Multiple measurements using electrochemical aptamer-based sensors

Abstract

This disclosure relates to a method using an electrochemical aptamer-based sensor for detecting and / or measuring at least two parameters of a solution or suspension.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority from Australian Provisional Application No. 2023902007, the entire content of which is incorporated herein by reference.

[0002] The present disclosure relates to a method of using an electrochemical aptamer - based (EAB) sensor for detecting and / or measuring at least two parameters of a solution or suspension.

Background Art

[0003] Lab - on - a - chip has emerged in recent years as a new tool for drug research, integrating microfluidics and microsensors with potential to provide a unique approach to significantly improve the understanding of human biology and enable accelerated cost - effective drug development. The pH and amino acids in the physiological environment are important parameters that affect the behavior of the micro - physiological system and how drugs are formulated, or the active ingredients in the formulation and the functions within the micro - physiological system.

[0004] Optical sensors and electrochemical sensors are currently at the forefront of the development of miniaturized lab - on - a - chip sensing technology. However, a drawback is that optical sensors are often limited by perturbations of the culture from complex cell culture / tissue environments (organisms on the wetted sensor surface generate structural and functional defects). In the case of electrical sensors, the reported measurement range is limited.

[0005] There is a need to detect multiple physiological parameters using sensors that are biocompatible, rapid, micro - sized, accurate, suitable for monitoring dynamic micro - physiological systems, and resistant to contamination due to non - specific adsorption of contaminants, even if they are nanoscale medical devices and have the potential to be miniaturized.

[0006] This invention aims to address one or more of the shortcomings of the prior art.

[0007] Any reference to prior art in this specification does not constitute an acknowledgment or suggestion that the prior art forms part of the general knowledge in any jurisdiction, or that it can be reasonably expected that the prior art will be understood, considered relevant, and / or combined with other prior art by those skilled in the art. [Overview of the project]

[0008] This disclosure provides a method for using an electrochemical aptamer-based sensor to detect and / or measure at least two parameters of a solution or suspension. Preferably, at least two parameters are measured using a single measurement. In a preferred embodiment, the present invention relates to a method for detecting and / or measuring pH and target ligand levels in a solution or suspension (e.g., a biological solution or suspension) using an electrochemical aptamer-based sensor. In some embodiments, the disclosure provides a method for measuring the presence of pH and target ligand in a biological solution or suspension by providing a sensor to the biological solution or suspension, wherein one or more signals in the sensor are proportional to the pH and concentration of target ligand in the biological solution or suspension.

[0009] In some embodiments, the solution or suspension may be a biological sample such as blood, urine, semen (seminal fluid), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage fluid), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal mucus, ear fluid, gastric juice, breast milk, and cell culture supernatant. In some embodiments, the solution or suspension may contain an active pharmaceutical ingredient, for example, a biological sample containing an active pharmaceutical ingredient. In other embodiments, the solution or suspension may be suitable for use in a bioreactor and may contain plant cultures, bacterial cultures, insect cultures, or mammalian cultures.

[0010] The sensor comprises an aptamer-functionalized working electrode. The surface of the working electrode may be functionalized with one or more aptamers. More specifically, the working electrode is functionalized with one type of aptamer and adapted to detect one type of target ligand. Preferably, the working electrode is functionalized with multiple single types of aptamers. More preferably, the surface of the working electrode is functionalized with a self-assembled monolayer of a single type of aptamer.

[0011] The aptamer may be a single-stranded or double-stranded oligonucleotide of DNA, RNA, or PNA. In certain embodiments, the aptamer is single-stranded. In certain embodiments, the aptamer is approximately 20 to approximately 100 nucleic acid lengths.

[0012] Aptamers bind to target ligands. Target ligands can be selected from analytes, amino acids, peptides, small proteins, metabolites, hormones, steroids, nucleic acid oligomers, sugars, cofactors, enzymes, metals, or carbohydrates. In some embodiments, the target ligand may be an active pharmaceutical ingredient.

[0013] The aptamer contains a redox label. Preferably, the aptamer is functionalized with a redox label located at the 3' or 5' end of the aptamer. More preferably, the aptamer is functionalized with a redox label via an amide linkage at the 3' end of the aptamer. In some embodiments, the redox label is selected from methylene blue, ferrocene, viologen, anthraquinone or any other quinone, daunomycin, organometallic redox labels, such as porphyrin complexes or crown ether rings or linear ethers, ruthenium, bis-pyridine, tris-pyridine, bis-imidazole, cytochrome c, plastocyanin, and ethylenetetraacetate metal complexes, or combinations thereof. Preferably, the redox label is methylene blue.

[0014] The working electrode can be selected from gold, gold-plated metal, aluminum, copper, palladium, titanium, tungsten, silver, platinum, chromium, nickel, carbon (including graphite, nanotubes, and graphene), mercury film, oxide-coated metal, conductive polymer, or any other conductive material. In a preferred embodiment, the working electrode is gold. In some embodiments, the gold working electrode has a thickness of about 1 nm to 1 mm (including about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, and 900 μm). In a particularly preferred embodiment, the working electrode has a thickness of about 200 μm. In some embodiments, the working electrode has a thickness of approximately 1 nm to 10 μm (including approximately 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, and 900 nm).

[0015] In some embodiments, the aptamer is fixed to the gold working electrode via a gold-sulfide bond provided by a thiol at the 3' or 5' end of the aptamer, preferably at the 5' end of the aptamer.

[0016] In some embodiments, the aptamer-functionalized electrode may include a polymer coating. The polymer coating may be any suitable antifouling polymer material, including but not limited to agarose.

[0017] The electrical sensing between the aptamer and the working electrode can be operably connected to an electrical measuring device such as a potentiator or voltameter.

[0018] In some embodiments, an aptamer may bind to a target ligand and induce a conformational change that alters the distance between the redox label and the working electrode. The electrical signal in the sensor may depend on the distance between the redox label and the working electrode.

[0019] One or more electrical signals in the sensor may be proportional to the pH of the solution or suspension and the concentration of the target ligand. These electrical signals may include peak potential, Faraday current, non-Faraday current, and combinations thereof. Preferably, the pH of the solution or suspension may be measured using a peak potential (V) electrical signal. Preferably, the target ligand may be detected and / or measured using a non-Faraday current (A) electrical signal. In a particularly preferred embodiment, pH and target ligand may be measured using a single measurement, preferably square wave voltammetry (SWV).

[0020] As used herein, unless otherwise required by the context, the term “comprise,” and variations thereof such as “comprising,” “comprises,” and “comprised,” are not intended to exclude further additives, components, items, or steps.

[0021] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, which is given by example and with reference to the accompanying drawings. [Brief explanation of the drawing]

[0022] [Figure 1] Structural description of the aptamer sequence of the present disclosure. Aptamer sequence with a thiol-bonded 5' terminus and a methylene blue-coupled 3' terminus. [Figure 2] This scheme demonstrates the detection of aptamer target molecules on a gold electrode, showing the structural changes of the SAM when the target molecule binds. The surface of the working electrode was functionalized with a monolayer of aptamer arrays via thiol bonding to the gold electrode surface. Changes in target analyte concentration, aptamer structure, and the distance between the redox reporter methylene blue and the gold surface altered the electron transfer rate. [Figure 3]a) Schematic diagram showing the connection of a potentiostat to the working electrode (WE), counter electrode (CE), and reference electrode (RE). b) Graph showing the change in peak potential due to high H+ concentration (low pH) and low H+ concentration (high pH). When the pH shifts to the higher side, the peak potential decreases, and when the pH shifts to the lower side, the peak potential increases. [Figure 4] Depiction of the potential waveform used in square wave voltammetry (SWV) measurements by combining a square wave potential and a staircase potential. In SWV, a potential corresponding to the combination of a square wave potential and a staircase potential was applied between the WE and the RE. The current signal between the CE and the WE was detected by such a method. [Figure 5] SWV was performed in an "operating buffer" containing 1 M phenylalanine (upper curve, dotted line) and an "operating buffer" without 10 μM of low-concentration phenylalanine (lower curve, solid line). The potential is the potential with respect to the Ag / AgCl reference electrode, and the current is the current generated by the reduction or oxidation of some chemicals at the electrode (Faradaic current). Such phenylalanine concentrations change the change in the efficiency of mass transfer, which can be measured by SWV. [Figure 6] Graph showing the titration measurement results for the aptamer sensor, where the dots are the measurement points and the solid line is the Langmuir-Hill fitting curve. Phenylalanine in the "operating buffer" was titrated from 1 nM to 3 mM, Di (dots) was calculated, and then it was fitted to the Langmuir-Hill fitting curve (solid line). Here, the equation of the solid line curve is as follows.

