Electrochemical capacitive biofouling resistant biosensors and methods of making and using same

Abstract

Described herein are electrochemical capacitive biosensors, methods of making same, and methods of using same. The electrochemical capacitive biosensors are especially useful for detecting pathogens, including viruses, bacteria, and allergens, in aerosols and complex biological media.

Description

ELECTROCHEMICAL CAPACITIVE BIOFOULING RESISTANT BIOSENSORS AND METHODS OF MAKING AND USING SAMERELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No.63 / 800,579, filed on May 6, 2025, and U.S. Provisional Application Serial No. 63 / 710,734, filed on October 23, 2024, the contents of which are hereby incorporated by reference in their entireties.FIELD OF INVENTION

[0002] Described herein are electrochemical capacitive biosensors, methods of making same, and methods of using same. The electrochemical capacitive biosensors are especially useful for rapid, label-free, and antifouling detecting respiratory pathogens and airborne pathogens, including bacteria, viruses, and allergens in both aerosol samples and biofluids.BACKGROUND

[0003] Airborne transmission is a primary transmission mode of respiratory pathogens, including the infectious influenza viruses, namely H5N1 and H1N1. These pathogens pose a high risk of morbidity and mortality, with the potential to cause serious threat to public health and pandemic outbreak. Despite the demonstrated risk associated with the transmission of these pathogens, techniques for their real time point-of-care detection have remained elusive.

[0004] Electrochemical biosensors have previously been used for the detection of influenza viruses and are rapidly emerging as an alternative to conventional clinical screening techniques. Electrochemical capacitive biosensors (ECBs) are one such emerging category of biosensors which focus on label -free pathogen detection, effectively eliminating the extra labeling step of applying additional reagents to generate detectable signals. The detection mechanism involves immobilizing redox-active materials on electrodes and monitoring changes in complex capacitance C® as represented:

[0005] Where j is an imaginary number, co is the angular frequency, and Zf., is the complex impedance when targets bind.

[0006] A major challenge associated with biosensing technology is signal stability in complex biological fluids and fouling-prone environments. In these complex environments, there is interference and difficulty in signal reproducibility. Additionally, for development of an effective point-of-need device for early disease prevention and intervention, at least the following biosensor properties are desired: rapid detection time (less than 10 min), high sensitivity and specificity with minimal signal degradation due to cross-interference in challenging environments, low Limit-of-Detection (LoD), and affordability.

[0007] In addition, countries worldwide, including the United States, have reported outbreaks of highly pathogenic avian influenza (HP Al), particularly H5 strains, often linked to direct contact with infected poultry, dairy cows, and other animals. Although avian H5N1 rarely transmits from human to human through airborne routes, it circulates among animals and frequently undergoes unpredictable mutations and reassortments. The potential for airborne transmission of H5N1 remains a concern, as even slight mutations could enable more efficient spread through respiratory droplets. Since humans lack immunity to H5N1, the virus poses a high risk of morbidity and mortality, with the potential to cause a pandemic on the scale of COVID-19. Given these risks, rapid and sensitive detection methods such as those disclosed herein are essential to monitor and mitigate the spread of influenza virus, including H5N1, as well as other pathogens.

[0008] Solutions involving electrochemical capacitance spectroscopy have been attempted, such as in US 2023 / 0081940. However, such technologies focus on conductive polymers and electro-polymerization and are limited at least in the electrical conductivity of the conductive polymers. Further, US 2023 / 0081940 relies on single redox-active polymer films, such as polyaniline, for capacitance spectroscopy, which requires that the polymers possess the capabilities of redox activity and receptor immobilization. These film-only compositions are not as versatile as other technologies, such as hybrid composites.

[0009] Hence, advances in biosensing technology are needed to achieve signal stability in complex biological fluids and fouling-prone environments and suitable detection properties.BRIEF DESCRIPTION

[0010] In one embodiment of the present disclosure, provided herein is an electrochemical capacitive biosensor (ECB) comprising an electrode and a capacitive composite disposed on the electrode. The capacitive composite comprises a capacitive material and a dielectric matrix. The ECB is configured for electrochemical capacitance spectroscopy.

[0011] In another embodiment of the present disclosure, provided herein is a method of making an electrochemical capacitive biosensor (ECB) comprising an electrode and a capacitive composite disposed on the electrode. The capacitive composite comprises a capacitive material and a dielectric matrix. The ECB is configured for electrochemical capacitance spectroscopy. The method comprises disposing the capacitive composite on the electrode.

[0012] In yet another embodiment of the present disclosure, provided herein is a method of using an electrochemical capacitive biosensor (ECB) comprising an electrode and a capacitive composite disposed on the electrode. The capacitive composite comprises a capacitive material and a dielectric matrix. The ECB is configured for electrochemical capacitance spectroscopy. The method comprises exposing the ECB to a sample; and analyzing the sample with the ECB using electrochemical capacitance spectroscopy to detect a target.BRIEF DESCRIPTION OF DRAWINGS

[0013] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings. These drawings are exemplary and are not to be construed as limiting.

[0014] Fig. 1 illustrates electrode modification and the biosensing mechanism in accordance with the present disclosure. (A) A dispersion of graphene oxide (GO) andPEDOT:PSS is prepared and drop-casted onto the working electrode (WE) of the screen-printed carbon electrode (SPCE). GO is then electrochemically reduced via cyclic voltammetry (CV) to form a PP / rGO hybrid composite. (B) A specific aptamer is immobilized on the modified surface to selectively capture the target influenza virus (lAVs). Virus binding reduces the availability of active surface sites, resulting in a measurable decrease in capacitance, as shown in the representative capacitance plot. This serves as the biosensor readout and confirms the presence of the virus in the sample.

