors were interrogated once every 20 s and the solution was mixed and purged with argon in between interrogation using a BASI C3 Cell Stand. Addition was performed sequentially (cocaine then dopamine) every 15-16 voltammograms.Blood experiment

[0071] Undiluted bovine whole-blood was obtained bi-monthly from the Sherbrooke Research and Development Centre in Lennoxville, Quebec that is part of the network of research centers from Agriculture and Agri-Food Canada (AAFC). The blood was collected directly from healthy cows into 10 ml_ BD vacutainer containing 158 USP units of lithium heparin to prevent coagulation. The tubes were kept at 4 °C and used within 1 day from collection. The blood was collected and used according to the recommendation of an ethics committee. The blood was manipulated in a containment level 1 lab (CL1 ) and decontaminated according to the present legislation.

[0072] The real-time measurements in blood were performed at an interrogating frequency of 250 Hz to maximize the sensor response of our sensor. Stock solutions of dopamine were diluted in PBS 1X at a concentration of 20 mM and diluted with blood to 2 mM prior to addition to prevent dopamine degradation in the complex matrix. Cocaine solution was diluted to 20 mM in blood from a solution of 130 mM in PBS 1X. Our sensors were interrogated once every 22 s and the blood was mixed in between each point using a BASI C3 Cell Stand. Addition was performed sequentially (cocaine then dopamine) every 15 voltammograms.In vivo experiment

[0073] The miniaturized sensors were inserted in a 22G catheter placed in the right jugular vein of Sprague-Dawley rats males (4-5 months old; acquired from Charles River Laboratories) as per past methodology (ACS Sens. 2019, 4, 10, 2832-2837). The rats weighed -300 g and were pair-housed in a standard light cycle room (12:12 regular light cycle with lights on at 8AM). They were allowed ad libitum access to food and water and the Institutional Animal Care Committee (CFPA) of the Universite de Sherbrooke approved our experimental protocol which adhered to the guidelines. Anesthesia was induced under 5% isofluorane in a Plexiglas anesthesia chamber and then maintained anesthesia during the entire length of the experiment using a 2-3% isofluorane / oxygen mixture. A catheter and infusion line were installed into the left jugular vein of the animal where injection of vancomycin or creatinine can be performed. To do so the area above each vein was first shaved and cleaned using betadine and 70% ethanol followed by a small incision done above each vein using spring-loaded micro scissors that allows to insert a silastic catheter and silastic tubing for infusions. Both the E-AB sensor and the infusion line were tied off using a sterile 6-0 silk suture. 30 units of heparin was infused in the rats prior to any recording. Gold- coated aptamer sensors, platinum and silver wires were used in a three-electrode setup where a minimal 20 min baseline was recorded prior to drug injection.

[0074] While the present description has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations and including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features herein before set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An electrochemical biosensor for determining an analyte concentration comprising: a sensing element comprising a redox reporter attached to at least one sensing receptor that interacts with the analyte, and a solid surface where the sensing element is attached thereto, wherein upon interacting with the analyte onto the sensing receptor, the redox reporter undergoes a change in electron transfer.

2. The electrochemical biosensor of claim 1 , wherein the surface is an electrode.

3. The electrochemical biosensor of claim 1 or 2, wherein the analyte is in a fluid, a tissue or an organ.

4. The electrochemical biosensor of claim 3, wherein the fluid is an environmental sample or a body fluid.

5. The electrochemical biosensor of claim 4, wherein the body fluid is an interstitial fluid, blood, plasma, saliva or urine.

6. The electrochemical biosensor of any one of claims 1-5, wherein the sensing receptor is an aptamer, an antibody, a nucleic acid molecule, a peptide, a biopolymer or a combination thereof.

7. The electrochemical biosensor of claim 6, wherein the nucleic acid molecule is a DNA molecule, a RNA molecule, a XNA oligonucleotide molecule, a spiegelmer, a peptide aptamer, or an affimer.

8. The electrochemical biosensor of any one of claims 1-7, wherein the redox reporter is ATTO 700.

9. The electrochemical biosensor of any one of claims 1-8, wherein the analyte can be a hormone, a cytokine, creatinine, glucose, adenosine triphosphate (ATP), carnitine, lactate, lactic acid, malate, malic acid, maltose, adenosine monophosphate (AMP), or a drug.