Number

[0023] This disclosure provides a method for using an electrochemical aptamer-based sensor to detect and / or measure at least two parameters of a solution or suspension. Preferably, at least two parameters are measured using a single measurement. In a preferred embodiment, the present invention relates to a method for detecting and / or measuring pH and target ligand levels in a solution or suspension (e.g., a biological solution) using an electrochemical aptamer-based sensor. In some embodiments, the disclosure provides a method for measuring the presence of pH and target ligand in a biological solution or suspension by providing a sensor to the biological solution or suspension, wherein one or more signals in the sensor are proportional to the pH and concentration of target ligand in the biological solution or suspension.

[0024] In some embodiments, the solution or suspension may be a biological sample such as blood, urine, semen, vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage solution), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal mucus, ear fluid, gastric juice, breast milk, and cell culture supernatant. In some embodiments, the solution may be a solution containing an active pharmaceutical ingredient, for example, a biological sample containing an active pharmaceutical ingredient. In other embodiments, the solution or suspension may be suitable for use in a bioreactor and may contain plant cultures, bacterial cultures, insect cultures, or mammalian cultures.

[0025] This disclosure relates to a sensor based on an electrochemical aptamer on an electrode interface that makes the sensor resistant to contamination resulting from the nonspecific adsorption of contaminants. This disclosure relates to the use of the sensor to detect both pH and a target ligand in complex media, including biological and environmental media, preferably using a single measurement.

[0026] In any embodiment, the solution or suspension is a turbid solution or suspension. In any embodiment, the turbid solution or suspension is 10 NTU or more, 15 NTU or more, 20 NTU or more, 25 NTU or more, 30 NTU or more, 35 NTU or more, 40 NTU or more, 45 NTU or more, 50 NTU or more, 55 NTU or more, 60 NTU or more, 65 NTU or more, 70 NTU or more, 75 NTU or more, 80 NTU or more, 85 NTU or more, 90 NTU or more, 95 NTU or more, 100 NTU or more, 150 NTU or more, 200 NTU or more, 250 NTU or more, 300 NTU or more, 350 NTU or more, 400 NTU or more, 450 NTU or more, 500 NTU or more, 550 NTU or more, 600 NTU or more, 650 NTU or more, 700 NTU It may contain turbidimetric analysis turbidity units (NTU) of U or higher, 750 NTU or higher, 800 NTU or higher, 850 NTU or higher, 900 NTU or higher, 950 NTU or higher, 1,000 NTU or higher, 1,500 NTU or higher, 2,000 NTU or higher, 2,500 NTU or higher, 3,000 NTU or higher, 3,500 NTU or higher, 4,000 NTU or higher, 4,500 NTU or higher, 5,000 NTU or higher, 5,500 NTU or higher, 6,000 NTU or higher, 6,500 NTU or higher, 7,000 NTU or higher, 7,500 NTU or higher, 8,000 NTU or higher, 8,500 NTU or higher, 9,000 NTU or higher, 9,500 NTU or higher, or 10,000 NTU or higher. In any embodiment, the turbid solution or suspension may have an NTU of 10NTU to 100NTU, 10NTU to 90NTU, 10NTU to 80NTU, 10NTU to 70NTU, 10NTU to 60NTU, 10NTU to 50NTU, 10NTU to 40NTU, 10NTU to 30NTU, 10NTU to 20NTU, 20NTU to 100NTU, 30NTU to 100NTU, 40NTU to 100NTU, 50NTU to 100NTU, 60NTU to 100NTU, 70NTU to 100NTU, 80NTU to 100NTU, or 90NTU to 100NTU.In any embodiment, the turbid solutions are 10,000 NTU, 9,500 NTU, 9,000 NTU, 8,500 NTU, 8,000 NTU, 7,500 NTU, 7,000 NTU, 6,500 NTU, 6,000 NTU, 5,500 NTU, 5,000 NTU, 4,500 NTU, 4,000 NTU, 3,500 NTU, 3,000 NTU, 2,500 NTU, 2,000 NTU. It may have a maximum NTU of 0 NTU, 1,500 NTU, 1,000 NTU, 950 NTU, 900 NTU, 850 NTU, 800 NTU, 750 NTU, 700 NTU, 650 NTU, 600 NTU, 550 NTU, 500 NTU, 450 NTU, 400 NTU, 350 NTU, 300 NTU, 250 NTU, 200 NTU, 150 NTU, 100 NTU, or 50 NTU.

[0027] This disclosure relates to a sensor comprising an aptamer-functionalized electrode. The aptamer-functionalized electrode provides an interface between the sensor body and the surrounding medium (e.g., a biological medium).

[0028] The working electrode is a material capable of transmitting or conducting electrochemical signals, such as signals generated by conformational changes in aptamers due to target binding, and / or signals generated by changes in the oxidation state of redox labels.

[0029] The working electrode may be selected from gold, gold-coated metal, aluminum, copper, palladium, titanium, tungsten, silver, platinum, chromium, nickel, carbon (including graphite, nanotubes, and graphene), mercury film, or oxide-coated metal, conductive polymer, or any other conductive material. In a preferred embodiment, the working electrode is gold. In some embodiments, the gold working electrode has a thickness of about 1 nm to 1 μm. In some embodiments, the working electrode is a gold working electrode having an aptamer present on the surface of the working electrode.

[0030] The working electrode may take any necessary or desired shape or size. For example, the working electrode may be a wire, a plane, or a porous material.

[0031] In some embodiments, the gold used in the gold working electrodes disclosed herein is of a specific level of purity or karat. In some embodiments, the karat of the gold is 7 to 24 (including 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23). In some embodiments, the gold is 9 karats. In other embodiments, the gold is 9 to 18 karats.

[0032] In some embodiments, the working electrode has a thickness of 1 nm to 1 mm (including about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, and 900 μm). In a particularly preferred embodiment, the working electrode has a thickness of about 200 μm. In some embodiments, the working electrode has a thickness of 1 nm to 10 μm (including about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, and 900 nm).

[0033] The aptamer-functionalized working electrode may include a polymer coating. The polymer coating may be any suitable antifouling polymer material, including but not limited to agarose. The surface of the working electrode may be functionalized with one or more aptamers. More specifically, the working electrode is functionalized with one type of aptamer and adapted to detect one type of target ligand. Preferably, the working electrode is functionalized with multiple single types of aptamers. Preferably, the surface of the working electrode is functionalized with a self-assembled monolayer of a single type of aptamer.

[0034] Aptamers may be known in the art and specific to most arbitrary targets, and may be generated, for example, by phylogenetic evolution of ligands through exponential enrichment (see, e.g., Wu et al., Anal. Chem. 91:15335-15344, 2019). Aptamers may include sequences or chains of oligonucleotides, such as DNA aptamers, RNA aptamers, and aptamers containing non-natural nucleic acids that may be used, as well as hybrids of the above with polymers such as PNA. In some embodiments, oligonucleotides may be able to form “stem-loop” or “hairpin” structures (also referred to as “stem-loop” or “hairpin” or simply “stem-loops” or “hairpins”) using electroactive labels to detect hybridization events. Typical aptamers are approximately 20-100 nucleotides long (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 (including 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100 amperes). However, aptamers of any size may also be used.Known existing aptamers for this technology include amino acids, chemotherapeutic agents, ATP (Zuo, X et al, JACS, 2009, 131(20), 6944-6945), lysozyme, thrombin (Xiao, Y et al, JACS, 2005, 127(51), 17990-17991), HIV, trans-acting response elements, hemin, interferon, vascular endothelial growth factor, prostate-specific antigen, dopamine, methotrexate (Flatebo, C et al, ACS Sensors, 2023, 8(1), 150-157), kanamycin (Ferguson, B et al, Sci Transl Med, 2013), platelet-derived growth factor (Lai, R et al, Analytical Chemistry, 2007, 79(1), 229-233), and aminoglycoside antibiotics (Rowe, A et Examples include those that can bind to target species such as (Baker, B. et al, Analytical Chemistry, 2010, 82(17), 7090-7095) and cocaine (Baker, B. et al, JACS, 2006, 128(10), 3138-3139).