[0015] Fig. 2 depicts analytical characterizations for the PP / rGO nanocomposite in accordance with the present disclosure, including: Environmental scanning electron microscopy (ESEM) images for (A) bare SPCE and (B) PP / rGO modified electrode. The bare SPCE shows a flat, porous surface with irregular graphite flakes. The PP / rGO modified electrode exhibits a typical wrinkled structure, likely due to the restacking of multiple rGO layers surface, and appears denser due to the incorporation of PEDOT:PSS. (All Scale bars of ESEM images represent 50 pm) (C) Raman spectra show the characteristic D and G bands of graphene-based materials, as well as distinct peaks corresponding to PSS deformation and PEDOT vibrational nodes. (D) XRD analysis confirms the successful formation of rGO and the emergence of a sharp peak near 26.5° in PP / rGO, corresponding to the crystalline 020 plane of PEDOT.

[0016] Fig. 3 depicts the electroanalysis results of the different electrode materials in accordance with the present disclosure: (A) Peak current obtained using cyclic voltammetry for different electrode materials in a solution of 5 mM potassium ferricyanide in 0.1 M potassium chloride at scan rate of 50 mV / sec; (B) Nyquist plots to measure impedance in the same solution. The impedance is fitted to a Randles circuit (inset) to obtain the resistance to charge transfer (Ret). (C) CV curves of PP / rGO at varying scan rates (5-400 mV / s) in potassium ferricyanide solution, demonstrating the scan rate-dependent behavior. (D) Linear relationship between peak current and the square root of scan rate, confirming diffusion-controlled charge transport as described by Randles-Sevcik equation. In all panels, bare SPCE is an unmodified carbon electrode, rGO refers to reduced graphene oxide and PP / rGO is modified with a dispersion of GO and PEDOT:PSS.

[0017] Fig. 4 depicts sensitive and specific detection of H5N1 and H1N1 with PP / rGO-based ECB in accordance with the present disclosure: (A) Complex capacitancecurve during different stages of the sensor modification, highlighting the excellent capacitive properties of PP / rGO electrode and the decrease in capacitance due to glutaraldehyde (Glu)-induced surface activation and final step of aptamer binding and BSA block on the ECB surface. (B) Example of a typical capacitance measurement demonstrating the notable decrease in capacitance with increasing viral load. Virus particles binding to surface-immobilized aptamers reduces the availability of active sites and induce a measurable change in capacitance. Sensitivity and specificity testing for (C) H5N1 and (D) H1N1 detection using PP / rGO modified ECB. The dotted line for both plots demonstrate the limit of detection (LoD) for specific virus. The data in calibration curves consist of at least n=3 independent ECB measurements.

[0018] Fig. 5 depicts robust performance of the ECB in accordance with the present disclosure in complex and fouling-prone environments. (A) Calibration curves for H5N1 detection in contaminated farm media and simulated chicken saliva. (B) Calibration curves for H1N1 detection in artificial nasal fluid and simulated human saliva. In both cases, the ECB shows a linear capacitance response with increasing virus concentration, indicating effective signal retention in challenging sample matrices. (C) Short-term stability of H5N1 and H1N1 sensors upon incubation in complex media, demonstrating minimal signal degradation over 2 hours. (D) Shelf-life analysis of PP / rGO-based electrodes stored at 4 °C, showing normalized capacitance values over time to assess material stability.

[0019] Fig. 6 depicts XPS analysis of PP / rGO hybrid composite in accordance with the present disclosure: (A) Survey spectrum and (B-D) elemental fitting for carbon, oxygen and sulphur respectively. Cis spectrum shows peaks at 284.5 eV (C=C, sp2carbon) and 288.5 eV (C=O). S2p spectrum exhibits peaks at 163 eV and 164.5 eV (thiophene sulfur, PEDOT) and 167.5 eV (oxidized sulfur, -SOs ", PSS). Ols peaks at 532 eV and 535.5 eV correspond to sulfonate groups (PSS) and oxygen functionalities from rGO. The results confirm successful hybrid film formation with some retained oxygen groups for aptamer immobilization.

[0020] Fig. 7 depicts a schematic of the capacitive biosensor platform in accordance with the present disclosure for pathogen aerosol monitoring. (A) Pathogen-laden aerosols from infected animals are collected using a wet-cyclone particle sampler. (B) The biosensingplatform detects the collected pathogens. (C) Fabrication highlights of PB / GO based capacitive biosensor.

[0021] Fig. 8 depicts characterization of a capacitive biosensor in accordance with the present disclosure. (A) EDX element analysis of the cross-section of the PB / GO codeposited SPCE. (B) Raman spectroscopy on sole-PB deposition, sole-GO deposition, and PB / GO co-deposition. (C) The electrochemical capacitive spectroscopy to compare the different deposition strategies. (D) The complex C* of aptamer-functionalized PB / GO capacitive biosensor for different concentrations of H5N1.

[0022] Fig. 9 depicts the performance of the capacitive biosensor in accordance with the present disclosure for H5N1 and E. coli. The sensitivity of the capacitive biosensors was evaluated for (A) H5N1 and (B) E. coli using serial dilutions of the targets in PBS. The specificity was also assessed for (C) H5N1 and (D)E. coli.

[0023] Fig. 10 depicts a quasi-quantitation of H5N1 and E. coli aerosols by the capacitive biosensor in accordance with the present disclosure integrated with a wet-cyclone bioaerosol sampler. (A) The flow chart of the process for quasi-quantification and (B) The quasi -quantification to screen H5N1 and E. coli aerosols collected by the wet-cyclone air sampler.The capacitive biosensor screened subdiluted samples, with results displayed as positive (O ) or negative (X) based on normalized AC% relative to the LoDs of each target.

[0024] Figure 11 shows the performance of the capacitive composite biosensor in accordance with the present disclosure to detect dust mite allergen Der fl. The LoD is calculated to be 0.012 ng / ml (S / N=3).

[0025] Figure 12 shows the capacitive property comparison with the addition of graphene oxide for capacitive composite biosensors in accordance with the present disclosure, namely: (a) The Ctot when different materials are electrodeposited on the electrode. The hybrid composite of PB / GO showed more than 10-times higher capacitance compared to that of single redox material, (b) The composite of PP / rGO showed improved capacitive value almost doubled that of a PEDOT:PSS-only film.

