Electrochemical waveforms for calibration-free and basal-level sensing using aptasensors

Abstract

A method and system for determining the concentration of a target in a medium using a target-binding aptamer sensor, which may include: dispersing the target in the medium, applying an intermittent pulse amperometric ("IPA") waveform to the target-binding aptamer sensor in the medium to sense the target, determining a reference point of the target-binding aptamer sensor to set a baseline level corresponding to this reference point, and determining the concentration of the target in the medium based on the baseline level of this reference point.

Description

[0001] Cross - reference to related applications

[0002] This specification claims priority to U.S. Provisional Patent Application No. 62 / 800,696, filed on February 4, 2019, entitled "Electrochemical Waveforms for Calibration - Free and Baseline - Level Sensing Using Aptasensors", which is hereby incorporated by reference in its entirety.

[0003] Statement regarding federally - sponsored research or development

[0004] This invention was made with government support under Award No. R01GM117159, awarded by the National Institute of General Medical Sciences of the National Institutes of Health. The government has certain rights in this invention. Technical field

[0005] This specification generally relates to determining the concentration of a target analyte in a medium by an electrochemical aptamer - based biosensor that is a target - binding aptasensor, and more particularly to determining the concentration of a target analyte in a medium without calibration by a target - binding aptamer using an applied potential waveform that employs an Intermittent Pulse Amperometry ("IPA") waveform. Background art

[0006] Sensor interrogation techniques that utilize aptasensors to determine the concentration of a target analyte in a medium may require prior knowledge of the amount of the target analyte in the medium or other calibration prior to use.

[0007] Accordingly, there is a need for alternative calibration - free sensor interrogation techniques that do not require prior knowledge of the amount of the target analyte in the medium. Summary of the invention

[0008] According to the subject matter of the present disclosure, and in one embodiment, a method of using a target - binding aptasensor to determine the concentration of a target in a medium can include: dispersing the target in the medium, applying an Intermittent Pulse Amperometry ("IPA") waveform to the target - binding aptasensor in the medium to sense the target, determining a reference point of the target - binding aptasensor to set a baseline level corresponding to this reference point, and determining the concentration of the target in the medium based on the baseline level of this reference point.

[0009] In another embodiment, a method of using a target-binding aptamer sensor to determine the concentration of a target in a medium can include: dispersing the target in the medium, applying an IPA waveform to the target-binding aptamer sensor in the medium to sense the target, where the IPA waveform is pulse-width-modulation duty-cycled at 1 ms and applied in the range of about 0.0 V to -0.4 V, determining a reference point of the target-binding aptamer sensor to set a baseline level corresponding to this reference point, and determining the concentration of the target in the medium based on the baseline level of this reference point. The concentration of the target can be determined based on the IPA waveform applied with a temporal resolution of 2 ms.

[0010] In yet another embodiment, a system for using a target-binding aptamer sensor to determine the concentration of a target in a medium can include: a medium, a target dispersed in the medium, a target-binding aptamer sensor configured to determine the concentration of this target dispersed in the medium, a processor communicatively coupled to the target-binding aptamer sensor, and a non-transitory computer-readable memory storing instructions. When executed by the processor, the instructions can cause the processor to apply an IPA waveform to the target-binding aptamer sensor in the medium to sense this target, determine a reference point of the target-binding aptamer sensor to set a baseline level corresponding to the reference point, and determine the concentration of this target in the medium based on the baseline level of the reference point. The IPA waveform can be pulse-width-modulation duty-cycled at 1 ms and applied in the range of about 0.0 V to -0.4 V. The concentration of this target can be determined based on the 2 ms temporal resolution of the applied IPA waveform.

[0011] These and additional features provided by the embodiments described herein will be more fully understood in conjunction with the accompanying drawings and in view of the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The embodiments illustrated in the drawings are illustrative and exemplary and are not intended to limit the subject matter defined by the claims. The following detailed description of the exemplary embodiments can be understood when read in conjunction with the following drawings, in which like structures are denoted by like reference numerals and in which:

[0013] Figure 1 is an electrochemical aptamer-based biosensor (“aptamer sensor”) according to one or more embodiments shown and described herein;

[0014] Figure 2 shows an Figure 1The aptamer sensor, where the electrode portion switches between a positively charged stage for sensing a target (left) and a negatively charged stage for resetting the aptamer sensor in a reset implementation (right).

[0015] Figure 3 is the applied potential waveform for sensor demodulation, sensor reset, and further sensor demodulation using Intermittent Pulse Amperometry (“IPA”) according to one or more embodiments shown and described herein;

[0016] Figure 4 is according to one or more embodiments shown and described herein, related to the application Figure 3 of a graph of the change in the current signal of the aptamer sensor over time corresponding to the waveform;

[0017] Figure 5 is a graphical description of the current crossover points when a target is sensed and when a target is not sensed, both cases using Figure 1 the aptamer sensor for the crossover point implementation according to one or more embodiments shown and described herein;

[0018] Figure 6 is a graphical description of the change in current at a specific time after applying a positive potential pulse to an aptamer sensor constructed with an aminoglycoside-binding aptamer and in response to the target aminoglycoside tobramycin according to one or more embodiments shown and described herein;

[0019] Figure 7 is a graphical description of the equilibrium calibration curve according to one or more embodiments shown and described herein, which shows the change in current at 400 microseconds (μs) for Figure 6 the aptamer sensor at a specific target concentration;

[0020] Figure 8 is according to one or more embodiments shown and described herein, for Figure 6 a graphical description of the change in current over time for the aptamer sensor represented on a logarithmic scale;

[0021] Figure 9 is according to one or more embodiments shown and described herein, for Figure 6 a graphical description of the change in current of the aptamer sensor over time and the crossover point (where the current is equal when there is no target and when there is a target);

[0022] Figure 10 is according to one or more embodiments shown and described herein, for Figure 6Graphical description of a crossover-based calibration / titration curve of current changes at a 30 μs crossover point and at a 400 μs measurement point at specific target concentrations for an aptamer sensor;

[0023] Figure 11 is, according to one or more embodiments shown and described herein, for Figure 6 Graphical description of the raw current decay curve over time for an aptamer sensor at different target concentrations (including at one or more crossover points);

[0024] Figure 12 is, according to one or more embodiments shown and described herein, for Figure 6 Graphical description of the current change for an aptamer sensor at a specific target concentration, corresponding to control targets of tobramycin and glucosamine, respectively;

[0025] Figure 13 is, according to one or more embodiments shown and described herein, for Figure 6 Graphical description of the current change for an aptamer sensor in response to the addition of a control target of glucosamine at a specific time;

[0026] Figure 14 is, according to one or more embodiments shown and described herein, for Figure 6 Graphical description of the average IPA titration curve relative to current change for an aptamer sensor at a specific target concentration at any time value up to 1 μs in response to a tobramycin target;

[0027] Figure 15 is, according to one or more embodiments shown and described herein, for Figure 6 Graphical description of the average IPA titration curve relative to current change for an aptamer sensor at a specific target concentration at 400 μs in response to a control target of glucosamine;

[0028] Figure 16 is, according to one or more embodiments shown and described herein, for Figure 6 Graphical description of the average IPA calibration-free curve relative to current change for an aptamer sensor at a specific target concentration at 400 μs in response to a control target of glucosamine;

[0029] Figure 17 is, according to one or more embodiments shown and described herein, a graphical description of the current change at a specific time after applying a positive potential pulse to an aptamer sensor constructed with an adenosine triphosphate (“ATP”)-binding aptamer and in response to the addition of target ATP;

[0030] Figure 18is a graphical depiction of a balance calibration curve according to one or more embodiments shown and described herein, showing the current change at 400 μs for an aptamer sensor for Figure 17 at a specific target concentration;