[0035] In this specification, the term "amino acid" refers to a molecule containing both an amino group and a carboxyl group. For example, in α-amino acids, there are "α-amino groups" directly bonded to a carbon atom having both an amino group and a carboxyl group, and "α-carboxyl groups" directly bonded to a carbon atom having both an amino group and a carboxyl group. The term "carboxyl" may refer to either a -COOH group or a -COO- group. α-amino acids are amino acids with the general formula H2N-CHR-COOH, where R is a side chain or H. The side chain is generally an alkyl chain, which is generally but not necessarily substituted at its distal end. The N-terminus of an amino acid (or peptide) is the end where an amine functional group (optionally ionized or substituted / protected) is located, and the C-terminus is the end where a carboxyl functional group (optionally ionized or substituted / protected) is located. A one-letter abbreviation system is often applied to specify the identity of 20 "canonical" or protein-constituting amino acid residues that are commonly incorporated into naturally occurring peptides and proteins (Table 1). Such one-letter abbreviations are semantically fully interchangeable with three-letter abbreviations or unabbreviated amino acid names. Non-canonical or non-protein-constituting amino acid residues include D-form or L-form amino acid residues that are not among the 20 canonical amino acids commonly incorporated into naturally occurring proteins, such as β-amino acids, homoamino acids, cyclic amino acids, selenoamino acids, thioamino acids, and amino acids with derivatized side chains. [Table 1]

[0036] Preferably, the aptamer can bind to a target species such as an amino acid or a chemotherapeutic agent. Any suitable chemotherapeutic agent can be used. Doxorubicin is a particularly preferred chemotherapeutic agent. More preferably, the aptamer can bind to Phe, Tyr, or doxorubicin.

[0037] In one embodiment, an aptamer capable of binding to Phe contains the sequence 5'-CGACC-GCGTT-TCCCA-AGAAA-GCAAG-TATTG-GTTGG-TCG-3'.

[0038] In one embodiment, an aptamer capable of binding to Trp contains the sequence 5'-CCGGT-GGTGT-AGTTC-CGGCG-TGGGG-AAGG-3'.

[0039] In one embodiment, an aptamer capable of binding to doxorubicin contains the sequence 5'-ACCAT-CTGTG-TAAGG-GGTAA-GGGGT-GGT-3'.

[0040] The aptamer may be a single-stranded or double-stranded oligonucleotide of DNA, RNA, or PNA. In certain embodiments, the aptamer is single-stranded. In certain embodiments, the aptamer is approximately 20 to approximately 100 nucleic acid lengths.

[0041] In some embodiments, the aptamer is immobilized to the working electrode via covalent interactions. In some embodiments, the aptamer is conjugated via thiol conjugations, alkanethiols, cyclic disulfides, dithiothreitols, dithiols, adenosine, or phosphorothioated adenosine. In certain embodiments, the aptamer is a single-stranded oligomer, such as ssDNA or RNA, functionalized with a thiol functional group at its 3' or 5' end, preferably at the 5' end. The thiol reacts with the gold of the working electrode to give a gold-sulfide bond, allowing the aptamer to be immobilized to the working electrode. (See, in general, Odeh et al., Molecules 25(1):3 (2020), Martinez-Jothar et al., J. Control Release 28:101-09 (2018), and Danesh et al., Int. J. Pharm. 489:311-17, (2015).) In one embodiment, the aptamer solution may be pretreated with phosphine, such as tris-(2-carboxyethyl)phosphine or dithiothreitol, for a period of about 30 minutes to 5 hours (including about 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240, 255, 270, and 285 minutes) to break any disulfide bonds. Some single-strand oligomeric aptamers may be physically adsorbed onto the gold surface, but the majority of aptamers on the surface are chemisorbed and fixed via gold-sulfide bonds.

[0042] Aptamers bind to target ligands. Target ligands can be selected from analytes, amino acids, peptides, small proteins, metabolites, hormones, steroids, nucleic acid oligomers, sugars, cofactors, enzymes, metals, or carbohydrates. In some embodiments, the target ligand may be an active pharmaceutical ingredient.

[0043] While the structures of some aptamers for known ligands are known in the art, further aptamers for other ligands can be readily identified by methods known in the art, including phylogenetic evolution of ligands by exponential enrichment (SELEX). In some embodiments, the aptamers may be amino acid-binding aptamers, aminoglycoside-binding aptamers, cocaine-binding aptamers, any small molecule-binding aptamers, thrombin-binding aptamers, platelet-derived growth factor-binding aptamers, neuropeptide Y-binding aptamers, any protein-binding aptamers, inorganic ion-binding aptamers, or DNAzyme-binding aptamers.

[0044] In some embodiments, the target ligand may be any inorganic or organic molecule, such as a small molecule drug, metabolite, hormone, amino acid, peptide, protein, carbohydrate, nucleic acid, analyte, steroid, nucleic acid oligomer, sugar, cofactor, enzyme, metal, or any other substance. The target ligand may be a therapeutic drug, antibiotic, illicit drug, antibiotic agent, or chemotherapy drug. Examples of target ligands include naturally occurring factors such as hormones, metabolites, growth factors, and neurotransmitters. The target ligand may be any other species of interest, such as pathogens (including pathogen-inducing factors or pathogen-derived factors), toxins, nutrients, and contaminants.

[0045] In some embodiments of the present invention, the aptamer is characterized by terminal redox labeling. Preferably, the aptamer is functionalized with a redox label located at the 3' or 5' end of the aptamer. More preferably, the aptamer is functionalized with a redox label via an amide linkage at the 3' end of the aptamer. Preferably, the aptamer is a single-stranded oligomer, such as ssDNA or RNA, containing a redox label at its 3' end. The redox label can be provided as an ester that reacts with an amine at the 3' end to link the two together. In some embodiments, the redox label is a methylene blue succinimide ester linked to the amine-terminated ssDNA or RNA. In other embodiments, the redox label may include a porphyrin-containing redox sensor capable of detecting and / or measuring physiological gases such as oxygen, NO, and CO.

[0046] In some embodiments, the end label may be an end label of a redox material, and as a result, a change in the distance between the label and the gold working electrode causes a detectable change in electron transfer between the two. Thus, when the ligand binds to the aptamer, its physical shape changes, the distance of the redox label changes, and a detectable change occurs in electron transfer and the resistance generated between the two.

[0047] In some embodiments, the redox label is capable of electron transfer to or from the electrode, with optional direct movement to the working electrode, and subsequently electron transfer to the sensor body. Sufficient proximity and accessibility of the redox label to the electrode allows for the generation of an electrical signal, such as current, voltage, or other measurable electrical interaction, between the redox label and the electrode. The redox label may be positioned such that binding of the target ligand to the aptamer causes a measurable change in the electrical signal generated by the redox label. In some embodiments, the redox label is positioned at the end of the aptamer, for example, as shown in Figure 1. In other embodiments, the redox label resides on a separate polynucleotide chain that binds to the aptamer in the absence of the target species and is replaced by binding of the target species to the aptamer (see Xiao et al., J.Am.Chem.Soc., 127:17990-91 (2005)). In some embodiments, the redox label may be configured for "turn-off" signaling, where the redox signal decreases upon binding to the target ligand. In other embodiments, the redox label may be configured for "turn-on" signaling, where the redox signal increases upon binding to the target ligand. Such sensing label arrangements can be selected using known methods for designing electrochemical sensors.

[0048] In some embodiments, the redox label is methylene blue. In other embodiments, the redox sensor is selected from known redox labels including methylene blue, ferrocene, viologen, anthraquinone or any other quinone, daunomycin, organometallic redox labels, such as porphyrin complexes or crown ether rings or linear ethers, ruthenium, bis-pyridine, tris-pyridine, bis-imidazole, cytochrome c, plastocyanin, and ethylenetetraacetate metal complexes, or combinations thereof. Preferably, the redox label is methylene blue.

[0049] In some embodiments, the redox label is linked to the aptamer, and the aptamer-labeled unit is then immobilized on the working electrode using a carbon chain linker or directly using thymine. In some embodiments, the aptamer is immobilized on the working electrode by incubation with an aptamer solution at a concentration of about 100 nM to 500 nM (including about 200 nm, 300 nm, and 400 nm) for a period of about 1 to 5 hours (including about 2, 3, and 4 hours). In further embodiments, incubation can be carried out at a temperature suitable for incubation of plant cultures, bacterial cultures, insect cultures, or mammalian cultures, for example, about 0 to 100°C (including any range or value within that range). Preferably, incubation can be carried out at a temperature of about 20 to 45°C (including about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, and 43°C).

[0050] The target ligand binds to an aptamer, and the aptamer is assumed to have a structure such that the redox label is close to the sensor body and / or working electrode, thereby generating a flow, Faraday current, or other measurable electrical interaction. In a sensor containing multiple recognition elements, the bulk dynamics of target binding and dissociation, as well as the resulting electrical interaction with the substrate, generate a measurable electronic signal proportional to the concentration of the target species in the sample solution or suspension.

[0051] One or more electrical signals in the sensor may be proportional to the pH of the solution or suspension and the concentration of the target ligand. These electrical signals may include peak potential, Faraday current, non-Faraday current, and combinations thereof.

[0052] Preferably, the pH of a solution or suspension can be measured using an electrical signal of peak potential (V). In a preferred embodiment, a sensor based on an electrochemical aptamer can measure the pH of a solution or suspension within a physiological pH range. Preferably, the pH is about 4 to about 8 (including any value or range within that range, including about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, and about 8.0).