[0026] Figure 13 shows the performance of the capacitive composite biosensor in accordance with the present disclosure for different pathogens, namely, (a) H5N1, (b) E. coli and (c) Der fl at a single frequency (10 Hz).

[0027] Unless otherwise indicated, the drawings provided herein are meant to illustrate features of embodiments of the disclosure. These features are believed to be applicable in a wide variety of systems comprising one or more embodiments of the disclosure. As such, the drawings are not meant to include all conventional features known by those of ordinary skill in the art to be required for the practice of the embodiments disclosed herein.DETAILED DESCRIPTION

[0028] The present disclosure relates to an electrochemical capacitive biosensor (ECB) including an electrode and a capacitive composite disposed on the electrode. The present disclosure also relates to methods of making same and using same.

[0029] The capacitive composite in accordance with the present disclosure may take a variety of forms. Two particular forms are described in detail and exemplified herein: (1) a PP / rGO modified electrode, and (2) an interlocked Prussian blue (PB) / graphene oxide (GO) network on a screen-printed carbon electrode (SPCE). The particular hybrid composites disclosed herein uniquely integrate a PB / GO nano-network or PP / rGO film for high capacitance and to resist nonspecific adsorption in complex biological and environmental samples. This synergistic architecture not only stabilizes the baseline signal but also enables rapid label-free detection of pathogens in both aerosols and biofluids with limits of detection (LoDs) <50 RNA copies / mL or <10 bacterial cells / mL. The present ECB system addresses the versatility of composite ingredients, antifouling, aerosol integration, and multiplexed pathogen detection. However, these particular forms are non-limiting and merely illustrative.

[0030] In some particular embodiments, the present disclosure relates to the integration of a PP / rGO modified electrode with electrochemical capacitance spectroscopy (ECS) technique to develop an electrochemical capacitive biosensor (ECB). The ECB provides a rapid and suitable alternative for the label-free detection of respiratory pathogens,specifically influenza strains H5N1 and H1N1. The detection mechanism is based on measuring the change in capacitance, which relates to the presence of viral particles in samples. The ECB enables rapid detection within 5 minutes and has demonstrated excellent results for H5N1 and H1N1 detection in PBS, with a linear range of 10— 105copies / mL, excellent high sensitivity and specificity, and low detection limits of 39 copies / mL for H5N1 and 10 copies / mL for H1N1 respectively. Additionally, interference testing in complex biological fluids and environmental samples demonstrated minimal signal degradation, highlighting the robust, biofouling-resistant nature of the PP / rGO interface. The synergistic combination of electrode material with ECS leverages enhanced capacitive characteristics, thereby enabling label-free detection. This is the first known report of a PP / rGO nanocomposite-based ECB for rapid detection of influenza strains.

[0031] The present disclosure explores the properties of conductive polymeric material matrix; specifically, Poly(3,4 ethylenedi oxythiophene) :Poly-styrene-sulfonic acid (PEDOT :PSS), and studies its applicability for rapid influenza detection. PEDOT is a unique example of a conducting polymer, as it possesses low oxidation potential and good chemical stability, making it highly durable under various operating conditions. PEDOT:PSS possesses intrinsic anti-fouling capabilities due to the hydrophilic nature and negatively charged sulfonate groups associated with PSS, which repel nonspecific protein adsorption and other contaminants. By forming a stable conductive film on the electrode surface, PEDOT:PSS minimizes biofouling and enables signal stability even in dirty environments. This anti-fouling property is critical for achieving reliable, real-time pathogen detection in challenging settings such as poultry farm environments and clinical biological fluids, addressing a significant research gap in biosensor performance under interference-prone conditions. Additionally, graphene oxide (GO) is introduced to a polymer dispersion to enhance effective sensing surface area, leveraging its functional groups that enable covalent conjugations. Upon reduction, reduced GO offers enhanced electrical conductivity and mechanical stability, making it highly suitable for sensitive electrochemical measurements.

[0032] In other particular embodiments, described herein is a label-free capacitive biosensor using an interlocked Prussian blue (PB) / graphene oxide (GO) network on a screen-printed carbon electrode (SPCE) for direct detection of avian H5N 1 virus and E. coli bacteria. A single-step electro-co-deposition process grows GO branches on the SPCE surface, while the PB nano-crystals simultaneously decorate around the GO branches, forming anultrasensitive capacitive response at nanofarad levels. The biosensor was tested for H5N1 concentrations from 2.0 copies / mL to 1.6 x 105viral RNA copies / mL, with a limit of detection (LoD) of 56 viral RNA copies / mL for the H5N1 strain. Similarly, E. coli concentrations were tested from 2.0 CFU colony forming unit (CFU) / mL to 1.8 x 104CFU / mL, with a LoD of 5 CFU / mL, achieving results in under 5 minutes. This biosensor was integrated with a custom wet-cyclone bioaerosol sampler to successfully detect and quasi -quantitatively estimate H5N1 and E. coli concentrations in air (=93 viral RNA copies / m3and =8 CFU / mL, respectively). The quasi-quantification method, based on dilution and binary detection (positive / negative), enabled accurate risk assessment of airborne pathogens. This platform is adaptable for multiplexed detection of other respiratory pathogens, such as H1N1, making it a versatile tool for real-time airborne pathogen monitoring.

[0033] Turning now to the more general embodiments, disclosed herein is an electrochemical capacitive biosensor (ECB) comprising an electrode and a capacitive composite disposed on the electrode. The capacitive composite comprises a capacitive material and a dielectric matrix. The ECB is configured for electrochemical capacitance spectroscopy.

[0034] Generally, the electrode may include any electrode known in the art suitable to facilitate the ECB. In some embodiments, the electrode is selected from the group consisting of carbon electrodes (e.g., carbon paste, graphite, glassy carbon, carbon nanotubes, graphene, graphene oxide, screen printed carbon, etc.), metal electrodes (e.g., platinum, gold, silver, copper, nickel, stainless steel, etc.) , metal oxide electrodes (e.g., indium tin oxide (ITO), TiO?, SnO2, RuO?, MnCh, etc.), conductive polymer electrodes (e.g., PEDOT:PSS, polyaniline, polypyrrole, etc.), and combinations thereof.