[0031] Figure 19 is according to one or more embodiments shown and described herein, a graphical depiction of the current change over time for an aptamer sensor for Figure 17 represented on a logarithmic scale;

[0032] Figure 20 is according to one or more embodiments shown and described herein, a graphical depiction of the current change of an aptamer sensor for Figure 17 relative to time and the crossover point (where the current without target and the current with target are equal);

[0033] Figure 21 is according to one or more embodiments shown and described herein, a graphical depiction of a calibration / titration curve of the current change based on the crossover point at a specific target concentration at the 30 μs crossover point and the 400 μs measurement point for an aptamer sensor for Figure 17 ;

[0034] Figure 22 is according to one or more embodiments shown and described herein, a graphical depiction of the raw current decay curve over time at different target concentrations (including at one or more crossover points) for an aptamer sensor for Figure 17 ;

[0035] Figure 23 is according to one or more embodiments shown and described herein, a graphical depiction of the current change at a specific target concentration for an aptamer sensor for Figure 17 corresponding to the ATP target and the control target of guanosine triphosphate, respectively;

[0036] Figure 24 is according to one or more embodiments shown and described herein, a graphical depiction of the current change at a specific time in response to the addition of the control target of guanosine triphosphate for an aptamer sensor for Figure 17 ;

[0037] Figure 25 is according to one or more embodiments shown and described herein, a graphical depiction of the average IPA titration curve over current change at any time value up to 1 μs at a specific target concentration in response to the ATP target for an aptamer sensor for Figure 17 ;

[0038] Figure 26 is according to one or more embodiments shown and described herein, forFigure 17 Graphical description of the average IPA titration curve of an aptamer sensor in response to a guanosine triphosphate control target at a specific target concentration at 400 μs with respect to current change;

[0039] Figure 27 is, according to one or more embodiments shown and described herein, for Figure 17 Graphical description of the average IPA calibration-free curve of an aptamer sensor in response to a guanosine triphosphate control target at a specific target concentration at 400 μs with respect to current change;

[0040] Figure 28 is, according to one or more embodiments shown and described herein, using an aptamer sensor (e.g., Figure 6 aptamer sensor of Figure 17 aptamer sensor of Figure 2 reset implementation of Figure 5 flowchart of the process of the intersection implementation of ; and

[0041] Figure 29 Schematically shows, according to one or more embodiments shown and described herein, a computer and software-based method for implementing the use of Figure 6 and / or Figure 17 aptamer sensor of Figure 28 method of DETAILED DESCRIPTION OF THE INVENTION

[0043] Generally referring to the accompanying drawings, embodiments of the present disclosure relate to methods and systems for determining the concentration of a target in a medium using an aptamer sensor that binds to the target, which senses the target by applying an Intermittent Pulse Amperometry ("IPA") waveform to the aptamer sensor in the medium. Embodiments of such target-binding aptamer sensors and the method and system of applying the IPA waveform will now be described in detail, and examples thereof together with components and systems are also shown in the accompanying drawings. Whenever possible, the same reference numerals will be used throughout all the drawings to refer to the same or similar parts. Various embodiments of the aptamer sensor will be described in further detail herein with specific reference to the accompanying drawings.

[0044] Referring to Figure 1, an electrochemical aptamer-based switchable biosensor (“aptasensor”) 100, comprising: a passivation layer 101, an electrode 106, and an aptamer portion 102 including a plurality of tethers 108. Each tether 108 includes a target-binding aptamer 112 that is coupled to the surface of the electrode 106 at a first end and to a redox (“redox”) label 114 at a second end opposite the first end. The passivation layer 101 can be formed by passivation via a non-electrolyte finishing process that uses an acid to remove free iron from the sensor surface of the electrode 106, thereby providing an inert, protective oxide layer that is less likely to be corroded by chemical reactions with air and can thus be used more effectively for redox reactions.

[0045] In an embodiment, the target-binding aptamer 112 is an oligonucleotide or peptide molecule configured to bind to a specific target molecule. Oligonucleotides include short deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules, oligomers of molecular complexes of chemicals including several repeating units. For example, the target-binding aptamer 112 can be composed of nucleic acids such as DNA or RNA. Refer to Figure 2 , each tether 108A, 108B of the aptamer portion 102 of the aptasensor 100 includes a target-binding aptamer 112 that extends from the surface of the electrode 106 and terminates at the redox label 114. Each tether 108A, 108B includes a negative charge 116 surrounding the respective target-binding aptamer 112. As Figure 2 shown in the left side view of, each tether 108A, 108B is configured to detect and bind to a target 110 via the target-binding aptamer 112 for detection. As described herein, by using intermittent pulse amperometry (“IPA”), ions in a solution are detected via the aptasensor 100 based on a current or a change in current to detect the target 110. Detection of the target 110 from measurements of the electrode 106 of the aptasensor 100 is based on the oxidation reaction of the vesicle cargo of the target 110 released into the medium. When the cargo of the vesicle from the target 110 is fused to the target-binding aptamer 112 of the aptasensor 100, the oxidation of the cargo transfers electrons to the electrode 106, thereby causing a spike in electrons, which can be used to estimate the number of vesicles and thus the concentration of the target 110 in the medium. In one embodiment, such vesicle cargo of the target 110 can include nucleic acids, proteins, and / or enzymes.

[0046] Accordingly, the aptamer sensor 100 described herein is configured to allow for specific target recognition of one or more targets 110 with high sensitivity and to allow for facile preparation based on the specific target 110. In one embodiment, the sensing mechanism for sensing the target 110 using the aptamer sensor 100 is based on a change in the charge transfer rate between a redox label 114 (attached to the 3'-end of the target-binding aptamer 112) and the sensor surface of the electrode 106 upon addition of the target 110. The electrode 106 can be a 2 mm gold electrode, and other measurement means and / or electrode compositions for the electrode 106 applicable to the aptamer sensor 100 are also encompassed within the scope of the present disclosure. The concentration of the target 110 can be determined based on the difference between the signal associated with the target-free state of the electrode 106 and the signal associated with the target-bound state.

[0047] Sensor demodulation techniques can include conventional square wave voltammetry (SWV) and chronoamperometry-based IPA. SWV allows for highly selective target recognition with adjustable sensitivity, which is based on the applied signal frequency and has suppression of the double layer charging current, although the time resolution may be within a few seconds. IPA can include more double layer charging current and faradaic current, but is configured to allow for detection of the target 110 in solution with a time resolution of 2 ms.

[0048] Aptamer sensors may require calibration of each individual sensor prior to measurement, and it may be difficult to pre-determine the target concentration if the target 110 is already present in the solution. Calibration-free SWV can employ a dual-frequency approach, which is based on potential sweeps at two different frequencies, one a non-responsive frequency and the other the optimal frequency selected for each aptamer type. The ratio between these two peak currents is independent of differences between sensors. Another calibration-free means can be used for chronoamperometric measurements, which is based on lifetime-concentration measurements and single exponential fitting of the current decay curve and extraction of the lifetime parameter from the fitting. Additionally, a dual reporter approach can use two redox labels, one of which is used as an internal reference to correct for signal differences between sensors of the primary redox label.