10. The electrochemical biosensor of claim 9, wherein the drug is cocaine, vancomycin, methotrexate, or dopamine.

11. The electrochemical biosensor of claim 9, wherein the hormone is epinephrine, norepinephrine, testosterone, or thyroid hormone.

12. A co-functionalized electrochemical biosensor for determining analytes concentrations in a matrix comprising: a first sensing element comprising a first redox reporter attached to a first sensing receptor that interacts with a first analyte, at least one other sensing element comprising another redox reporter attached to at least one other sensing receptor that binds to another analyte, and a solid surface where the first and at least one other sensing elements are attached thereto, wherein upon interaction the first or another analyte onto the first or the at least one other sensing receptor, the redox reporter and / or other redox reporters undergoes a change in electron transfer, and wherein the first redox reporter and the other redox reporters have a distinct electrochemical signature.

13. The co-functionalized electrochemical biosensor of claim 12, wherein another redox reporter is from ferrocene, or anthraquinone.

14. The co-functionalized electrochemical biosensor of claim 12, wherein the another redox reporter can comprise a thiazine core, a thiafulvalene core, or a phenylenediamine core.

15. The co-functionalized electrochemical biosensor of any one of claims 12-14, wherein a redox reporter is an oxazine.

16. The co-functionalized electrochemical biosensor of any one of claims 12-15, wherein the surface is an electrode.

17. The co-functionalized electrochemical biosensor of any one of claims 12-16, wherein the matrix is a fluid, a tissue or an organ.

18. The co-functionalized electrochemical biosensor of any one of claims 12-17, wherein the first sensing receptor and at least one other sensing receptor are an aptamer, an antibody, a nucleic acid molecule, a peptide, a biopolymer or a combination thereof.

19. The co-functionalized electrochemical biosensor of claim 18, wherein the nucleic acid molecule is a DNA molecule, a RNA molecule, a XNA oligonucleotide molecule, a spiegelmer, a peptide aptamer, or an affimer.

20. The co-functionalized electrochemical biosensor of any one of claims 12-19, wherein the analytes are a hormone, a cytokine, a biomarker like creatinine, glucose, adenosine triphosphate (ATP), carnitine, lactate, lactic acid, malate, malic acid, maltose, adenosine monophosphate (AMP), or a drug.

21. The co-functionalized electrochemical biosensor of claim 20, wherein the drug is cocaine, methotrexate, vancomycin, or dopamine.

22. The co-functionalized electrochemical biosensor of claim 21 , wherein the hormone is epinephrine, norepinephrine, testosterone, or thyroid hormone.

23. A kit comprising the electrochemical biosensor of any one of claims 1-11 or the co- functionalized electrochemical biosensor of any one of claims 12-22 and instruction for use.

24. The kit of claim 23, further comprising a monitoring device.

25. The kit of claim 24, wherein the monitoring device monitors the change in electron transfer.

26. The kit of claim 25, wherein the electron transfer is monitored via a change in the peak height of square-wave voltammograms.

27. The kit of claim 24, wherein the monitoring device monitors the change in electron transfer voltametrically, chronoamperometrically or impedimetrically.

28. A method of measuring at least one analyte level in a sample comprising the steps of: contacting the sample with the electrochemical biosensor of any one of claims 1-11 or the co-functionalized electrochemical biosensor of any one of claims 12-22; and monitoring the change in electron transfer indicating the presence of the at least one analyte in the sample.

29. The method of claim 28, wherein the sample is a fluid, a tissue or an organ.

30. The method of claim 28 or 29, wherein at least one analyte is a hormone, a cytokine, a biomarker like creatinine, glucose, adenosine triphosphate (ATP), carnitine, lactate, lactic acid, malate, malic acid, maltose, adenosine monophosphate (AMP), or a drug.

31. The method of claim 30, wherein the drug is cocaine, methotrexate, vancomycin, or dopamine.

32. The method of claim 31 , wherein the hormone is epinephrine, norepinephrine, testosterone, or thyroid hormone.

33. The method of any one of claims 28-32, wherein the electron transfer is monitored via a change in the peak height of square-wave voltammograms.

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