[0035] Generally, the fabricating of the electrode may include any technique known in the art suitable to facilitate the method. In some embodiments, the fabricating comprises a technique selected from the group consisting of drop-casting, spin-coating, spray-coating, screen printing, inkjet printing, aerosol jet / 3D printing, photolithography, thin-film deposition (e.g., physical / chemical vapor deposition, atomic layer deposition, etc.), electrochemical growth (e.g., electrodeposition, electropolymerization, etc.), mechanicalfabrication (e.g., extrusion, machine cut, foil, mesh, etc.), nano- / surface- engineering (e.g., electrospinning, self-assembly, laser scribing, etc.), and combinations thereof.

[0036] Generally, the electrode layouts may include any design known in the art suitable to facilitate the ECB. In some embodiments, the electrode layout is selected from the design including single electrode (circular, rectangular, concentric ring), dual electrodes, three electrodes, quadra-electrodes, parallel-digit interdigitated, concentric interdigitated, microstripes, stacked electrodes, multi el ectrode arrays, microneedle electrodes, nano-brush electrodes, and combinations thereof.

[0037] Generally, the dielectric matrix may include any dielectric matrix material known in the art suitable to facilitate the ECB. In some embodiments, the dielectric matrix is selected from the group consisting of graphene oxide (GO), carbon nanotubes, metal nanowires, metal oxides, nanocellulose, hexagonal boron nitride, transition metal dichalcogenides, polymer dielectrics (e.g., polyvinyl alcohol, polyethylene glycol, Nafion, zwitterionic polymers, etc.), and combinations thereof.

[0038] In some embodiments, the dielectric matrix is a dielectric scaffold.

[0039] Generally, the capacitive material may include any capacitive material known in the art suitable to facilitate the ECB. In some embodiments, the capacitive material comprises a conducting metal salt and / or a conducting polymer.

[0040] In some embodiments, the capacitive material comprises a conducting metal salt selected from the group consisting of iron(III) hexacyanoferrate(II), Prussian Blue (PB) analogues (e.g., cobalt hexacyanoferrate, nickel hexacyanoferrate, manganese hexacyanoferrate, copper hexacyanoferrate, zinc hexacyanoferrate, iron hexacyanomanganate, vanadium hexacyanoferrate, mixed-metal PB analogues, etc.), transition metal oxides (e.g., MnCE, RuCE, TiCE, NiO, C03CE, Fe2C , FesCE, ZnO, V2O5, WCE, MoCE, CuO, CU2O, etc.), conductive metal-organic frameworks (e.g., MIL-lOO(Fe), MIL-lOl(Cr), MIL-53(A1), ZIF-8 (Zn-based), ZIF-67 (Co-based), HKUST-1 (Cu3(BTC)2), UiO-66 (Zr-based), MOF-74 (Mg, Ni, Co variants), Co-MOF-74, Ni-MOF-74 (conductive variants), Cu3(HHTP)2 (2D conductive MOF), Fe-TCPP MOF (porphyrin-based, redoxactive), MOF-derived TMOs (e.g., pyrolyzed MOFs forming porous C03CE or Fe2C ), etc.), and combinations thereof.

[0041] In some embodiments, the capacitive material comprises a conducting polymer selected from the group consisting of Poly(3,4 ethylenedi oxythiophene) Toly-styrene-sulfonic acid (PEDOT:PSS), polyaniline (PANI), polypyrrole (PPy), polyimide, poly(thiophene), poly(3 -hexylthiophene), poly(phenylene vinylene), poly(acetylene), poly(fluorene) derivatives, Poly(benzimidazole), poly(anthracene), poly(3,4-porpylenedi oxythiophene), poly(3 -thiopheneacetic acid), poly(ethyleneimine), zwitterionic polymers (PEDOT-sulfobetaine, PEDOT-carboxybetaine, PEDOT-phosphorylcholine, PPy-sulfobetaine, PPy-phosphorylcholine, Poly(sulfobetaine methacrylate) hybrids, Poly(carboxybetaine methacrylate) hybrids, zwitterionic Poly(thiophene) derivatives), and combinations thereof. In some embodiments, one or more dopants or additives (e.g., in low quantity), such as ethylene glycol (EG), dodecylbenzenesulfonic acid, camphorsulfonic acid, perchlorate, sulfonated polyethers or sulfonated pegs, hydrochloric acid, phytic acid, sulfonic acids, polyacrylic acid, dodecyl sulfate, tetrafluoro-tetracyanoquinodimethane, ferric chloride, etc. may be incorporated into the conducting polymer composition to enhance its electrical conductivity.

[0042] In some embodiments, the capacitive material comprises a surface area enhancer selected from the group consisting of nanocomposites, graphene oxide, metal organic frameworks (MOFs), metal nanoparticles, metal oxides, gold nanoparticles (AuNP), nickel nanoparticles (NiNP), titanium / cerium oxide (TiCE / CeCE), and combinations thereof. In these embodiments, it is understood that the capacitive material is different than the dielectric matrix.

[0043] In some embodiments, for signal amplification and greater signal stability, incorporation of metal organic frameworks (MOFs) and / or metal nanoparticles and oxides including, but not limited to, gold nanoparticles (AuNP), nickel nanoparticles (NiNP), and titanium / cerium oxide (TiO2 / CeO2) is contemplated.

[0044] In some embodiments, the capacitive material is in the form of a film.

[0045] In some embodiments, the capacitive composite is deposited on the electrode.