[0049] The calibration-free embodiments described herein for the aptamer sensor 100 include applying an IPA waveform to sense the target 110 and determine a reference point for the aptamer sensor 100, which is used to determine the concentration of the target 110 with time resolution based on the applied IPA waveform. For example, the time resolution can be 2 ms, and the reference point can be a reset point determined via a reset embodiment, which will be described in more detail below with reference to Figure 2-4 or a crossover point determined via a crossover embodiment, which will be described in more detail below with reference to Figure 5-27 ​

[0050] Construction of the aptamer sensor

[0051] In one embodiment, a 2 mm polycrystalline gold working electrode (commercially available, e.g., from CH Instruments, USA) can be used; a platinum (Pt) wire counter electrode with a diameter of 0.5 mm (commercially available, e.g., from Alfa Aesar, USA); and a silver / silver chloride (Ag / AgCl) reference electrode (commercially available, e.g., from BASi, USA) to construct the aptamer sensor 100. The working electrode can be hand-polished for 2 minutes on a MicroCloth polishing cloth (commercially available, e.g., from Buehler, USA) in a diamond and alumina solution with a figure-eight motion, rinsed with ultrapure DI water between polishing steps, and an electrochemical cleaning procedure can be employed. An ultrapure water (18.0 MΩ·cm at 25 °C) can be prepared using a Biopak Polisher Millipore ultrapurification system (commercially available, e.g., from Millipore, Billerica, USA).

[0052] Before sensor construction, a disulfide bond reduction step can be carried out using 2 μM of 100 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (commercially available, e.g., from Aldrich, USA). The sensor construction process can start with incubation in a 200 nM aptamer solution for 1 hour (1 h), rinsing, then incubation in a 30 mM 6-mercapto-1-hexanol solution (commercially available, e.g., from Sigma-Aldrich, USA) for 1 h, and a equilibration step in a Tris buffer containing 100 mM NaCl (commercially available, e.g., from Fisher Chemical, USA), 20 mM Trizma base (commercially available, e.g., from Sigma, USA), and 5 mM MgCl 2 (commercially available, e.g., from Sigma, USA) at pH = 7.4 for 1 h.

[0053] Two small molecules can serve as targets 110: (1) the aminoglycoside antibiotic tobramycin and (2) adenosine triphosphate (ATP). The oligonucleotide sequences for tobramycin and ATP can be HSC6-GGGACTTGGTTTAGGTAATGAGTCCC (SEQ ID NO:1)-MB (parent tobramycin aptamer) and HSC6-CTGGGGGAGTATTGCGGAGGAAA (SEQ ID NO:2)-MB (destabilized ATP aptamer). HSC6 is mercaptohexanol and MB is methylene blue. Mercaptohexanol passivates the electrode surface 106, while MB is a signal transduction molecule on the DNA. A target solution for the target-binding aptamers 112 for tobramycin and ATP can be prepared using the tobramycin target 110 and 5'-adenosine triphosphate disodium hydrate (both of which are commercially available via Sigma, USA and both in Tris buffer at pH = 7.4). As described in more detail below, the control target D(+)-glucosamine hydrochloride (commercially available via Sigma, USA) at pH = 7.4 and 5'-guanosine triphosphate disodium hydrate (commercially available via Sigma, USA) at pH = 7.4 can be used respectively for control experiments of the tobramycin and ATP targets 110.

[0054] Electrochemical measurements

[0055] Electrochemical measurements of the aptasensor 100 can be performed in a three-electrode electrochemical cell configuration on a 620D potentiostat (commercially available via CH Instruments, USA) under equilibrium conditions in Tris buffer, and an external signal applied IPA waveform can be used via this CH Instrument. The external signal source can be an NI-6255 controlled by LabVIEW code, and the IPA parameters can be a high voltage of 0.0 V, a low voltage of -0.4 V, and a pulse width of 1 ms as the time resolution.

[0056] Figure 2-4 Reset implementation

[0057] Figure 2-4 involves the use of the aptasensor 100 in a reset implementation. Referring to Figure 2 , the left view shows the aptamer portion 102 of the aptasensor 100 in the bound sensor state 100A, where each tether 108A, 108B extending from the surface of the electrode 106 in the positive potential electrode state 106A binds to the target 110 in each corresponding target-binding aptamer 112.

[0058] Applying a negative potential to the electrode 106 such that at Figure 2The electrode 106 in the right - hand view has a negative - potential electrode state 106B. Thus, after reset in the reset implementation, the negative charges 116 in the electrode 106 in the negative - potential electrode state 106B repel the negative charges 116 of the target - binding aptamer 112 of each tether 108A, 108B in the direction 118 and generate an electric field 104. Therefore, in the right - hand view, the aptamer portion 102 of the aptamer sensor 100 in the unbound sensor state 100B is shown, where each tether 108A, 108B extending from the surface of the electrode 106 in the negative - potential electrode state 106B is not bound to the target 110 in each corresponding target - binding aptamer 112. Removing the negative potential from the electrode 106 in the negative - potential electrode state 106B in the right - hand view causes the aptamer sensor 100 to change from the unbound sensor state 100B back to Figure 2 the bound sensor state 100A in the left - hand view, where each target - binding aptamer 112 is configured to bind to a corresponding target 110.

[0059] Figure 3 The IPA waveform 200, which is used as the applied potential waveform in the reset implementation, is depicted. This applied potential waveform is applied as an intermittent pulse to sensor demodulation 204, as a negative potential to sensor reset 206, and as an intermittent pulse again to sensor demodulation 208. Thus, the IPA waveform 200 is configured to demodulate the aptamer sensor 100 when applied with pulse - width modulation having a predetermined duty cycle and, as described below, does not require a baseline scan due to the use of sensor reset 206. In Figure 3 it, the IPA waveform 200 is shown as a pulse between a negative voltage and a positive voltage relative to a predetermined duty cycle. The voltage range of the predetermined duty cycle can be based on the redox potential of the redox marker 114, above and below. As a non - limiting example, the redox potential of methylene blue, which can be used for the redox marker 114, is 250 mV. In the implementations described herein, the voltage range of the predetermined duty cycle can be from 0 V to - 4 V. Other ranges are also covered within the scope of the present disclosure, which cover redox voltages sufficient to allow the redox reaction of the redox marker 114 to detect the target 110.

[0060] Figure 4 is a graph 300 showing the relationship between the change in the current signal of the aptamer sensor 100 corresponding to the applied Figure 3 IPA waveform 200 and time. In Figure 3During sensor demodulation 204, the current in the corresponding sensor demodulation section 302 remains constant through the aptamer sensor 100. During sensor reset 206, the current in the corresponding sensor reset section 304 is reset simultaneously. During further sensor demodulation 208, the current in the corresponding sensor demodulation section 306 changes from being faster to less fast, and ultimately returns to the initial state of the sensor demodulation section 302. The rate of decrease in its change decreases along the change amplitude 308, and this rate of change is via the kinetic signal change K obs Shown.

[0061] By using the sensor reset 206 and Figure 4 the corresponding sensor reset section 304 of, the Figure 3 IPA waveform 200 is configured to allow the aptamer sensor 100 to measure the basal (i.e., baseline) level of the target 110 as the target analyte without prior knowledge of the concentration of the target analyte. The IPA waveform 200 can be used to rapidly (2 ms) demodulate the surface of one or more aptamer sensors 100. As described herein, the IPA waveform 200 is directly used in the sample matrix to reset the sensor surface of the electrode 106 of the aptamer sensor 100 before probing the sensor surface to measure the concentration of the target analyte. This reset is configured to achieve a balanced signal change as the first measurement mode, while achieving the kinetic signal change K obs as the second measurement mode. For the first measurement mode, the reset is configured to achieve a balanced signal change from the initial state when the current is balanced in the sensor demodulation section 302 to an unbalanced state in the sensor demodulation section 306. For the second measurement mode, the reset is configured to achieve the kinetic signal change K obs in the sensor demodulation section 306, which represents the current change from the unbalanced state to the balanced state along the change amplitude 308. By measuring the amount of decrease from balance caused by the reset in the sensor demodulation section 306 and the rate of the kinetic signal change K obs returning to balance, the corresponding concentration of the target 110 detected by the aptamer sensor 100 as described herein can be determined as a function of these first and second measurement modes. Thus, by alternatively using the first and second measurement modes for determination in a calibration-free process, the corresponding concentration of the target 110 can be detected without prior knowledge of the prior amount of the specific target concentration.