[0053] Preferably, the target ligand can be detected and / or measured using an electrical signal of non-Faraday current (A). In a preferred embodiment, a sensor based on an electrochemical aptamer can measure the concentration of the target ligand in a solution or suspension within a physiological range. Preferably, the target ligand concentration is about 0 M to about 10 M (including any range or value therein). Preferably, the target ligand concentration is about 0 mM to about 100 mM (including, but not limited to, any range or value therein, including about 0 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM and about 100 mM). Preferably, the target ligand concentration is about 0 mM to about 10 mM (including, but not limited to, any range or value within that range, including about 0 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, and about 10 mM). Preferably, the target ligand concentration is about 0 μM to 100 μM (including, but not limited to, any range or value within that range, including about 0 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, and about 100 μM).

[0054] In a particularly preferred embodiment, pH and target ligand may be measured using a single measurement, preferably square wave voltammetry (SWV).

[0055] In some embodiments, the working electrode is further functionalized with a carbon chain material to prevent nonspecific binding and / or to provide a buffer between the redox label and the electrode / sensor body. In some embodiments, the carbon chain comprises 6-mercapto-1-hexanol or 6-aminohexanol. The carbon chain can be bound by incubation at concentrations of about 100 nM to 500 nm (including about 200, 300, and 400 nm) for a period of about 30 minutes to 2 hours (including 45, 60, 75, 90, and 105 minutes). The bound carbon chain can prevent nonspecific adsorption of the aptamer to the electrode surface, thus providing more reliable signal measurement.

[0056] The working electrode can be immersed in or brought into contact with a biological fluid, which alters the detected current. As shown in Figure 1, the presence of aptamers on the surface of the working electrode allows small ligands to freely associate.

[0057] The aptamer-functionalized electrode can be fixed to the sensor body. In some embodiments, the sensor body is made of a conductive material, and as a result, any electrical signal generated by the aptamer can be transmitted through the sensor body to a measuring or detecting device connected thereto. The sensor body may be made of any suitable conductive material for electrochemical sensing, including, for example, gold or any gold-coated metal or material, aluminum, copper, palladium, titanium, tungsten, silver, platinum, carbon (including graphite, nanotubes, and graphene), mercury film, oxide-coated metal, semiconductor material, and any other conductive material.

[0058] The sensor body can take the form of any necessary or desired shape or size. For example, the sensor body may be a disk, cylinder, wire, sphere, paddle, rectangle, strip, array, screen print, etc. Those skilled in the art will understand that even narrower sensor bodies, such as thin wires, may offer even less invasive advantages for in vivo applications in some embodiments.

[0059] In some embodiments, the sensor body communicates electrically with a measuring or detecting device and auxiliary components such as a power supply or connector to it. Such communication may include lines or other conductive elements connecting the sensor body to the detector, measuring device, controller, power supply, voltage regulator, and other control elements that operate the sensing element. The sensor body may be further connected to a structure designed and positioned to house or support the sensor body during operation in order to ensure proper operation.

[0060] In some embodiments, the sensor body is part of an overall sensor electrode sensing system for detecting and / or measuring the pH and the presence of a target ligand or molecule in a culture medium, such as a biological culture medium. In addition to the sensor body, which can function as a biosensor electrode, the sensing system may also comprise further elements such as a reference electrode, a counter electrode, a voltage and / or current source, a control element, and means for reading or detecting changes in electrical conductivity through the sensor body. The sensor body and / or other electrodes may, in some cases, be configured for various electrochemical observation techniques, including potentiometer, current measurement, and voltage measurement, such as differential pulse voltammetry, cyclic voltammetry, AC voltammetry, and square wave voltammetry. In some embodiments, the sensing system may further comprise a controller for providing current and / or voltage to the working electrode and / or reference electrode within appropriate operating parameters. Further components may include readout circuits, data acquisition, and data storage components.

[0061] Preferably, the sensor comprises a working electrode, a reference electrode, and a counter electrode.

[0062] The reference electrode may be selected from Ag / AgCl, Cu / CuSO4, and Hg / HgSO4. Preferably, the reference electrode is Ag / AgCl.

[0063] The counter electrode may be selected from Pt (including Pt wire or mesh) and graphite (including graphite rod). Preferably, the counter electrode is Pt.

[0064] In some embodiments, the sensor body is enclosed, sealed, covered, or partially covered to provide a substantially leak-free or leak-less reference electrode. Preferably, the sensor body is enclosed, sealed, covered, or partially covered in glass, plastic, or a combination thereof, most preferably plastic. In some embodiments, a substantially leak-free reference electrode may refer to an enclosed, sealed, covered, or partially covered reference electrode that is stable over time, resistant to ethanol contact, or a combination thereof. A leak-free reference electrode that is stable over time may exhibit potential drifts of less than about 100mV, 80mV, 70mV, 60mV, 50mV, 40mV, 30mV, 20mV, or 10mV over a period of at least about 30, 40, 50, or 60 days. Leakless reference electrodes resistant to ethanol exposure may exhibit potential drifts of approximately 100mV, 80mV, 70mV, 60mV, 50mV, 40mV, 30mV, 20mV, and less than 10mV after contact with at least approximately 60%, 70%, or 80% ethanol.

[0065] The sensor may have a micron, submicron, or nanoscale structure. Preferably, the sensor may be fitted to fit the wells of a 24-well culture plate (less than approximately 16 mm in diameter).

[0066] The sensor may be part of a flow system in which a solution or suspension flows over or through the sensor. The solution or suspension may be supplied to the sensor in a continuous or discontinuous flow, preferably a continuous flow.

[0067] In one embodiment, the sensor may comprise one working electrode. In another embodiment, the sensor may comprise multiple aptamer-functionalized working electrodes, each working electrode may comprise a different type of aptamer. In embodiments where the sensor comprises two or more working electrodes, each working electrode comprising a different type of aptamer, the sensor may be adapted to detect two or more different types of target ligands.

[0068] In a particularly preferred embodiment, the sensor may comprise a leakless reference electrode, a counter electrode, and one or more working electrodes. The one or more working electrodes may include, but are not limited to, one, two, three, four, five, six, seven, eight, nine, ten, or more working electrodes. Each working electrode may contain the same or different types of aptamers. Preferably, each working electrode contains different types of aptamers so that the sensor can be adapted to detect and / or measure one, two, three, four, five, six, seven, eight, nine, ten, or more different types of target ligands.

[0069] In some embodiments, an array comprising multiple sensors is provided. Each sensor may be the same or different. Preferably, the sensors are different. Preferably, each sensor comprises a working electrode having a different type of aptamer. An array comprising 24 sensors, each sensor comprising up to 3 working electrodes, each working electrode comprising a different type of aptamer, may be adapted to detect and / or measure up to 72 different types of target ligands.

[0070] The array may be mounted on a printed circuit board adapted to connect to a measuring or detecting device. Preferably, the board is adapted to fit a 24-well culture plate. Preferably, each sensor in the array is embedded in or fixed to the board and adapted to fit the wells of the plate, thereby enabling readings of all 24 wells of the plate with a single measuring or detecting device.

[0071] All publications, including patents, published patent applications, and non-patent literature, referenced herein are incorporated in their entirety by reference.

[0072] It will be understood that the present invention, as disclosed and defined herein, extends to all alternative combinations of two or more of the individual features referred to or evident from the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention. [Examples]

[0073] Materials and methods We purchased a platinum wire (1 mm diameter) counter electrode, an Ag / AgCl (3M NaCl) reference electrode, a gold wire (0.20 mm diameter, 0.5 m length, 99.99% high purity) working electrode, and 6-mercapto-1-hexanol from Sigma-Aldrich. We purchased the aptamer sequence from Integrated DNA Technologies (Coralville, IA, USA). We purchased IDTE, EDTA, TCEP solution, Na2HPO4, NaCl, MgCl2, NaOH, KCl, KH2PO4, and H2SO4 from commercial sources.

[0074] The potentiostat used was the CH1000C, purchased from CH Instruments (Austin, TX, USA).

[0075] In vitro measurement for characterization of phenylalanine Signal measurements were performed using a ChI1040C 8-channel potentiostat in square-wave voltammetry (SWV) mode and a standard three-electrode cell. SWV experiments were conducted with a potential step of 0.001, a potential window of -0.1 to -0.4 V, and an amplitude of 0.05 V at a frequency of 300 Hz. The experiments were performed in working buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 at pH 7.3).

[0076] Example 1: Aptamer Sequence For the electrochemical aptamer-based sensor, the aptamer sequence used for phenylalanine detection was as shown below. 5'-CGACC-GCGTT-TCCCA-AGAAA-GCAAG-TATTG-GTTGG-TCG-3'.

[0077] The aptamer sequence may be modified at its 5' end with a thiol-C6-SS group to form a thiol-Au bond and connect to the surface of the gold electrode. Alternatively, the 3' end of the aptamer sequence may be connected to methylene blue (redox reporter) via a six-carbon linker, as shown in Figure 1.