[0046] In some embodiments, the capacitive material has anti-fouling properties. In some embodiments, the anti-fouling properties are inherent. In some embodiments, the antifouling properties result from further inclusion of an anti-fouling coating. In someembodiments, the anti-fouling coating includes an anti-fouling component selected from the group consisting of 4-nitrobenzenediazonium, zwitterionic polymers, (poly(sulfobetaine methacrylate), poly(carboxybetaine methacrylate), poly(phosphorylcholine), poly(trimethylamine N-oxide methacrylate), poly(oxonorbornene)-based zwitterions), polyethylene glycol (PEG) based polymers, hydrogels (e.g., acrylamide, polyacrylamide, poly(2-hydroxyethyl methacrylate), etc.), nanoparticles (e.g., TiCE, ZrCE, AI2O3 thin films, etc.), polydopamine / PEG or zwitterions hybrid film, and combinations thereof.

[0047] In some embodiments, the ECB does not comprise a chemical label or a biochemical label. In these embodiments, the ECB is configured for label-free detection.

[0048] In some embodiments, the ECB further comprises a chemical label or a biochemical label selected from the group consisting of aptamers, antibodies, nanobodies, enzymes, oligonucleotides, peptides, cells, tissues, organelles, lectins, molecularly imprinted polymers, and combinations thereof. In these embodiments, the ECB is configured for labelbased detection.

[0049] In some embodiments, the chemical label or the biochemical label is a chemical receptor or a biochemical receptor.

[0050] Generally, the ECB may be configured for any use known in the art suitable to facilitate the ECB. In some embodiments, the ECB is configured for detection of a target.

[0051] In some embodiments, the ECB is configured for detection of a target selected from the group consisting of viruses, bacteria, allergens, fungi, parasites, biomarkers (e.g., inflammatory, cardiac, cancer, coagulation, enzymatic, etc.), nucleic acid (e.g., DNA or RNA, etc.), small molecules and metabolites (e.g., glucose, lactate, urea, creatinine, neurotransmitters, hormones, drugs, etc.), pollutants (e.g., heavy metals, toxins, pesticides, herbicides, etc.), whole cells (e.g., tumor cells, immune cells, exosomes, extracellular vesicles, etc.), antimicrobial resistant (AMR) agents (e.g., AMR genes, AMR pathogens, etc.), and combinations thereof.

[0052] In some embodiments, the ECB is configured for detection of pathogens.

[0053] In some embodiments, the ECB is configured for detection of respiratory pathogens. In these embodiments, it is understood that pathogens may be biological pathogens and / or chemical pathogens.

[0054] In some embodiments, the respiratory pathogens are selected from the group consisting of viruses (Influenza A strains (H1N1, H3N2, H5N1, H5N2, H7N2, H7N9), Influenza B strains (Yamagata, Victoria lineages), Coronavirus stains (229E, NL63, OC43, HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2), Paramyxoviridae (respiratory syncytial virus (RSV), Human Metapneumovirus (hMPV), Parainfluenza viruses (HPIV)), Picornaviridae (rhinoviruses, enteroviruses), Adenoviridae, Parvoviridae, Measles, Varicella-zoster virus, Cytomegalovirus), bacteria (Streptococus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Moraxella catarrhalis, Neisseria meningitidis, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Legionella pneumophila, Bordetella pertussis, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Chlamydophila psittaci, Mycobacterium tuberculosis, Non-tuberculous mycobacteria, Corynebacterium diphtheriae, Bacillus anthracis, Nocardia species), fungi (Aspergillus fumigatus, Histoplasma capsulatum, Coccidioides immitis / posadasii, Blastomyces dermatitidis, Cryptococcus neoformans, Pneumocystis jirovecii), parasites (Strongyloides stercoralis, Paragonimus westermani, Toxoplasma gondii, Echinococcus granulosus), other strains identified now or in the future, and combinations thereof.

[0055] In some embodiments, the ECB comprises a limit of detection of less than 50 copies / mL.

[0056] The ECB demonstrates signal stability even in fouling-prone environments, with no signal degradation observed during testing with environmental farm samples and complex biological fluids. In preliminary experiments, ECB demonstrated sensitivity of ~ 45 and 49 nF per logiocopies per mm2for H5N1 and H1N1 detection, respectively, a linear range of 10-105copies / mL, excellent specificity and negligible cross-interference from viruses like RSV, CoV-2, and H3N2.

[0057] Also disclosed herein is a method of making an electrochemical capacitive biosensor (ECB) comprising an electrode and a capacitive composite disposed on the electrode. The capacitive composite comprises a capacitive material and a dielectric matrix.The ECB is configured for electrochemical capacitance spectroscopy. The method comprises disposing the capacitive composite on the electrode.

[0058] Generally, the disposing may include any technique known in the art suitable to facilitate the method. In some embodiments, the disposing comprises a technique selected from the group consisting of drop-casting, spin-coating, spray -coati ng, dip-coating, electrodeposition, electrograft, electropolymerization, screen-printing, inkjet printing, 3D printing, electrophoretic deposition, layer-by-layer assembly, vacuum and physical deposition, self-assembly, sol-gel deposition, and combinations thereof.

[0059] In some embodiments, the method further comprises disposing on the capacitive composite a chemical label or a biochemical label receptor selected from the group consisting of aptamers, antibodies, nanobodies, enzymes, oligonucleotides, peptides, cells, tissues, organelles, lectins, molecularly imprinted polymers, and combinations thereof.

[0060] Also disclosed herein is a method of using an electrochemical capacitive biosensor (ECB) comprising an electrode and a capacitive composite disposed on the electrode. The capacitive composite comprises a capacitive material and a dielectric matrix. The ECB is configured for electrochemical capacitance spectroscopy. The method comprises exposing the ECB to a sample and analyzing the sample with the ECB using electrochemical capacitance spectroscopy to detect a target.

[0061] In some embodiments, the ECB further comprises target-specific bioreceptors deposited on the capacitive composite.