[0062] Thus, the IPA waveform 200 for the Figure 2-4 reset implementation is configured to utilize the ability of electrostatic interaction to unfold or denature the nucleic acid recognition element on the sensor surface of the electrode 106, thereby affecting the unbound sensor state 100B before measuring the response of the aptamer sensor 100 through the Figure 4 first and second measurement modesFigure 2 )。In one embodiment, prior to applying a number of short potential pulses to the sensor surface of the counter electrode 106 to restore the electrode 106 from the negative potential electrode state 106B to the positive potential electrode state 106A, the IPA waveform 200 of Figure 3 is configured to apply a negative potential pulse (shown relative to the zero charge potential) for a given amount of time (which may be adjusted according to the recognition element) to demodulate the response of the aptamer sensor 100 in the Figure 3 sensor demodulation 208 of

[0063] The resulting Figure 4 current-time trace (once in equilibrium) shown in obs can be analyzed by the absolute change in current as a percentage of the signal change and can be used to quantify the amount of target 110 present. Additionally or alternatively, the resulting Figure 4 current-time trace shown in

[0064] can be analyzed by the rise time of the current-time response monitored by combining kinetics and can be used to determine the amount of target 110 present. By way of example and not limitation, such kinetic combination applications can be used for the aptamer sensor 100 in complex media. Figure 2-4 Thus, the IPA waveform 200 in the reset embodiment of

[0065] can be compatible with the aptamer sensor 100 to enable in-situ measurement of the target 110 without prior knowledge of the underlying concentration level of the target 110 or without performing a baseline scan to calculate the percentage signal change. It is contemplated and within the scope of the present disclosure that the IPA waveform 200 and other waveforms described herein can be used for nucleic acid-based aptamer sensors 100 and / or other biomolecular recognition elements, such as peptides and proteins.

[0066] In the intersection embodiments described herein, the current decay curve characteristics of chronoamperometry are used to determine an intersection point at which the current decay point of the target-bound aptasensor 100 is the same as the current decay curve of the aptasensor 100 without a target. At this determined intersection point, which is a specific point of the decay curve, the current response of chronoamperometry is independent of the target concentration. The signal change can be quantified relative to this intersection point, which is the non-response point. For aptasensors 100 prepared under the same conditions, the calibration curve obtained is reproducible, and the conditions include but are not limited to solution pH value, aptamer solution concentration, passivation layer concentration, electrochemical cell configuration and settings, etc. Then, the obtained calibration curve with one or more determined intersection points can be used as a general means to determine the target concentration of a specific target via the aptasensor 100 prepared under the same conditions according to the signal change relative to the signal at the intersection point.

[0067] Thus, the calibration-free intersection method as described herein can approximate the IPA current response as a single-exponential decay curve. According to this model, the current decay curves of the aptasensor without a target and the aptasensor binding a target cross at the same specific point, i.e., the intersection point. Although the intersection points have different absolute current values, for all aptasensors 100 of each specific type and similar experimental conditions (such as buffer solution ionic strength, aptamer filling density, electrode material and diameter, applied waveform parameters, etc.), the intersection points occur at the same time variation (δt). As Figure 5 shown, this calibration-free intersection method can be based on the comparison of the current at several selected time variation values (δts) with the signal at the intersection point.

[0068] Figure 5 FIG. 500 shows a current-time graph of the current intersection points 408A, 408B when using the IPA waveform 402 to sense a target as shown in the current decay curve 404 of the target-bound aptasensor and when not sensing a target as shown in the current decay curve 406 of the aptasensor without a target, both cases being sensed using the intersection embodiment with the Figure 1 aptasensor 100. At the current intersection points 408A, 408B, the current decay curve 404 of the target-bound aptasensor and the current decay curve 406 of the aptasensor without a target cross at the first time t (ms). The current decay curve 404 of the target-bound aptasensor and the current decay curve 406 of the aptasensor without a target also respectively show their respective comparison points 410A, 410B at which the curves 404, 406 do not cross at the second time t (ms). Therefore, Figure 5 this calibration-free sensing of the intersection embodiment as shown can be based on the comparison of the current signals when the currents cross. If the intersection point δt 交叉 is consistent, then at δt 交叉The target current at any δt is compared and used to calculate the target concentration of target 110, independent of the baseline scan of the target concentration. In an embodiment of the intersection point, the intersection point can be determined via such a current decay curve and via a graph indicating the percentage change in signal (%SC), as Figure 6 and Figure 17 shown. The cases for aminoglycoside antibiotic tobramycin target detection and for ATP target detection are described in more detail below, respectively.

[0069] In an intersection point embodiment, the applied IPA waveform technique is configured to allow monitoring of the change in target concentration of target 110 with a time resolution of 2 ms. As a non-limiting example, Figure 5 the IPA waveform 402 of includes a series of potential steps (i.e., in pulse width modulation (PWM) with a duty cycle of 1 ms) between two specifically selected voltage values V. This PWM repetition is configured to continue the redox cycle of the redox marker 114 of the aptamer molecule attached to the target-binding aptamer 112. In one embodiment, a potential duty cycle step from 0.0 V to -0.4 V is defined herein as a "forward" step, while a potential duty cycle step from -0.4 V to 0.0 V is defined herein as a "reverse" step.

[0070] Each 2 ms pulse with a time resolution of 2 ms can include a forward step and a reverse step, each step being 1 ms. The current can be sampled every 10 μs. Thus, each forward and each reverse step can each include 100 data points, so that there are a total of 200 data points in each pulse. The amount of time from the start of the pulse can be referred to as the time variation (δt), which can be monitored in milliseconds (ms) with high, fast, and effective time resolution.

[0071] Determining the intersection point of the target-binding aptamer sensor 100 for a specific target 110 can utilize the percentage change in signal (%SC) and the signal without target as a reference point. The following formula can be applied as Formula 1 to calculate the percentage change in signal (%SC) compared to the state without target.

[0072]

[0073] where i[0] and i[T] are the currents in the states without target and with target binding, respectively, and the percentage change in signal (%SC) is a function of the time variation (δt), and can be calculated for all data points in each pulse.

[0074] For the following, corresponding respectively Figure 6-16 and Figure 17-27In the aforementioned Examples 1 and 2, two types of aptamer sensors 100 for the corresponding first target 110 and the corresponding second target 110 were demodulated using the applied IPA waveform technique. In Example 1, the corresponding first target 110 was the aminoglycoside antibiotic tobramycin, and detection was carried out via the aptamer sensor 100 of Figure 6-16 . In Example 2, the corresponding second target 110 was ATP, and detection was carried out via the aptamer sensor 100 of Figure 6-16 . In one embodiment, such detection can be carried out under a positive pulse.

[0075] Example 1

[0076] Figure 6-16 Involved the detection of the target 110 of the aminoglycoside antibiotic tobramycin via the tobramycin target-binding aptamer 112 of the aptamer sensor 100 for the cross-point embodiment of Example 1.

[0077] As Figure 6 shown, Figure 500 shows the current change expressed as a percentage of signal change at a specific time (δt in ms) up to 1 ms after applying a positive (from 0.0 V to -0.4 V) potential to the aptamer sensor 100 constructed with the aminoglycoside antibiotic-binding aptamer and in response to the addition of the target aminoglycoside antibiotic tobramycin as the target 110. Figure 500 shows the current change at a specific time for multiple target concentrations, including 0 millimolar (mM), 0.001 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM, which are the concentration values shown in the other specific concentration curves described in Example 1 herein. Figure 500 shows the Figure 6 quantitative response of the aptamer sensor 100 to the increasing amounts of the target aminoglycoside antibiotic tobramycin as the target 110. The measured current in Figure 600 showed an increase at the time change (δt) value between 30 and 170 μs and a decrease at the time change (δt) > ~170 μs compared to the current in the absence of the target. The data provided represent the mean and standard deviation of at least three independently constructed aptamer sensors 100.

