US-20260126439-A1 - METHODS AND APPARATUS FOR DETERMINING THE AMOUNT OF AN ANALYTE IN A FLUID USING A PERIODIC WAVEFORM

Abstract

Methods for interrogating aptamer-based electrochemical sensors using square wave voltammetry. A redox reporter is attached to each of the aptamers. Currents resulting from the interrogation are measured and used to determine an initial distribution of redox reporter above the surface of the aptamer-coated working electrode of the sensor. The measured currents are used to determine a location or a distribution of the redox-active species in relation to the surface, and in turn the amount of analyte about the electrode.

Inventors

• Alastair Hodges
• STANLEY TEO
• Jiezhen LI

Assignees

• Nutromics Technology Pty Ltd

Dates

Publication Date

20260507

Application Date

20230802

Claims (20)

1. 1 . A method for determining an amount of an analyte in a fluid, the method comprising: providing an electrochemical sensor working electrode, the working electrode having associated therewith a plurality of analyte recognition elements each of which has a redox-active species associated therewith, applying a potential to the working electrode according to a periodic waveform, and measuring a current value resulting from the application of the potential at one or more time points within a cycle of the periodic waveform.
2. 2 . The method of claim 1 , wherein each of the plurality of analyte recognition elements is associated with a surface of the working electrode, and the measured current value(s) is/are used to determine a location or a distribution of the redox-active species in relation to the surface.
3. 3 . The method of claim 2 , wherein the location or the distribution is an initial location or an initial distribution.
4. 4 . The method of claim 2 , wherein the location or the distribution of the redox-active species is used to determine the extent to which the redox-active species mobilize toward the surface of the working electrode, and in turn the amount of analyte recognized by the plurality of analyte recognition elements.
5. 5 . The method of claim 1 , wherein the measured current value(s) is/are used to determine the extent to which the redox-active species mobilize toward the surface of the working electrode, and in turn the amount of analyte recognized by the plurality of analyte recognition elements
6. 6 . The method of claim 1 , wherein the measured current value(s) are used to generate one or more current-potential relationships.
7. 7 . The method of claim 6 , wherein each of the one or more current-potential relationships is a differential current-potential relationship.
8. 8 . The method of claim 6 , wherein the one or more current-potential relationships each provide a peak current, and the peak currents are used to calculate one or more f values, each f value being indicative of the fraction of the redox-active species that is at or proximal to a surface of the working electrode.
9. 9 . The method of claim 8 , wherein the f value is calculated according to Equation (3).
10. 10 . The method of claim 1 , wherein the periodic waveform has a substantially fixed frequency, the periodic waveform being superimposed on an underlying swept potential.
11. 11 . (canceled)
12. 12 . (canceled)
13. 13 . (canceled)
14. 14 . (canceled)
15. 15 . (canceled)
16. 16 . (canceled)
17. 17 . (canceled)
18. 18 . The method of claim 1 , wherein the periodic waveform is applied according to a square wave voltammetry method.
19. 19 . The method of claim 1 , wherein the measured current value(s) are used to provide a time-current relationship
20. 20 . (canceled)

Description

FIELD OF THE INVENTION The present invention relates generally to electrochemical sensors useful in determining the amount of an analyte in a fluid, including biological fluids such as blood and interstitial fluid. More particularly, the invention provides an electrochemical sensor that is operated by way of an improved method which provides for higher reliability analyte determination and extended sensor life. BACKGROUND TO THE INVENTION Some classes of electrochemical sensors are selective and capable of the real-time, continuous detection of target analytes, as well as single point measurements, including agents which are exogenous (e.g., pharmaceutical compounds and toxins) and also those which are endogenous (e.g., metabolites, proteins, hormones, and the like). Electrochemical sensors show significant promise in the field of human and animal health. In that context, a sensor may be embodied in the form of a microneedle-based patch applied to skin. The microneedle forms a working electrode which is inserted into the skin so as to contact the interstitial fluid. The tip of the microneedle functions as the sensor electrode, with the analyte recognition element (such as an aptamer) being associated with the tip. This arrangement provides a minimally invasive platform for real-time, continuous in vivo target analyte detection, which is sufficiently sensitive and selective to function in the complex matrix of the interstitial fluid. An electrochemical sensor typically comprises a working electrode being coated in an analyte recognition element that undergoes a conformational change upon analyte binding. A redox reporter (such as methylene blue) may be covalently linked to the analyte recognition element. The conformational change in the analyte recognition element alters the accessibility of the redox reporter to the electrode surface, thereby producing an analyte-induced change in the level of electron transport between the redox reporter and the electrode. In some circumstances, binding of the analyte brings the redox reporter proximal to the electrode surface, thereby increasing the level of electron transport and in turn increasing current through the electrode. In other circumstances, binding moves the reporter to distal to the electrode surface resulting in the opposite effects. Regardless, binding of the analyte results in a detectable change in electrode current. Electrochemical sensors are typically interrogated by the application of an electrical potential across the working electrode and a counter electrode, and then measuring current flow after the potential is removed or changed. One method of sensor interrogation is square wave voltammetry, whereby an electrical potential is applied to the working electrode in the form of a square wave. Voltammograms (i.e., current versus voltage) are generated, and peak current through the working electrode is determined. Analyte amount is determined by way of an earlier generated calibration curve defining a relationship between analyte amount and peak current. Square wave voltammetry can be customized for a particular application to provide an increase (“signal-on”) or a decrease (“signal-off”) in peak current in the presence of target analyte. Signal drift and diminishing signal gain can be problems, which are addressed by taking measurements at two square wave frequencies which are then used to generate kinetic differential measurement (KDM) values. KDM values are calculated by subtracting the normalized peak currents measured at signal-on and signal-off frequencies, then dividing by their average. Averaged KDM values collected over a range of target concentrations to create a calibration curve, fitted using a Hill-Langmuir isotherm. Another method used for interrogation of electrochemical sensors is cyclic voltammetry. In this method, the voltage across the working electrode and reference electrode is modulated between two values (V1 and V2) at a fixed rate. When the voltage reaches V2 the scan is reversed, and the voltage is modulated back to V1. The voltage is measured between the reference electrode and the working electrode, while the current is measured between the working electrode and the counter electrode. The obtained measurements are plotted as a voltammogram. As for square wave voltammetry, peak current is used to determine the amount of analyte about the working electrode. A further prior art interrogation method is chronoamperometry, which has been shown to achieve drift-free and sub-second-resolved aptamer-based sensing. The difference in electron transfer rate between bound and unbound analyte can be measured as differences in current decay lifetimes. Such lifetimes can be related to the concentration of the target in the sample. Because chronoamperometric lifetimes are a function of the fractional population of bound vs unbound receptors, they are less sensitive to progressive changes of the sensor interface relative to total current ampl