[0062] In some embodiments, the ECB is configured for detection of a target selected from the group consisting of viruses, bacteria, allergens, fungi, parasites, biomarkers (e.g., inflammatory, cardiac, cancer, coagulation, enzymatic, etc.), nucleic acid (e.g., DNA or RNA, etc.), small molecules and metabolites (e.g., glucose, lactate, urea, creatinine, neurotransmitters, hormones, drugs, etc.), pollutants (e.g., heavy metals, toxins, pesticides, herbicides, etc.), whole cells (e.g., tumor cells, immune cells, exosomes, extracellular vesicles, etc.), antimicrobial resistant (AMR) agents (e.g., AMR genes, AMR pathogens, etc.), and combinations thereof

[0063] In some embodiments, the ECB is configured for detection of pathogens.

[0064] In some embodiments, the ECB is configured for detection of respiratory pathogens. In these embodiments, it is understood that pathogens may be biological pathogens and / or chemical pathogens.

[0065] In some embodiments, the respiratory pathogens are selected from the group consisting of viruses (e.g., Influenza A strains (H1N1, H3N2, H5N1, H5N2, H7N2, H7N9, etc.), Influenza B strains (e.g., Yamagata, Victoria lineages, etc.), Coronavirus stains (e.g., 229E, NL63, OC43, HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, etc ), Paramyxoviridae (e.g., respiratory syncytial virus (RSV), Human Metapneumovirus (hMPV), Parainfluenza viruses (HPIV), etc.), Picornaviridae (e.g., rhinoviruses, enteroviruses, etc.), Adenoviridae, Parvoviridae, Measles, Varicella-zoster virus, Cytomegalovirus, bacteria (e.g., Streptococus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Moraxella catarrhalis, Neisseria meningitidis, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Legionella pneumophila, Bordetella pertussis, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Chlamydophila psittaci, Mycobacterium tuberculosis, Non-tuberculous mycobacteria, Cory neb acterium diphtheriae, Bacillus anthracis, Nocardia species, etc.), fungi (e.g., Aspergillus fumigatus, Histoplasma capsulatum, Coccidioides immitis / posadasii, Blastomyces dermatitidis, Cryptococcus neoformans, Pneumocystis jirovecii, etc.), parasites (e.g., Strongyloides stercoralis, Paragonimus westermani, Toxoplasma gondii, Echinococcus granulosus, etc.), other strains identified now or in the future, and combinations thereof.

[0066] In some embodiments, the respiratory pathogens are selected strains of pathogenic avian flu, including influenza strain H5N1, influenza strain H1N1, H7N9, H5N2, H7N2, other strains identified now or in the future, strains of RSV, CoV-2, and other respiratory viruses and bacteria, and combinations thereof. In other embodiments, the respiratory pathogens include other influenza strains adapted for humans, including those identified in the future, and combinations thereof. In other embodiments, the respiratory pathogens include respiratory diseases for animals including humans, birds, livestock, and combinations thereof.

[0067] In some embodiments, the sample is selected from the group consisting of bodily fluids, saliva, nasal fluid, blood, poultry farm samples, air samples, breath samples, waste water, environmental samples and combinations thereof.

[0068] In some embodiments, the ECB does not comprise a chemical label or a biochemical label. In these embodiments, the ECB is configured for label-free detection.

[0069] In some embodiments, the ECB further comprises a chemical label or a biochemical label selected from the group consisting of enzymes (e.g., horseradish peroxidase, alkaline phosphatase, glucose oxidase, urease, etc.), redox-active labels (e.g., ferrocene and derivatives, methylene blue, anthraquinone, etc.), nanoparticles (e.g., gold, silver, magnetic, etc.), quantum dots (e.g., CdSe, CdTe, ZnS, etc.), polystyrene beads, polymer / conductive agents, and combinations thereof. In these embodiments, the ECB is configured for label-based detection.

[0070] Generally, exposing the ECB to a sample may include any technique known in the art suitable to facilitate the method. In some embodiments, the exposing the ECB to a sample is achieved via a sampler selected from the group consisting of particle-to-liquid sampling systems, wet-cyclone particle samplers, exhaled breath condensate (EBC) devices, condensation growth samplers, clinical and diagnostic sampling (e.g., swabs, sputum, blood / serum / plasma, urine, etc.), filter membranes, precipitators, surface swabs / wipes, microfluidic devices, lab-on-a-chip devices, sweat patches, tear fluid sampling, wearable sampler, breath masks / filters and combinations thereof.

[0071] In some embodiments, the exposing the ECB to a sample is achieved via a wet-cyclone particle sampler.

[0072] Generally, analyzing the sample with the ECB using electrochemical capacitance spectroscopy to detect a target may include any technique known in the art suitable to facilitate the method. In some embodiments, the electrochemical capacitance spectroscopy comprises a whole-scan frequency capacitance measurement (e.g., a frequency range scanning from 1 mHz to 1 GHz). In some embodiments, the amplitude of the applied sinusoidal ac potential varies from 1 mV to 100 mV, optionally from 5 mV to 50 mV, optionally from 5 mV to 20 mV, optionally from 5 mV to 15 mV, optionally 8 mV to 12 mV, optionally about 10 mV. In some embodiments, the electrochemical capacitance spectroscopy comprises a partial scan frequency capacitance measurement.

[0073] In some embodiments, the electrochemical capacitance spectroscopycomprises a single-frequency capacitance measurement. A single-frequency scan offers a practical balance between sensitivity and simplicity. Instead of sweeping across a wide frequency range as in full ECS spectra, monitoring the system at one carefully chosen frequency reduces acquisition time, minimizes data complexity, and enables real-time or portable sensing. A skilled person can select the optimal frequency by identifying the region where the capacitive response is most sensitive to interfacial changes caused by target binding, while minimizing contributions from bulk solution resistance or faradaic processes. The skilled person can routinely observe the broad frequency sweep to map the ECS spectra, then choosing a frequency near the plateau of the capacitive region (typically mid-kHz to low-MHz, depending on electrode geometry and dielectric scaffold) where the signal-to-noise ratio is highest and the response is dominated by double-layer or interfacial capacitance rather than diffusion or charge transfer.