[0078] Example 2: Signal Measurement The sensor used consists of a classic three-electrode configuration: a working electrode (WE), a counter electrode (CE), and a reference electrode (RE). The high potential stability of the Ag / AgCl electrode allows for the determination of the potential of the other half-cell. A Pt electrode was used as the counter electrode, cleaned with fire, and then rinsed with Milli-Q water before use. A gold electrode was used as the working electrode. A self-assembled monolayer (SAM) of aptamer arrays was formed on the surface of the gold working electrode. The thiol-C6-SS group at the 5' end of the aptamer array forms a thiol-Au bond with the gold electrode, thereby connecting the aptamer to the gold electrode. The binding of the target molecule (phenylalanine) to the aptamer (i.e., oligoprobe) induces a structural / stereoconjugate change in the aptamer, which in turn affects the electron transfer rate of the redox reporter bound to the 3' end of the aptamer to the gold electrode, thereby altering the observed Faraday current. The structural changes are schematically shown in Figure 2, and these are reflected by methylene blue, a redox reporter at the 3' end of the aptamer sequence.

[0079] Variations in electron transfer efficiency can be measured by Faraday current. To detect the Faraday current, all three electrodes were connected to a potentiostat, as shown in Figure 3a. The potential of the working electrode (WE) was controlled relative to a constant potential of the reference electrode (RE). Current flows between the working electrode and the counter electrode (CE).

[0080] When square wave voltammetry (SWV) (equal to the sum of the square wave potential and step potential) is applied to the WE, the Faraday current can be measured between the WE and CE. The corresponding peak potential shifts relative to the electrode mechanism. In electrochemically reversible processes, the MB is 1e at different pH levels, particularly at pH 6.0–10.7. - / nH + The process can be oxidized or reduced (Figure 3b). Thus, pH was reflected by changes in peak potential shift within the physiologically relevant pH range for humans (6.5–7.4).

[0081] After connecting to the potentiostat, square wave voltammetry (SWV) measurements were performed on the device using the following method. 1. As shown in Figure 4, the potentiostat applied a combination of a step-level potential and a square-wave potential between the working electrode and the reference electrode. 2. The current was measured twice in a single step between the counter electrode and the working electrode: once when pulsed in the forward direction and once when pulsed in the reverse direction. The Faraday current was calculated from the difference between the forward and reverse currents. Theoretically, the magnitude of the peak current difference is proportional to the concentration of methylene blue, which is the redox reporter.

number

number

number

number

[0082] Example 3: Manufacturing of a sensor based on an electrochemical aptamer A platinum electrode (1 mm in diameter) was used as the counter electrode. An Ag / AgCl solution containing NaCl was used as the reference electrode. A gold wire (200 μm in diameter) was used as the working electrode.

[0083] Before bonding the aptamers, the gold wire was cut to a length of approximately 4-5 cm, and then the center of the gold wire was covered with heat-shrinkable PTFE tubing (McMaster-Carr Supply Company, Elmhurst, Illinois).

[0084] The following steps were used to coat the gold electrode with an aptamer. a) DNA oligos (modified aptamer sequences) were resuspended in TE buffer (IDTE: 10 mM Tris, 0.1 mM EDTA, pH 8.0). b) The thiol-modified oligoprobe (100 μM, 2 μL) is reduced by treating it in 2 μL of 10 mM TCEP solution at room temperature in the dark for 1 hour. c) Dissolve the modified oligonucleotide in "assembly buffer" (10 mM Na2HPO4 at pH 7.3 containing 1 M NaCl and 1 mM MgCl2) to a final concentration of 500 nM. d) Electrochemical cleaning of gold using a potentiostat: For 1500 segments, pulses of -1V to -1.6V are applied in a 0.5m NaOH solution at a scan speed of 1V / s to remove organic residue from the gold electrode surface. e) Increase the roughness of the gold electrode surface: Increase the electrode roughness by scanning at 0.35-1.6V for 20 cycles at a scan speed of 0.1V / s with a sample spacing of 0.0001s (no waiting time between pulses) for 0.5M H2SO4. f) Immerse the cleaned gold electrode in a 500 nM oligo probe in the dark for 1 hour. g) Finally, in the dark, incubate the gold electrode overnight in 5 mL of assembling buffer containing 5 mM 6-mercaptoethanol.

[0085] Example 4: Measurement in the working buffer Response curve The experimental titration curves were obtained in the working buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 at pH 7.3). For each measurement, the sensor was scanned by a potentiostat under two different frequencies in SWV mode: 300 Hz (initial E: -0.1 V, final E: -0.4 V, increment: 0.001 V) and 10 Hz (initial E: -0.1 V, final E: -0.4 V, increment: 0.003 V).

[0086] After setting up the connection, measurements were performed in the absence of phenylalanine (14.985 mL of working buffer) by interrogating the sensor more than 100 times until a stable peak current was obtained at both frequencies, and this was taken as the stabilized Faraday peak current difference.

number

[0087] After the measurement is complete, the raw signals for each concentration are normalized to the current change difference ratio D i It was converted to [this].

number

number

[0088] Figure 6 shows the titration results for the aptamer sensor, where the dots represent measurement points and the solid line is the Langmuir-Hill fitting curve. The titration curve indicates that the sensor has the potential to measure physiological phenylalanine concentrations in the range of 30 μM to 300 μM.

[0089] Key features of the sensor The sensor hysteresis effect was investigated, and the results are shown in Figure 7. After gold electrode fabrication and overnight incubation, the sensor was measured under SWV from high to low phenylalanine concentrations (forward direction, 3 mM to 10 μM) and from low to high phenylalanine concentrations (reverse direction, 10 μM to 3 mM). Twenty measurements were performed at each concentration point. The results indicate that the hysteresis to phenylalanine concentration fluctuations is negligible for this sensor to detect phenylalanine.

[0090] Sensor reproducibility The reproducibility of the sensors was tested using six gold electrodes (sensors 1-6) that underwent three aptamer recoating (manufacturing and overnight incubation), and the titration process described herein was performed simultaneously on the six gold electrodes. Figure 8 shows the reproducibility of the electrodes with respect to aptamer recoating. Figure 9 shows the reproducibility for different electrodes. The results indicate that calibration is not required after each manufacturing, as the aptamer sensors can achieve a stable response curve under a standard manufacturing process.

[0091] Example 5: Aptamer-based dual pH sensor pH is a crucial physiological factor that affects both cellular metabolic activity and drug activity, and it is becoming increasingly popular and integrated into lab-on-a-chip (LoC) devices for drug screening.

[0092] In vitro measurement for pH characterization Signal measurements were performed using a ChI1040C 8-Channel Potentiostat SWV mode and a standard 3-electrode cell, as described in Example 4. pH was reflected by the variation in peak potential, as shown in Figure 10. SWV was performed in “working buffer” with different pH values ​​(7.97 and 6.20). The number of protons involved in the reversible redox reprocessing of methylene blue is pH-dependent. At higher pH values ​​(pH=7.97 in the plot), the peak potential increased, and at lower pH values ​​(pH=6.20), the peak potential decreased.

[0093] Titration and calibration curves for pH measurement To further confirm the pH detection range, response curves were obtained by adjusting the pH level of 20 ml of "working buffer" to pH=7.4 by adding 0.5 ml of hydrogen chloride or sodium hydroxide after gold electrode fabrication and incubation. After each addition, the reference pH was measured using a commercially available pH meter, and each dot in the plot was the average of 20 consecutive measurements performed in the same pH solution. The results are shown in Figure 11.

[0094] pH calibration curves were measured in "working buffers" with various pH values ​​ranging from 6 to 8. The solutions were pre-sterilized and prepared. In Figure 12, the forward direction measured from low to high pH, ​​and the reverse direction measured from high to low pH. The sensor was rinsed with deionized water before moving between solutions with different pH values. The LR reverse and LR forward curves are lines fitted by logistic regression to the reverse and forward data, respectively. Each solution was measured under SWV 300HZ using 20 groups of data.

[0095] The results in Figure 12 show that the sensor peak potential measurement has a good linear relationship with pH, ​​and hysteresis over pH fluctuations can be ignored.

[0096] Functionality across different media Figure 13 shows the evaluation of functional compatibility in different types of cell culture media using the same sensor. After the fabrication of the gold electrode, pH calibration curves were performed at various pH values ​​from 6 to 8, measured in "working buffer" and "Melbourne medium". The solutions were sterilized and pre-prepared at different pH values. The sensor was rinsed with deionized water before moving it between different pH solutions. LR MM and LR WB are lines fitted by logistic regression to "working buffer" data and "Melbourne medium" data, respectively. Each solution was measured under SWV 300HZ using 100 data sets.