[0078] Titration curves can be plotted for each time change (δt) value. The titration results at the time change (δt) = 0.4 ms in Figure 6 were plotted in Figure 7 . The time change (δt) values selected for further analysis (such as by plotting titration curves) can be determined by the achieved sensitivity, signal amplitude, and error values from all the aptamer sensors 100 analyzed in the group.

[0079] For example,Figure 7 FIG. 502 showing the equilibrium calibration curve, which shows for the aptasensor 100 of Example 1 Figure 6 the current change at 400 microseconds (μs) (i.e., 0.4 ms) expressed as a percentage change in signal at a specific target concentration in mM.

[0080] To establish the equilibrium binding curve, a baseline measurement can be generated in a solution without the target analyte. For the target aminoglycoside antibiotic tobramycin, which is the target 110 for the aptasensor 100 used Figure 6 Table 1 below presents the different concentrations in mM of the target 110 at three separate times of 240, 300, and 400 μs, expressed as the K d value of the target 110. Such data represent the mean and standard deviation of at least three independently constructed aptasensors 100.

[0081] Time (δt, μs) <![CDATA[Tobramycin target 110 (K d , mM)]]> 240 0.097±0.022 300 0.083±0.016 400 0.060±0.012

[0082] Table 1

[0083] This technique allows a time resolution of 2 ms, and the use of the intersection determination methodology as described herein allows for target concentration analysis and is independent of individual sensor calibration and / or prior knowledge of the amount of target already present in the solution. For example, the IPA current response can be approximated as a single exponential decay curve. According to the intersection determination model described herein, the current decay curves for the target-free and target-bound cases cross at the same specific point. This point has different absolute current values, however, for all aptasensors 100 of each specific type and similar experimental conditions such as but not limited to buffer solution ionic strength, aptamer filling density, electrode material and diameter, and / or applied waveform parameters, it occurs at the same time variation (δt) value. As shown above in Figure 5 this calibration-free intersection method is based on the comparison of the current at several selected time variation values (represented as δt) with the signal at the intersection point. This determination process employed for Example 1 can additionally be used for other intersection determinations, such as Example 2 described in more detail below.

[0084] In Example 1, two intersection points were determined for the current decay curve and the percentage change in signal plot at time variation (δt) values of approximately 30 ms and 170 ms. Figure 8 FIG. 504 showing the current (log(i)) as a function of time variation (δt in ms) on a logarithmic scale at a specific target concentration (in mM) using the aptasensor 100 Figure 6 including the currents at these intersection points represented as logarithmic scale values.

[0085] Figure 9 Figure 506 shows the current change (in microamperes (μA)) relative to the change in time (δt in ms), which includes an intersection point at which the target-free current (i.e., percentage change in signal) and the target-present current (i.e., with percentage change in signal) are equal for the aptamer sensor 100 for Figure 6 . For the aptamer sensor 100 for tobramycin, there are two values of the change in time (δt) at which the target-free current and the target-present current are equal, thus defining the intersection points (δt 交叉 ). The intersection point (δt 交叉 ) values as described herein do not vary with concentration. The sensor constructed for tobramycin exhibits two intersection point (δt 交叉 ) values at 30 and 170 μs. Figure 506 further represents the current difference, defined as Δi = i[T] - i[0].

[0086] For the intersection point determination as described herein, the reference point for the target 110(T) at the change in time (δt) value to determine the corresponding intersection point (cp) can utilize the following formula as shown in Equation 2:

[0087]

[0088] Figure 10 Figure 508 shows the intersection point-based calibration / titration curve of the current change expressed as a percentage change in signal at a specific target concentration (in mM) at the 30 μs intersection point and the 400 μs measurement point for the aptamer sensor 100 for Figure 6 . Prior to using an aptamer sensor 100 that is substantially similar and / or identical to Figure 6 , an intersection point-based calibration curve can be pre-created to obtain calibration-free measurements from the curve created for the baseline aptamer sensor 100 for Figure 6 . In Example 1, for the aptamer sensor 100 for tobramycin, the calibration was created with the intersection point (δt 交叉 ) = 30 μs as the reference point and the change in time (δt) = 400 μs as the measurement point. This calibration-free IPA technique can be applied to the monitoring of target diffusion in large batches of solutions, and Example 1 shows how this technique can be used with a time resolution of 2 ms and calibration-free for large batches of solutions to monitor Figure 6The changes in target diffusion and target concentration of the target 110 at the sensor surface of the electrode 106 of the aptamer sensor 100. Thus, the calibration-free IPA technique described herein is a highly sensitive and selective electrochemical technique that is configured to monitor changes in target concentration in a large number of solutions with high time resolution (e.g., at 2 ms), without the need to calibrate each individual aptamer sensor 100 for the same target type and conditions. This technique can be further used for kinetic measurements under flow conditions and can be applied to the aptamer sensor 100 of Example 2 or other similar aptamer sensors.

[0089] Figure 11 Shows the raw current decay curves at different target concentrations, including the presence of crossover points. For Figure 8-9 respectively, the crossover points are presented on a scale of log(i) and Δi = i[T] - i[0]. Figure 11 Represents the actual current decay curves measured at different target concentrations and in the absence of a target.

[0090] Specifically, Figure 11 Shows FIG. 510 of the raw current decay curves (current i in mA) as a function of time (δt in ms) at different target concentrations in mM using the Figure 6 aptamer sensor 100, including at one or more crossover points. The crossover point (δt 交叉 ) values all occur on the raw current decay curve of the tobramycin aptamer sensor 100 of Figure 6 . The corresponding crossover point (δt 交叉 ) values of the tobramycin aptamer sensor 100 are 30 and 170 μs, respectively.

[0091] Figure 12-16 Shows a comparison of the control with the glucosamine control target in a control experiment of the tobramycin aptamer sensor 100. Regarding such control experiments described herein, the charging current can have a significant impact on the measured current in the IPA setup. To eliminate the possibility of non-specific sources of measurement signal changes upon target addition due to such potential significant variations in the charging current, control experiments were performed for Example 1 with targets that are structurally similar but do not bind to the parent tobramycin aptamer. The control experiments were conducted as SWV control experiments, as Figure 12-16 shown, and it was found that the response amplitude for the control target was several times lower in terms of the percentage change in signal compared to the response to the aptamer-specific target.

[0092] Figure 12 Shows, respectively, for the tobramycin target in the target curve 514 and the glucosamine control target in the control curve 516, using Figure 6Figure 512 showing the current change of the aptamer sensor 100 as a percentage of signal change at a specific target concentration (in mM). Compared with the specific target response of the target curve 514, the response level to the control target shown in the control curve 516 as a percentage of signal change is negligible.

[0093] Figure 13 shows the use of Figure 6 the aptamer sensor 100, Figure 520 showing the current change as a percentage of signal change at a specific time (δt in 0.01 ms) for the response to the addition of the glucosamine control target. This can be compared with the Figure 6 Figure 500 showing the current change as a percentage of signal change at a specific time for the response to the addition of the tobramycin target 110, and is shown to be negligible relative to this Figure 500. Thus, the IPA titration shows that the glucosamine control target does not cause a significant signal change in the IPA signal for Figure 6 the tobramycin aptamer sensor 100.