[0074] Also provided herein are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to the biosensor components, system components, reagents, etc., as described herein. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0075] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral nonreacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consistof foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0076] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or another substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0077] A control sample or a reference sample as described herein can be a sample from a healthy subject or sample, a wild-type subject or sample, or from populations thereof. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects or a wild-type subject or sample. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0078] The methods and algorithms of the invention may be enclosed in a controller or processor. Furthermore, methods and algorithms of the present invention, can be embodied as a computer-implemented method or methods for performing such computer-implemented method or methods, and can also be embodied in the form of a tangible or non-transitory computer-readable storage medium containing a computer program or other machine-readable instructions (herein “computer program”), wherein when the computer program is loaded into a computer or other processor (herein “computer”) and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. Storage media for containing such computer program include, for example, floppy disks and diskettes, compact disk (CD)-ROMs (whether or not writeable), DVD digital disks, RAM and ROM memories, computer hard drives and back-up drives, external hard drives, “thumb” drives, and any other storage medium readable by a computer. The method or methods can also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted over a transmission medium such as electrical conductors,fiber optics or other light conductors, or by electromagnetic radiation, wherein when the computer program is loaded into a computer and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. The method or methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice the process or processes. When a general -purpose microprocessor is employed, the computer program code configures the circuitry of the microprocessor to create specific logic circuit arrangements. Storage medium readable by a computer includes medium being readable by a computer per se or by another machine that reads the computer instructions for providing those instructions to a computer for controlling its operation. Such machines may include, for example, machines for reading the storage media mentioned above.

[0079] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel etal. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. andWolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studi er (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).EXAMPLES

[0080] Without further elaboration, it is believed that one skilled in the art using the preceding description can utilize the present invention to its fullest extent. The following Examples are, therefore, to be construed as merely illustrative, and not limiting of the disclosure in any way whatsoever. The starting material for the following Examples may not have necessarily been prepared by a particular preparative run whose procedure is described in other Examples. It also is understood that any numerical range recited hereinincludes all values from the lower value to the upper value. For example, if a range is stated as 10-50, it is intended that values such as 12-30, 20-40, or 30-50, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

[0081] Example 1. PP / rGO electrode.

[0082] Fabrication and characterization.

[0083] Preparation of PP / rGO electrode.

[0084] The first step in sensor preparation is modification of the screen-printed carbon electrode (SPCE) surface with a nanocomposite of graphene oxide and PEDOT:PSS. Commercial round-shape SPCEs with 2 mm WE diameter were used. A dispersion of GO (2 mg / mL) and PEDOT:PSS is prepared in 1:5 weight ratio, followed by sonication for 90 minutes to prevent agglomeration. After sonication, about 4 pL of the dispersion is drop-casted onto the surface of bare SPCE and dried at 45 °C for 15 minutes in a conduction oven followed by RT drying for 45 minutes. The drop-casted electrodes are then subjected to electrodeposition using cyclic voltammetry (CV) to obtain a rGO / PEDOT:PSS (PP / rGO) modified electrode. Cyclic voltammetry (CV) is carried out from a potential range of 0 to -1.5 V at a scan rate of 50 mV / sec for 20 cycles. The electrodes are then rinsed with DI water, dried, and stored at RT for short-term use (<2 days) and at 4 °C for long-term storage. The PP / rGO electrode acts as a capacitive layer that combines the high conductivity of reduced graphene oxide (rGO) with the electroactive stability of PEDOT:PSS.

[0085] Sensor fabrication for virus detection.

[0086] For preparing H5N1 and H1N1 sensors, the PP / rGO electrodes are dipped into a 1% (W / V) glutaraldehyde solution and gently shaken at room temperature (RT) for 4 h to activate the WE of the modified electrodes for the aptamer immobilization. After washing with deionized water and dried by N2, the aptamer (or antibody) solution is drop-casted onto the activated WE. The SPCEs are maintained still at RT for 60 min before being stored at 4 °C overnight to allow sufficient cross-linking of the aptamer (or antibody) to theoxygen moieties on the capacitive layer. On the following day, the SPCEs were gently washed with deionized water and dried. To block nonspecific binding, 1% (W / V) BSA solution is drop-casted on the WE and incubated for 30 min at RT.

[0087] Electrochemical measurements.

[0088] All electrochemical measurements are performed using a potentiostat. For electroanalysis to compare electrode performance, the parameters for CV and EIS are as follows:

[0089] CV - potential range from 0.8 to -0.4 V with scan rate of 50 mV / sec. For iR compensation, the potential data in CV is corrected by accounting for the solution resistance (Rs) obtained in Electrochemical Impedance Spectroscopy (EIS) tests.V’ = V - iRs

[0090] where V’ is the corrected potential and Rs is solution resistance.

[0091] EIS - Edc = 0.17 V, Eac= 10 mV, scanning frequency range of 100 kHz to 1 Hz. The resulting EIS data is fitted to a Simplified Randles Circuit and the corresponding parameters are obtained: solution resistance (Rs), resistance to charge transfer (Ret) and total capacitance (C).

[0092] Pathogen detection using electrochemical measurements.

[0093] For pathogen detection using the capacitive sensor, serial dilutions of the stock pathogen solution were first prepared. Then, the aptamer (or antibody) functionalized sensors were immersed in the solution for 5 min. After the incubation, EIS measurement (Edc = 0.2 V, Eac= 10 mV, scanning frequency range of 100 kHz to 0.5 Hz) was directly performed in the pathogen-containing media. The same procedures were conducted across the different dilutions and the different pathogens of interest. The Ctot value was obtained by fitting it into a simplified Randles circuit.

[0094] The presence of virus in samples is determined by measuring the change in capacitance of PBS blank runs and the samples.AC (ftot)bla?ifc (.Cfot)Sample

[0095] Using this formula, the 4 C values for different virus concentrations are obtained and plotted to obtain the calibration curves for specific influenza strains. Capacitance values are obtained after data-fitting.

[0096] Analytical Characterization.