[0097] The results confirm the pH detection capability in both complex Melbourne media and working buffer. The similar response curves in the working buffer and Melbourne media suggest that the pH sensor may function in complex cell cultures.

[0098] Evaluation of sensor stability To evaluate the stability of the sensor, after overnight manufacturing, 100 consecutive SWV readings were measured within 10 minutes at five different pH values ​​pre-adjusted with "working buffer" having pH=6.27, pH=6.59, pH=7.05, pH=7.58, and pH=8.07. 100 consecutive SWV readings were also measured within 10 minutes at five different pH values ​​pre-adjusted with "Melbourne medium" having pH=6.23, pH=6.50, pH=7.09, pH=7.51, and pH=8.00. The measured potentials were relatively stable for each pH solution.

[0099] Figure 14 shows the sensor reproducibility for individual sensors measuring pH in the "working buffer." 100 consecutive SWV values ​​were measured within 10 minutes at five different pre-adjusted pH values ​​in the "working buffer" with pH values ​​of pH=6.27, pH=6.59, pH=7.05, pH=7.58, and pH=8.07 (from bottom to top).

[0100] Figure 15 shows the sensor reproducibility for individual sensors measuring pH in the "Melbourne medium." 100 consecutive SWV measurements were taken within 10 minutes at five different pre-adjusted pH values ​​in the "Melbourne medium," with pH values ​​of pH=6.23, pH=6.50, pH=7.09, pH=7.51, and pH=8.00 (from bottom to top).

[0101] Evaluation of response curves using different aptamer sequences To demonstrate that the device was not specific to the phenylalanine aptamer sequence, and to show that other aptamer sequences modified with methylene blue were pH-responsive, two further experiments were performed. Two electrodes coated with distinct aptamer sequences (doxorubicin aptamer and tryptophan aptamer) were fabricated according to the manufacturing process described in Example 3.

[0102] Figure 16 shows the potential results of the doxorubicin aptamer probe at various pH levels. This was performed using six “working buffer” solutions with different pH values ​​(pH = 7.87, 7.78, 7.61, 7.36, 6.88, and 6.65). Each solution was measured using SWV with a frequency of 300 Hz, with 10 groups of data.

[0103] Figure 17 shows the potential results of the L-tryptophan aptamer probe at various pH levels. This was performed using six "working buffer" solutions with different pH values ​​(pH = 7.84, 7.71, 7.56, 7.28, 6.79, and 6.62). Each solution was measured using 10 groups of data at a SWV with a frequency of 300 Hz.

[0104] Example 6: On-chip device On-chip sensor devices can be fabricated to directly measure cell culture pH and phenylalanine levels in real time, as schematically shown in Figures 18 and 19.

[0105] Figure 18 shows the bottom view (left) and side view (right) of the lid of the on-chip sensor well. The central cylinder is a support and holder for the Ag / AgCl electrode, Au electrode, and Pt electrode, into which they are inserted to create a nearly flat surface. On the opposite side of the cylinder are two holes for inserting tubes for the flow of nutrients and waste.

[0106] Figure 19 shows an overview of the on-chip sensor well. The perfusion well plate has three flutes. One flute is for nutrient inlet, and when the liquid level in this flute rises above that of the second intermediate round flute, the medium gradually flows through the cell culture flute. When the medium in the second intermediate round flute overflows its flute wall, the waste medium flows out and is discharged into the waste reservoir. The sensor well lid covers the perfusion well plate with a distance of 1 mm between cells on the bottom well, accurately measuring bioinformation of the cell environment and protecting the SAM layer on the gold electrode surface. The flat sensor lid surface and the small distance between the cells and the sensor allow laminar flow on the cell surface.

[0107] The on-chip device can be used for real-time monitoring of the cellular environment and for detecting cell pH and phenylalanine concentration in the flowing medium supply. Using the on-chip sensor, different target molecules can be measured by replacing aptamer probes in a modular manner. Furthermore, the sensor housing assembly can be modified to accommodate up to eight sensors, enabling real-time measurement of multiple analyte targets.

[0108] Example 7: Sensitivity Prediction 1000 measurements were performed in Melbourne medium and working buffer under different pH conditions. The average potential was measured for each pH and used to extract noisy potentials for each pH condition by subtracting the measured potential from the average potential. The noisy potential signals were fitted to a Gaussian noise distribution model. From the fitted model, the standard deviation (σ) and mean (μ) of the noise potentials were calculated. The resolution was predicted based on the standard deviation (σ) and sensor gradient (0.037 mV / pH), which is 0.08 pH (3-sigma rule, 99.7% prediction accuracy) (Figure 20).

[0109] Example 8: Measurement of at least two analytes simultaneously using a dual biosensor. Two experiments were conducted to confirm that the sensor could simultaneously measure at least two analytes. In Experiment 1, two electrodes coated with phenylalanine aptamer and two other electrodes coated with L-tryptophan aptamer were prepared and incubated overnight. All four electrodes were tested simultaneously under the same conditions. In the titration process, phenylalanine (1 nM to 3 mM) was first titrated in the "working buffer," followed by L-tryptophan (1 nM to 3 mM) in the "working buffer." The results show that there is only a negligible difference between the phenylalanine aptamer sensor and the L-tryptophan aptamer sensor, particularly in the target measurement range (Figure 21).

[0110] After the titration process in Part 1 of Experiment 1, the sensor was placed in working buffer at different pH values ​​(6.61, 7.05, 7.31, 7.54, 7.81), and 10 measurements were taken at each pH. The results show that both PHE and L-TRP aptamers indicate pH measurements after PHE and L-TRP titration (Figure 22).

[0111] In Experiment 2, two electrodes coated with phenylalanine aptamers and two other electrodes coated with L-tryptophan aptamers were prepared and incubated overnight. All four electrodes were tested simultaneously under the same conditions. In the titration process, L-tryptophan (1 nM to 3 mM) in the "working buffer" was titrated first, followed by phenylalanine (1 nM to 3 mM) in the "working buffer" as a second titration. The results show that there is only a negligible difference between the phenylalanine aptamer sensor and the L-tryptophan aptamer sensor in the measurement range of interest (Figure 23).

[0112] After the titration process in Experiment 2, the sensor was placed in working buffer at different pH values ​​(6.81, 6.99, 7.21, 7.38, 7.54), and 10 measurements were taken at each pH. The results show that both PHE and L-TRP aptamers exhibit pH measurements after PHE and L-TRYP titration (Figure 24).

[0113] Example 9: Dynamic Condition Test To integrate the sensor into a dynamic in vitro system, the inventors of this invention investigated whether flow rate affects the measurement. An experiment was designed to allow the sensor in the on-chip device to perform 10 groups of measurements in a working buffer at pH=7.3 at the following six different flow rates: 0 μl / min, 0.5 μl / min, 1 μl / min, 2 μl / min, 4 μl / min, and 8 μl / min. The flow rate was achieved and controlled by a microfluidic pump, and the flow rate varied accordingly from low to high and high to low. From the results in Figure 25, the peak potential was relatively stable within the acceptable range of -0.281 V to -0.279 V, and the converted pH was in the range of 7.26 to 7.32, demonstrating the sensor's ability to maintain measurement stability with flow rate fluctuations.

[0114] To assess the stability of the target analyte under dynamic conditions, electrodes coated with PHE and L-TRP aptamers were placed in 24 cell wells. The experiment began under static conditions, then increased the flow rate to 0.5 μl / min, 1 μl / min, 2 μl / min, 4 μl / min, and 8 μl / min. Two concentrations of the target analyte (0 μM and 100 μM) were tested. Five measurements were taken at each concentration and flow rate. The results in Figure 26 show that the "forward" dynamic condition (change in flow rate from low to high) has a negligible effect on the sensor's measurement capability.

[0115] In another experiment, the flow velocity was applied in the "reverse direction," starting at 8 μl / min and decreasing to 4 μl / min, 2 μl / min, 1 μl / min, 0.5 μl / min, and then to static conditions. In this experiment, two concentrations of the target analyte (0 μM and 100 μM) were tested. Five measurements were taken at each concentration and flow velocity. The results in Figure 27 show that the "reverse direction" dynamic condition (flow velocity changing from high to low) has only a negligible effect on the sensor's measurement capability.

[0116] For the flow velocity hysteresis test on the target analyte, PHE aptamer electrodes and L-TRP aptamer electrodes were placed in a 100 μM "working buffer." The flow velocity changed from "forward" (from low to high) to "reverse" (from high to low). Five measurements were taken for each flower rate. The results showed that the flow velocity did not produce any significant hysteresis effect in the aptamer sensor measurements for flow velocity changes from "forward" to "reverse" (Figure 28).