[0094] Figure 14-16 A detailed figure further shows that the glucosamine control target does not cause a significant signal change in the IPA signal for Figure 6 the tobramycin aptamer sensor 100. Figure 14 shows the use of Figure 6 the aptamer sensor 100, Figure 522 showing the average IPA titration curve of the current change as a percentage of signal change at any time value up to 1 μs at a specific target concentration (in mM) for the response to the tobramycin target in curve 524 and the response to the glucosamine control target in the control curve 526. For increasing concentrations of the target 110, the percentage of signal change in the control curve 526 is negligible compared to the percentage of signal change in the target curve 524.

[0095] Figure 15 shows the use of Figure 6 the aptamer sensor, Figure 534 showing the average IPA titration curve of the current change as a percentage of signal change at a specific target concentration (in mM) at 400 μs for the response to the glucosamine control target ( Figure 14 a more detailed view of the control curve 526). Figure 16 shows the use of Figure 6 the aptamer sensor 100, another Figure 536 showing the average IPA uncalibrated curve of the current change as a percentage of signal change at a specific target concentration (in mM) at 400 μs for the response to the glucosamine control target.

[0096] Example 2

[0097] Figure 17-27 Detection of target 110 for ATP by ATP target-binding aptamer 112 via aptamer sensor 100 for the cross-point implementation of Example 2.

[0098] Figure 17 Figure 600 is shown, which shows the current change expressed as a percentage of signal change at a specific time (δt in ms) up to 1 ms after applying a positive potential pulse to aptamer sensor 100 constructed with adenosine triphosphate ("ATP")-binding aptamer and in response to the addition of target ATP as target 110. Figure 600 shows the current change at a specific time for multiple target concentrations, including 0 millimolar (mM), 0.001 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM, and these concentrations are the concentration values shown in other specific concentration graphs described in Example 2 herein. As Figure 17 shown in Figure 600 of, aptamer sensor 100 constructed with ATP-binding aptamer shows a quantitative increase in current with increasing ATP concentration, and its maximum current change occurs at a time change (δt) = 230 μs. The data provided represent the mean and standard deviation of at least three independently constructed aptamer sensors 100.

[0099] Titration curves can be plotted for each time change (δt) value. Figure 17 The titration results at a time change (δt) = 0.4 ms in Figure 18 are plotted in. The time change (δt) values selected for further analysis (such as by plotting titration curves) can be determined by the achieved sensitivity, signal amplitude, and error values from all aptamer sensors 100 analyzed in the group.

[0100] For example, Figure 18 Figure 602 showing the equilibrium calibration curve is shown, which shows the current change expressed as a percentage of signal change at 400 μs (i.e., 0.4 ms) at a specific target concentration in mM for aptamer sensor 100 of Figure 17 Example 2.

[0101] To establish an equilibrium binding curve, baseline measurements can be generated in a solution without the target analyte. For target ATP as target 110 for aptamer sensor 100 used in Figure 17 the following Table 2 presents the concentrations in mM expressed as the K d value of target 110 at a time of 400 μs. Such data represent the mean and standard deviation of at least three independently constructed aptamer sensors 100.

[0102] Time (δt, μs) <![CDATA[ATP target 110 (K d , mM)]]> 400 0.218±0.098

[0103] Table 2

[0104] This technique allows for a time resolution of 2 ms, and the use of the intersection determination methodology as described herein allows for target concentration analysis and is independent of individual sensor calibration and / or prior knowledge of the amount of target already present in the solution.

[0105] In Example 2, at a time variation (δt) value of approximately 30 ms, an intersection was determined for the current decay curve and the percent signal change plot. Figure 19 Shows Figure 17 Figure 604 of the variation of current (log(i)) with time (δt in ms) on a logarithmic scale for an aptasensor 100 using

[0106] Figure 20 Figure 606 shows the variation of current (in μA) with respect to time variation (δt in ms), which includes the intersection point at which the target-free current (i.e., percent signal change = 0) and the target-present current (i.e., with percent signal change) of the aptasensor 100 using Figure 17 are equal. For the aptasensor 100 for ATP, there is a time variation (δt) value at which the target-free current and the target-present current are equal, thus defining the intersection point (δt 交叉 ) value. The aptasensor 100 for ATP shows an intersection point (δt 交叉 ) = 30 μs. Figure 606 further represents the current difference, defined as Δi = i[T] - i[0].

[0107] Figure 21 Shows Figure 17 Figure 608 of a cross-point-based calibration / titration curve of the current change expressed as percent signal change for an aptasensor 100 using Figure 17 at specific target concentrations (in mM) at the 30 μs intersection point and at the 400 μs measurement point. A cross-point-based calibration curve can be created in advance in order to utilize an aptasensor 100 that is substantially similar and / or identical to Figure 17 to obtain calibration-free measurements from the created curve for the baseline aptasensor 100 of 交叉) = 30 μs as a reference point and created with a time variation (δt) = 400 μs as a measurement point. This calibration-free IPA technique can be applied to the monitoring of target diffusion in a large number of solutions, and Example 2 shows how this technique can be used with a time resolution of 2 ms and calibration-free for a large number of solutions to monitor Figure 17 changes in target diffusion and target concentration of target 110 at the sensor surface of electrode 106 of aptasensor 100.

[0108] Figure 22 shows the original current decay curves at different target concentrations, including the presence of crossover points. Respectively for Figure 19-20 , the crossover points are shown as a scale of log(i) and Δi = i[T] - i[0]. Figure 11 represents the actual current decay curves measured at different target concentrations and in the absence of a target.

[0109] Specifically, Figure 22 shows Fig. 610 of the original current decay curves (current i in mA) over time (δt in ms) at different target concentrations in mM using Figure 17 the aptasensor 100, including at one or more crossover points. The crossover point (δt 交叉 ) values occur on Figure 17 the original current decay curve of the ATP aptasensor 100. The crossover point (δt 交叉 ) value of the ATP aptasensor 100 is 30 μs.

[0110] Figure 23-27 shows a comparison with a control as a guanosine triphosphate control target in a control experiment of the ATP aptasensor 100. Regarding such control experiments described herein, the charging current can have a significant impact on the measured current in the IPA setup. To eliminate the possibility of a non-specific source of measurement signal variation upon target addition due to such potential significant variations in the charging current, control experiments were performed for Example 2 with targets that are structurally similar but do not bind to destabilize the ATP aptamer. The control experiments were performed as SWV control experiments, as Figure 23-27 shown, and it was found that the response amplitude for the control target was several times lower in terms of the percentage of signal variation compared to the response to the aptamer-specific target.

[0111] Figure 23 shows, respectively, for the ATP target in the target curve 614 and the guanosine triphosphate control target in the control curve 616, using Figure 17Figure 612 of the current change of the aptamer sensor 100 expressed as a percentage change in signal at a specific target concentration (in mM). Compared with the specific target response of the target curve 614, the response level to the control target expressed as a percentage change in signal shown in the control curve 616 is negligible.

[0112] Figure 24-26 A detailed figure shows that the control target of glucosamine does not cause a significant signal change in the IPA signal for Figure 6 the tobramycin aptamer sensor 100. Figure 24 Shows the use of Figure 17 the aptamer sensor 100, and Figure 620 of the current change expressed as a percentage change in signal in response to the addition of the guanosine triphosphate control target at a specific time (δt in 0.01 ms). This can be compared with Figure 17 Figure 600 of the current change expressed as a percentage change in signal in response to the addition of the ATP target as target 110 at a specific time, and is shown as negligible relative to this Figure 600. Thus, the IPA titration shows that the control target of guanosine triphosphate does not cause a significant signal change in the IPA signal for Figure 6 the ATP aptamer sensor 100.