[0117] In further experiments, PHE aptamer electrodes and L-TRP aptamer electrodes were placed in a 100 μM "working buffer." The flow rate was changed from "reverse direction" (high to low flow rate change) to "forward direction" (low to high flow rate change). Five measurements were taken at each flow rate. The results showed that the flow rate did not produce any significant hysteresis effect in the measurement of the aptamer sensor from "reverse direction" to "forward direction" (Figure 29).

[0118] Example 10: Measurement of Cellular Metabolism To confirm the measurement capabilities and biocompatibility of the dual pH sensor in vitro, cellular metabolic measurements were performed.

[0119] Before the experiment, a leak-free reference electrode was fabricated. Using a torch, one end of a capillary glass tube was sealed with a platinum wire. The other end was then filled with 3M NaCl, and an electrochemically coated Ag / AgCl wire was inserted into the glass tube. The end with the Ag / AgCl wire was sealed with Parafilm, and the exposed Ag wire was covered with copper tape, as illustrated in Figure 30. To confirm the stability of the leak-free reference electrode, the open-circuit potential was measured in 3M NaCl against a commercially available reference electrode and then used for cell culture measurements (Figure 31).

[0120] Figure 32 shows an overview of the measurement timeline. To ensure complete confluence before the measurement day, cells were initially seeded on day 0. Meanwhile, on day 0, gold electrodes coated with phenylalanine aptamer were fabricated and incubated overnight. All fabrication kits were sterilized, filtered before fabrication, and all other components were sterilized with ethanol and UV for 1 hour before being placed in the measurement incubator (5% CO2, 37°C). On day 1, the cell medium was changed to Melbourne medium, the cell condition was confirmed under a microscope, and then the incubator was used for the initial calibration of electrode 1. For each calibration, the sensor was calibrated in the incubator under two different pH buffer solutions in Melbourne medium and two concentrations of phenylalanine: 0 μM, 10 μM, and 100 μM. On days 2 and 3, the calibration process and cell microscopy were repeated for electrodes 2 and 3. Between each day, continuous measurements were performed for 5 hours at 15-minute intervals. The experiment was stopped on day 4, and cell microscopy was performed to confirm the health of the cells.

[0121] A comparison of cell microscopy images of cells containing sensor wells and those without sensor wells (Figure 33) revealed no significant differences in morphology or viability. These data confirmed the biocompatibility and non-cytotoxicity of the sensor.

[0122] Measurements of phenylalanine during the first five hours of the first day (Figure 34, left) indicate that the cell culture remains closely within the physiological range to maintain cell growth and division. On the second day, as cell growth and division require amino acid consumption, phenylalanine levels decrease, as expected, to the low physiological range of 1 μM.

[0123] pH measurements (Figure 34, right) show that when cells consume nutrients from the culture medium, metabolic byproducts and waste products are generated, including organic acids (e.g., lactic acid). If the rate of nutrient consumption exceeds the supply of fresh medium, the accumulation of metabolic byproducts may cause the pH to decrease.

[0124] The ability of sensors to reflect cellular metabolism has been demonstrated, confirming that regular monitoring of nutrient levels, pH, and waste accumulation is crucial to ensure optimal cell growth and maintain healthy cell cultures in long-term experiments, thereby improving predictive efficacy.

[0125] Example 11: Continuous flow sensor To develop an example of real-time continuous flow measurement of a dual-purpose sensor, two syringe pumps containing solutions of different pH values ​​were loaded (Figure 35) and connected to a manually controlled valve. The flow rate of each syringe pump was 1 mL / min, and the pump solutions were manually switched every 10 minutes. The valve output was connected to the input of one well of a 24-well culture plate. The output of the well was aspirated by a line with a flow rate higher than the input flow rate. To ensure sufficient media volume inside the well covering the sensor, the input tube was positioned lower than the output tube but higher than the sensor level.

[0126] The results were obtained from two sensor electrodes (from the same well) (black and gray). The gray curve represents the theoretical pH (Figure 36). The pH measurements provided by the two separate electrodes closely agree with the theoretical predictions. However, the signals showed some fluctuations, which may be due to high flow rates that generate perturbations on the sensor surface. After Savitzky-Golay filtering, the pH estimates were more stable (Figure 37).

[0127] Real-time measurement of phenylalanine was achieved using the same settings as those used for real-time pH measurement. The syringe pump contained either 10 μM or 100 μM phenylalanine in the working buffer. The flow rate was 0.25 mL / min (because the scanning speed for phenylalanine measurement is slower than that for pH measurement), and the syringe pump perfusing the well containing the sensor was manually switched every 40 minutes (Figure 38).

[0128] Real-time measurements of phenylalanine from two separate electrodes (black and gray) closely matched the predicted value line (gray) (Figure 39).

[0129] Example 12: pH Sensing Temperature Dependence Experiment The sensors were equilibrated in an incubator (5% CO2, 37°C) with Melbourne media (human plasma-like media) and then exposed to a stepwise increase in pH. The pH response curve in the incubator with Melbourne media was tested first. Both the working buffer and Melbourne media were prepared with three different pH values ​​(working buffer: pH 6.48, pH 7.42, pH 7.91) and three different pH values ​​(Melbourne media: pH 6.55, pH 7.50, pH 7.91). The same sensors were first tested in the working buffer at ambient temperature, and then in the Melbourne media in the incubator. For each pH value, 10 SWV scans were performed at ≤300 Hz. The two fitted curves showed no significant difference (Figure 40).

[0130] Example 13: Sensor phenylalanine response curve under human plasma-like conditions To find the optimal response frequency of the sensor for measuring phenylalanine under human plasma-like conditions, the sensor was scanned five times in Melbourne medium at different frequencies (6 Hz to 1000 Hz) with phenylalanine concentrations ranging from 1 μM to 1 mM. 1000 Hz and 10 Hz were considered to be the optimal signal-on and signal-off frequencies, respectively, under human plasma-like conditions (Figure 41).

[0131] After obtaining the optimal frequency, the response curves for phenylalanine were obtained by preparing different stock solutions of phenylalanine in Melbourne medium with the following phenylalanine concentrations: 10 mM, 1 mM, 300 μM, 100 μM, 30 μM, 10 μM, 1 μM, 100 nM, and 10 nM. For the titration curves, a pre-set pod containing 14 ml of sterile Melbourne medium in an incubator (37°C, 5% CO2) was equilibrated for 1 hour in the incubator environment. Measurements were then performed using an apparatus including a three-electrode structure in the pod connected to a potentiostat, with a long silicone tube connected between the pod in a syringe in the incubator fitted with a filter to maintain sterility and the Melbourne medium reservoir. Syringe titration (Table 3) was then performed. Sensor phenylalanine detection using a turn-on frequency of 1000 Hz is performed with higher stability and sensitivity compared to a turn-on frequency of 300 Hz (Figures 42 and 43). [Table 3]

[0132] Example 14: Characteristics of a leakless reference electrode The long-term stability of a leak-free glass reference electrode was tested. After the first day of manufacture, the potential of the leak-free reference electrode was compared to that of a commercially available reference electrode in 3M NaCl. The leak-free reference electrode was then stored under ambient conditions. After two months, another potential measurement was performed. The leak-free reference electrode was observed to have a potential drift of less than 9 mV (Figure 44).

[0133] Ethanol is often used to sterilize biosafety laboratory equipment. The stability of leakless reference electrodes exposed to ethanol was tested. Open-circuit potential (OCP) measurements were first performed on both the leakless and commercially available reference electrodes, and then 80% ethanol was sprayed onto the reference electrodes. Next, 80% ethanol was sprayed onto both the leakless and commercially available reference electrodes. Another OCP measurement was performed on both electrodes to compare the effects of ethanol spraying. The potential of the leakless electrode appeared unaffected by ethanol spraying, while the potential of the commercially available reference electrode became unstable (Figure 45). These observations confirmed that the leakless reference electrode possesses a greater sealing capability.

[0134] Example 15: Design of a sensor holder for measurement in cell cultures The sensor detection element was miniaturized to fit a single well (15.6 mm in diameter) of a 24-cell well plate (CoStar, Washington, USA) (Figure 46). The sensor detection element includes three electrodes: a counter electrode (Pt), a reference electrode (Ag / AgCl), and a working electrode (Au). The 3D printed sensor holder secures the counter and working electrodes with screws, seals the reference electrode, and fixes it with paraffin. The stable arrangement of the three electrodes ensures that the sensor remains in a fixed position and prevents direct contact between the sensor and the cells (Figure 46). This configuration minimizes the possibility of interference during system operation, such as sensor scratches that could affect cell growth.

[0135] Example 16: Real-time cell culture measurement The timeline and methods are shown in Figure 47. Phenylalanine calibration was performed on the Melbourne medium, and curves for 0.10 μM and 100 μM were generated. Working buffers with pH 6.5 and 7.5 were prepared for pH calibration. A549 cells (human adenocarcinoma alveolar basal epithelial cells) were seeded on a 24-well plate at a seeding density of 10,000 cells / well. On day 1, the Melbourne medium was pre-equilibriumized over 2 hours. Next, the first group of sensors was calibrated with working buffer at a scan frequency of 1000 Hz. Then, the phenylalanine sensing of the sensors was also calibrated in the Melbourne medium at signal-on frequencies of 1000 Hz and signal-off frequencies of 10 Hz (0 μM, 10 μM, 100 μM). The A549 cell culture medium was changed to 1.5 mL of Melbourne medium, and the cells were observed and imaged under a bright-field optical microscope. Real-time in-situ measurements in the cell culture were started at hourly intervals. The electrodes were recalibrated daily using daily cell imaging. This entire calibration measurement process was repeated for three days.

[0136] The pH of the A549 cell culture remained relatively stable over 72 hours, as benchmarked by a pH strip (Figure 49) (Figure 48, n=3).

[0137] Sensor results, such as those shown for phenylalanine measurement, reflect a decrease in phenylalanine levels, consistent with the consumption of cell medium for cellular metabolism (Figure 50, n=3). Initial and final levels of phenylalanine detected by the sensor were benchmarked using GC-MS analysis. The final / initial values ​​were compared to reflect the phenylalanine consumption from day 0 observed in similar ratios using two different analytical procedures (Figure 51).

[0138] Figure 33 compares cell microscopy images over a 4-day measurement period between cell wells with and without sensors. Optical microscopy was performed using an Olympus IX53 inverted microscope equipped with a 10x objective lens. Images were taken directly from the center of each well in a 24-well plate. Imaging was performed at room temperature. There were no significant differences in cell morphology or density between the two conditions, which validate the sensor's superior biocompatibility and non-toxicity for long-term experiments.

[0139] After exploring the optimal scan frequency under human plasma-like conditions, more reliable and accurate real-time measurement results were achieved in cell cultures under human plasma-like conditions.

[0140] Example 17: Agarose gel coating to improve the antifouling ability of sensors The use of an agarose hydrogel protective layer for electrochemical aptamer-based sensors enhances signal transmission stability. Gel-protected sensors exhibit significantly improved stability, enabling accurate and continuous molecular measurements in vivo (Li, S et al., 2023, ACS Nano 17(18):18525-38). Following the fabrication of the sensor's gold working electrode, the following process was performed after agarose coating (Figure 52). 1. Dissolve the agarose powder in 1x PBS at near-boiling temperature. (1.5 wt%) 2. The agarose solution was left at 60°C for 1 hour. 3. Next, the sensor was gently immersed in agarose solution 3 to 5 times, with each immersion lasting approximately 3 seconds, and the sensor was stored in PBS buffer before use.

[0141] Four phenylalanine working electrodes were prepared, three sensors were coated with agarose gel, and one was left uncoated. Fifty groups of SWV scans (300 Hz and 10 Hz) were completed for each electrode in working buffer without phenylalanine. Then, phenylalanine in the working buffer was titrated from 1 nM to 3 mM, and one group of SWV scans was completed for each electrode at each concentration.

[0142] The agarose coating did not affect the ability to measure aptamer phenylalanine (Figure 53).

[0143] Example 18: Design of a Multiplexed Sensor Array System The proposed design is shown in Figure 54. This array is designed to accommodate 24-well culture plate multiplexing monitoring. The sensors are embedded in the lid of the culture plate, with each well having its corresponding sensor element. To enable sensor array multiplexing, the entire lid board is suitable for connecting to a bus connector, which can then be connected to a multiplexer and an 8-channel potentiostat, allowing real-time reading of the 24-well culture plate with a single potentiostat.

[0144] The finalized printed circuit board (PCB) is shown in Figure 55, with a board size identical to that of the 24-well plate, and holes drilled at the location of each well for bright-field imaging and CMOS sensor integration. Three connectors for multiplexer connections were embedded in one long edge of the PCB, each connected to two rows of sensors (8 wells). The PCB provided arrays of working and counter electrodes aligned in each well, each with a small hole for connecting a leakless reference electrode.

[0145] The sensor electrode array PCB design is shown in Figure 56. A 3mm x 6mm gold area was printed on the first side of the PCB that functioned as the counter electrode. Two 1mm x 1mm gold working electrodes were printed on the first side, and one 1mm x 1mm gold working electrode was printed on the second side. All four electrodes were connected to copper tracks. The copper tracks were covered with an insulating layer that had only the connection ends exposed for electronic signal transmission to the PCB.

[0146] Figure 57 shows an alternative configuration for the reference electrode (V2). The previous glass capillary tube (V1) was replaced with a plastic capillary tube (V2) to improve the rigidity of the reference electrode. One end of the plastic capillary tube was sealed with a platinum wire by pinching the other end, and then completely sealed with a biocompatible UV gel. Next, the plastic capillary tube was filled with 3M NaCl, an Ag / AgCl wire was inserted, and the other end was sealed with Parafilm and gel.

[0147] The design for the 3D printed sensor array lid is shown in Figure 58. The lid for the sensor array system was customized to match the size of a 24-well cell culture plate. The inner dimensions were customized for PCB assembly. Three holes were left on one side of the 3D printed lid for connecting the multiplexer and potentiostat.

[0148] Example 19: Testing of a Multiplexed Sensor Array System The sensor array system was manufactured and assembled as shown in Figure 59.

[0149] To ensure that the gold counter electrode has the same measurement capability as the platinum counter electrode, a sensor was manufactured using gold as the counter electrode instead of platinum. Testing the sensor response curves in working buffers without phenylalanine and with phenylalanine (Figure 60) showed that the use of gold as the counter electrode did not affect the sensor measurement.

[0150] The potential stability of the second form of a leak-free plastic reference electrode (plastic capillary tube) was tested against a commercially available reference electrode in 3M NaCl. Figure 61 shows the comparison results of the two forms of the leak-free reference electrode. The potential of the second form of the leak-free reference electrode is relatively stable compared to the commercially available reference electrode. In summary, the multiplexed sensor system offers one or more of the following advantages: 1. Multiplexed high-throughput monitoring function: The multiplexed sensor system enables monitoring of multiple analytes based on the number of gold working electrodes printed on the PCB. Each working electrode can perform dual-purpose monitoring of pH and aptamer target analytes. 2. Non-cytotoxic: The integration of a leak-free reference electrode prevented the leakage of silver ions from the NaCl and AgCl containers into the cell culture medium. 3. Novel analytes introduced into cell culture monitoring: Aptamer sequences can be customized to detect a wide range of target analytes, including phenylalanine, tryptophan, and doxorubicin.

Claims

1. The use of electrochemical aptamer-based sensors for detecting or measuring pH and target ligand levels in solutions or suspensions.

2. The use according to claim 1, wherein the sensor comprises an aptamer-functionalized electrode.

3. The use according to claim 2, wherein the surface of the working electrode is functionalized with a self-assembled monolayer of a single type of aptamer.

4. The use according to claim 2 or 3, wherein the aptamer includes a redox label.

5. The use according to claim 4, wherein the redox label is selected from the group comprising methylene blue, ferrocene, viologen, anthraquinone or any other quinone, daunomycin, organometallic redox labels, such as porphyrin complexes or crown ether rings or linear ethers, ruthenium, bis-pyridine, tris-pyridine, bis-imidazole, cytochrome c, plastocyanin, and ethylenetetraacetate metal complexes, or combinations thereof.

6. The use according to claim 5, wherein the redox label is methylene blue.

7. The use according to any one of claims 2 to 6, wherein the working electrode includes an antifouling polymer coating.

8. The use according to any one of claims 2 to 7, wherein the sensor comprises two or more working electrodes, and each working electrode contains a different type of aptamer.

9. The use according to any one of claims 1 to 8, wherein the sensor comprises a body and a reference electrode, and the sensor body is enclosed, sealed, covered, or partially covered so as to provide a substantially leak-less or leak-free reference electrode.

10. The use according to any one of claims 1 to 9, wherein the sensor is adapted to fit the wells of a 24-well culture plate.

11. The use according to any one of claims 1 to 10, wherein the pH and the concentration of the target ligand in the solution or suspension are proportional to one or more electrical signals from the sensor.

12. The use according to claim 11, wherein one or more electrical signals are selected from a peak potential, a Faraday current, and a non-Faraday current.

13. The use according to claim 12, wherein the pH of the solution or suspension is measured from the electrical signal of the peak potential.

14. The use according to claim 12 or 13, wherein the target ligand is detected or measured from the electrical signal of the Faraday current.

15. The use according to any one of claims 12 to 14, wherein the pH and target ligand are detected or measured using a single measurement.

16. The use according to claim 15, wherein the single measurement is square wave voltammetry.

17. Use of an array comprising multiple sensors according to any one of claims 1 to 16 for detecting or measuring the pH and multiple target ligand levels in a solution or suspension.

Images