[0113] Figure 25-27 A detailed figure further shows that the control target of guanosine triphosphate does not cause a significant signal change in the IPA signal for Figure 17 the ATP aptamer sensor 100. Figure 25 Shows for Figure 17 the aptamer sensor 100, Figure 622 of the average IPA titration curve of the current change expressed as a percentage change in signal in response to the ATP target in curve 624 and the guanosine triphosphate control target in the control curve 626 at any time value up to 1 μs at a specific target concentration (in mM). For the target 110 with increasing concentration, compared with the percentage change in signal of the target curve 624, the percentage change in signal of the control curve 626 is negligible.

[0114] Figure 26 Shows the use of Figure 17 the aptamer sensor 100, Figure 634 of the average IPA titration curve of the current change expressed as a percentage change in signal in response to the guanosine triphosphate control target at 400 μs at a specific target concentration (in mM) (a more detailed view of the control curve 626 for Figure 25 ). Figure 27 Shows the use of Figure 17Another graph 626 of the average IPA uncalibrated curve of the aptamer sensor 100 in response to the GTP control target at a specific target concentration at 400 μs, with respect to the current change expressed as a percentage of signal change.

[0115] Determination of reference points for uncalibrated methodology

[0116] Figure 28 Method 700 shows a process for determining a reference point via the target-binding aptamer sensor 100 described herein to further determine the target concentration of target 110. It can be done through Figure 2-4 the reset implementation or Figure 5-27 the crossover implementation to determine the reference point. Method 700 is to use the target-binding aptamer sensor 100 to determine the concentration of target 110 in the medium as described herein. Method 700 can first disperse target 110 in the medium.

[0117] Apply the IPA waveform to the aptamer sensor 100 in the medium to sense target 110. For example, in block 702, apply the IPA waveforms 200, 402 (corresponding to Figure 2 and Figure 5 for the respective reset and crossover implementations) to the target-binding aptamer sensor 100 to sense target 110. The aptamer sensor 100 senses the target as described in Figure 1-2 . In one implementation, the IPA waveform is applied with a pulse width modulation duty cycle of 1 ms and in the range of approximately 0.0 V to -0.4 V.

[0118] Determine the reference point of the aptamer sensor 100 to set a baseline level corresponding to this reference point and based on the applied IPA waveform. In block 704, in the uncalibrated IPA method, determine the reference point of the aptamer sensor 100 through Figure 2-4 the reset implementation or Figure 5-27 the crossover implementation of

[0119] In the reset implementation, the reference point can be the reset point indicating the equilibrium change when applying a negative potential through the applied IPA waveform 200. This negative potential can be a constant -0.4 V to reset the aptamer sensor 100. The concentration of the target in the medium can be determined based on the reset point and the kinetic change rate of restoring equilibrium with respect to the Figure 4 reset point as described herein.

[0120] In an embodiment of the intersection, the reference point is the intersection indicating the time point at which the percentage change in current for the aptamer sensor 100 when there is no target 110 in the medium is equal to the percentage change in current for the aptamer sensor 100 when there is a target 110 in the medium. Moreover, the reference point is the intersection indicating the time point at which the current for the aptamer sensor 100 when there is no target 110 in the medium is equal to the current for the aptamer sensor 100 when there is a target 110 in the medium.

[0121] The concentration of the target 110 in the medium is determined by the baseline level of the reference point and can be based on the time resolution of the applied IPA waveforms 200, 402. In one embodiment and as shown in block 706, the reference point is used to determine the target concentration of the target 110, the time resolution of which is based on the time resolution of the applied IPA waveforms 200, 402. Such time resolution can be 2 ms.

[0122] Referring Figure 29 , for implementing Figure 28 the computer- and software-based method to utilize the system 800 of the aptamer sensor 100 as shown in Figure 1 and Figure 2 , a graphical user interface (GUI) can be used together, which presents, for example, the determined reference point and graphical analysis related to the target concentration as described herein. For example, the GUI can be accessed on the display of a user workstation (such as, computing device 824). The system 800 includes a communication path 802, one or more processors 804, memory components 806, one or more IPA waveform generators 812 for applying IPA waveforms to, for example, the aptamer sensor 100, a storage or database 814, an aptamer sensor 816 (corresponding to the aptamer sensor 100 described herein), network interface hardware 818, a network 822, a server 820, and at least one computing device 824. Each component of the system 800 and their interactions will be described in detail below.

[0123] In some embodiments, a wide area network (WAN) or network 822, such as an intranet or the Internet, is used to implement the system 800. The computing device 824 can include digital systems and other devices that allow network connection and network navigation. Other variations of the system 800 that allow communication between various components at different geographical locations are also possible. Figure 29 The lines depicted in

[0124] As described above, system 800 includes communication path 802. Communication path 802 can be formed by any medium capable of transmitting signals, such as conductive wires, conductive traces, optical waveguides, etc., or by a combination of media capable of transmitting signals. Communication path 802 communicatively couples the various components of the system 800. As used herein, the term "communicatively coupled" means that the coupled components are capable of exchanging data signals with each other, e.g., electrical signals via a conductive medium, electromagnetic signals via air, optical signals via an optical waveguide, and the like.

[0125] As described above, system 800 includes processor 804. Processor 804 can be any device capable of executing machine-readable instructions. Accordingly, processor 804 can be a controller, integrated circuit, microchip, computer, or any other computing device. Processor 804 is communicatively coupled via communication path 802 to the other components of system 800. Accordingly, communication path 802 can communicatively couple any number of processors to each other and allow the modules coupled to communication path 802 to operate in a distributed computing environment. Specifically, each module can operate as a node that can send and / or receive data.

[0126] As described above, system 800 includes memory component 806 coupled to communication path 802 and communicatively coupled to processor 804. Memory component 806 can be a non-transitory computer-readable medium or non-transitory computer-readable memory and can be configured as a non-volatile or volatile computer-readable medium. Memory component 806 can include RAM, ROM, flash memory, a hard disk, or any device capable of storing machine-readable instructions such that the machine-readable instructions can be accessed and executed by processor 804. The machine-readable instructions can include logic or algorithms written in any programming language, such as machine language that can be directly executed by the processor, or assembly language, object-oriented programming (OOP), scripting languages, microcode, etc., which can be compiled or assembled into machine-readable instructions and stored on memory component 806. Optionally, the machine-readable instructions can be written in a hardware description language (HDL), such as logic implemented via a field-programmable gate array (FPGA) configuration or application-specific integrated circuit (ASIC) or the like. Accordingly, the methods described herein can be implemented in any conventional computer programming language, as pre-programmed hardware elements, or as a combination of hardware and software components. In an embodiment, system 800 can include processor 804 communicatively coupled to memory component 806 storing instructions that, when executed by processor 804, cause the processor to perform one or more of the functions described herein.

[0127] Continuing to refer to Figure 29, as described above, system 800 includes a display, such as a GUI on the screen of computing device 824, which is used to provide visual output, such as information, the target concentration determination as described above, graphical reports, messages, alerts, or combinations thereof. The display on the screen of computing device 824 is coupled to communication path 802 and communicatively coupled to processor 804. Accordingly, communication path 802 communicatively couples the display to other modules of system 800. The display may include any medium capable of transmitting light output, such as a cathode ray tube, light emitting diodes, liquid crystal displays, plasma displays, etc. Additionally, it is noted that the display or smart device 824 may include at least one of processor 804 and memory component 806. Although system 800 is shown as a single integrated system in Figure 29 , in other embodiments, the system may also be a stand-alone system.

[0128] System 800 includes an IPA waveform generator 812 for generating IPA waveforms 200, 402 to be applied to aptamer sensor 816 (i.e., Figure 1-2 the target-binding aptamer sensor 100), thereby determining the target concentration of target 110 as described herein. IPA waveform generator 812 and aptamer sensor 816 are coupled to communication path 802 and communicatively coupled to processor 804. As will be described in further detail below, processor 804 may process input signals received from system modules and / or extract information from such signals.

[0129] System 800 includes network interface hardware 818 for communicatively coupling system 800 to a computer network such as network 822. Network interface hardware 818 is coupled to communication path 802, such that communication path 802 communicatively couples network interface hardware 818 to other modules of system 800. Network interface hardware 818 may be any device capable of transmitting and / or receiving data via a wireless network. Accordingly, network interface hardware 818 may include a communication transceiver for transmitting and / or receiving data according to any wireless communication standard. For example, network interface hardware 818 may include a chipset (e.g., antenna, processor, machine-readable instructions, etc.) for communicating via a wired and / or wireless computer network, such as Wi-Fi, WiMax, Bluetooth, IrDA, Wireless USB, Z-Wave, ZigBee, etc.

[0130] Still referring to Figure 29, data of various applications running on computing device 824 can be provided to system 800 via network interface hardware 818 from computing device 824. Computing device 824 can be any device having hardware (such as, a chipset, a processor, a memory, etc.) for communicatively coupling with network interface hardware 818 and network 822. Specifically, computing device 824 can include an input device having an antenna for communicating via one or more of the above wireless computer networks.

[0131] Network 822 can include any wired and / or wireless network, such as a wide area network, a metropolitan area network, the Internet, an intranet, a satellite network, etc. Accordingly, computing device 824 can use network 822 as a wireless access point to access one or more servers (such as, server 820). Server 820 and any other servers generally include a processor, a memory, and a chipset for transmitting resources via network 822. Resources can include, for example, processing, storage, software, and information provided from server 820 to system 800 via network 822. In addition, it should be noted that server 820 and any other servers can share resources with each other via network 822, for example, via the wired portion of the network, the wireless portion of the network, or a combination thereof.

[0132] It should be noted that the components of the present disclosure described herein being “configured” or “programmed” in a particular manner (to embody a particular property or to operate in a particular manner) is a structural recitation, rather than a recitation of intended use. More specifically, the manner in which a component herein is “configured” or “programmed” represents the current physical state of that component and, thus, should be regarded as an express recitation of a structural feature of that component.

[0133] It should be noted that the terms “substantially” and “about” and “approximate” may be used herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. These terms are also used herein to represent the degree to which a quantitative expression may vary from a claimed reference value without causing a change in the basic function of the subject matter being discussed.

[0134] Although specific embodiments have been shown and described herein, it should be understood that various other changes and modifications can be made without departing from the spirit and scope of the claimed subject matter. In addition, although multiple aspects of the claimed subject matter have been described herein, these aspects need not be used in combination. Accordingly, the appended claims are intended to cover all such changes and modifications within the scope of the claimed subject matter. Sequence Listing <110> University of Cincinnati <120> Electrochemical Waveforms for Calibration-Free and Baseline Sensing Using Aptamer Sensors <130> CIN0295WO / 2019-004 / 10739-771 <150> 62 / 800696 <151> 2019-02-04 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Parent tobramycin aptamer <400> 1 gggacttggt ttaggtaatg agtccc 26 <210> 2 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Destabilized ATP aptamer <400> 2 ctgggggagt attgcggagg aaa 23

Claims

1. A method for determining the concentration of a target in a medium using a target-binding aptamer sensor, the method comprises: Dispersing the target in the medium; Applying an Intermittent Pulse Amperometry (IPA) waveform to the target-binding aptamer sensor in the medium to sense the target; Determining a reference point of the target-binding aptamer sensor to set a baseline level corresponding to the reference point; and Determining the concentration of the target in the medium according to the baseline level of the reference point; wherein the reference point is: (i) A reset point indicating the equilibrium change when applying a negative potential through the applied IPA waveform, (ii) An intersection point indicating the time point at which the percentage change in current for the target-binding aptamer sensor when there is no target in the medium is equal to the percentage change in current for the target-binding aptamer sensor when there is a target in the medium, or (iii) An intersection point indicating the time point at which the current for the target-binding aptamer sensor when there is no target in the medium is equal to the current for the target-binding aptamer sensor when there is a target in the medium.

2. The method according to claim 1, wherein the concentration of the target is determined based on the time resolution of the applied Intermittent Pulse Amperometry (IPA) waveform.

3. The method according to claim 2, wherein the time resolution is 2 ms.

4. The method according to claim 1, wherein the Intermittent Pulse Amperometry (IPA) waveform is applied with a pulse width modulation duty cycle of 1 ms and in the range of 0.0 V to -0.4 V.

5. The method according to claim 1, wherein the negative potential is -0.4 V.

6. The method according to claim 1, wherein the concentration of the target in the medium is determined based on the reset point and the kinetic rate of equilibrium recovery of the reset point.

7. A method for determining the concentration of a target in a medium using a target-binding aptamer sensor, the method comprises: Dispersing the target in the medium; Applying an Intermittent Pulse Amperometry (IPA) waveform to the target-binding aptamer sensor in the medium to sense the target, wherein the Intermittent Pulse Amperometry (IPA) waveform is applied with a pulse width modulation duty cycle of 1 ms and in the range of 0.0 V to -0.4 V; Determining a reference point of the target-binding aptamer sensor to set a baseline level corresponding to the reference point; and Determining the concentration of the target in the medium according to the baseline level of the reference point, wherein the concentration of the target is determined based on the 2 ms time resolution of the applied Intermittent Pulse Amperometry (IPA) waveform; wherein the reference point is: (i) A reset point indicating the equilibrium change when applying a negative potential through the applied IPA waveform, (ii) An intersection point indicating the time point at which the percentage change in current for the target-binding aptamer sensor when there is no target in the medium is equal to the percentage change in current for the target-binding aptamer sensor when there is a target in the medium, or (iii) indicate the intersection point at the time point at which the current of the target-binding aptamer sensor when there is no target in the medium is equal to the current of the target-binding aptamer sensor when there is a target in the medium.

8. The method according to claim 7, wherein the negative potential is -0.4V.

9. The method according to claim 7, wherein the concentration of the target in the medium is determined based on the reset point and the kinetic rate of change of the reset point to restore equilibrium.

10. A system for determining the concentration of a target in a medium using a target-binding aptamer sensor, the system comprising: a medium; a target dispersed in the medium; a target-binding aptamer sensor configured to determine the concentration of the target dispersed in the medium; a processor communicatively coupled to the target-binding aptamer sensor; and a non-transitory computer-readable memory storing instructions that, when executed by the processor, cause the processor to: apply an Intermittent Pulse Amperometry (IPA) waveform to the target-binding aptamer sensor in the medium to sense the target, wherein the duty cycle is modulated with a pulse width of 1 ms and the Intermittent Pulse Amperometry (IPA) waveform is applied in the range of 0.0V to -0.4V; determine a reference point of the target-binding aptamer sensor to set a baseline level corresponding to the reference point; and determine the concentration of the target in the medium based on the baseline level of the reference point, wherein the concentration of the target is determined based on a time resolution of 2 ms of the applied Intermittent Pulse Amperometry (IPA) waveform; wherein the reference point is: (i) an intersection point indicating a reset point of the equilibrium change when a negative potential is applied by the applied IPA waveform, (ii) an intersection point indicating the time point at which the percentage change in current of the target-binding aptamer sensor when there is no target in the medium is equal to the percentage change in current of the target-binding aptamer sensor when there is a target in the medium, or (iii) an intersection point indicating the time point at which the current of the target-binding aptamer sensor when there is no target in the medium is equal to the current of the target-binding aptamer sensor when there is a target in the medium.

11. The system according to claim 10, wherein the concentration of the target in the medium is determined based on the reset point and the kinetic rate of change of the reset point to restore equilibrium.

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