[0097] Characterization was performed using an environmental scanning electron microscope (ESEM) and energy-dispersive X-ray spectroscopy (EDX). The analyses were carried out at 2 / 5kV voltage and 3 spot size. Similarly, X-Ray / Ultraviolet photoelectron spectroscopy (XPS) was carried out. This setup enabled detailed characterization of the elemental composition and electronic states on the sample surfaces.

[0098] Electroanalysis of PP / rGO electrode.

[0099] CV and EIS measurements were also performed using a redox probe, specifically 5 mM potassium ferricyanide solution in 0.1 M potassium chloride. The average anodic peak current (Ipa) value increases from 10.2 pA for bare SPCE, to 14.6 A for rGO and 21.11 pA for PP / rGO electrode (Figure 3 Panel A and Table 1). EIS measurements in the same redox solution produced consistent Nyquist plot (Figure 3B). On fitting a Randles circuit to the plots, Rct values of 3320 Q were obtained for bare SPCE, which reduced significantly to 48 ohms for the PP / rGO composite. This highlights the significant reduction in impedance for the PP / rGO SPCE, which results in high conductivity and less resistance for the electron movement in solution. The modification of the electrodes with rGO and subsequently PP / rGO increases the surface roughness and number of electroactive sites, contributing to superior electrochemical performance across test solutions.

[0100] Analytical Characterization of PP / rGO electrode.

[0101] The surface morphology of the electrode material was studied using different analytical techniques, namely ESEM, EDX and XPS. As seen in Fig. 2, Panel B, the PP / rGO surface morphology is characterized by a texture typical of graphene-based layers embedded within a polymer matrix. This increased roughness can enhance the electrode active surface area, thereby improving sensitivity in biosensing applications.

[0102] Similarly, Fig. 6, Panel A presents the XPS survey spectrum, where a distinct sulfur peak (-165-170 eV) confirms the presence of PEDOT:PSS, and prominent carbon signals validate the successful incorporation of rGO within the PP / rGO nanocomposite.

[0103] Sensor performance.

[0104] Based on the aptamer, the PP / rGO modified electrodes were employed for both H5N1 and H1N1 detection using the specific aptamers for the different influenza strains. The specific aptamers were: 1) Anti-Avian influenza virus H5N1 aptamer (5’-amino modified) and 2) Anti-influenza virus A HA protein aptamer (5 ’-amino modified) for influenza virus A (H1N1).

[0105] Fig. 4 Panel B is a schematic demonstrating the decrease in capacitance with increasing virus concentration, highlighting the presence of virus in a sample. The binding on virus increases the complex impedance, decreasing the capacitance, and the change in capacitance is then plotted to analyze the sensor performance.

[0106] H5N1 detection.

[0107] The sensitivity of the ECB was evaluated by sequential dilution of a purified inactivated H5N1 stock solution and measuring the corresponding DC values for different virus concentrations (Fig. 3, Panel C). Initially, a control experiment run is performed using pure PBS (0.1 M) that acts as a blank measurement. The range of virus concentration is chosen to reflect real -world pathogen presence. The initial stock solution of the virus (105copies / mL) is diluted in series, and the change in capacitance was measured in each concentration using three independent electrodes. For the PP / rGO ECB, a sensitivity of 45.2 nF / logiocopies*mm2was achieved, with a linear detection range of 10-105copies / mL. The detection limit (LoD) of the biosensor is estimated by the formula:LoD = 3 X SDblank

[0108] Where SDbiank is the standard deviation for the electrode blank measurements. Using blank measurements of 5 independent electrodes, the LoD obtained was 39 copies / mL, which is less or comparable to the usual RT-PCR standards.

[0109] Additionally, as seen in Fig. 4, Panel C, the specificity test is performed using a mixture of 4 different viruses - H1N1, CoV-2, RSV and H3N2 - with concentrations varying from 10-104copies / mL. It is seen that the H5N1 biosensor produces negligible signal for the other interfering virus strains, which is below the LoD. Hence, the biosensor is highly specific to H5N1.

[0110] H1N1 detection.

[0111] For the PP / rGO based H1N1 biosensor, the same protocol was followed for sensitivity and specificity analysis as explained for the H5N1 biosensor above.

[0112] As seen in Fig. 4, Panel D, for the H1N1 sensor, a sensitivity of 49.5 nF / logiocopies*mm2was obtained, with a linear detection range of 10-105copies / mL. Also, a LoD of 10 copies / mL for H1N1 detection was obtained, highlighting the ultrasensitive nature of the ECB. The calibration experiments were performed using live H1N1 virus, illustrating the capability of ECB for real-world influenza detection. As seen in Fig 4, Panel D, the ECB showed negligible signal for other viruses with varying concentrations, minimizing the risk of cross-interference and highlighting its specificity for H1N1 detection.

[0113] The sensor characteristics for both H5N1 and H1N1 detection are summarized in Table 1.

[0114] Table 1. Performance characterization of H5N1 and H1N1 detection using PP / rGO-based ECB.H5N1 ECB H1N1 ECB Linear Sensitivity Medium LoD (RNA Linear Sensitivity Medium LoD (RNA Range (nF / logioCopies copies / mL) Range (nF / logioCopies copies / mL) per mL) per mL)106.3 PBS 30 142 PBS 41 96.6 Farm 55 134 Human 27 0 - 105Sample 10 - 105Saliva104.5 Chicken 46 129.3 Human 61 Saliva Nasal Fluid

[0115] Testing in fouling-prone environments.

[0116] PEDOT:PSS provides significant anti-fouling resistance, which is critical for ensuring stable sensor outputs in complex biological fluids and contaminated real-world environments. Accordingly, two tests were specifically designed to evaluate signal stability in these challenging environments and carried out.

[0117] Test 1: The H5N1 biosensor stability was tested using real -world poultry farm environment samples from Tyson. The samples are extracted in PBS (0.1 M) solution and spiked with inactivated H5N 1 virus, followed by the same testing procedure described above. As seen in Fig. 5, Panel A, a linear correlation was seen with increasing virus concentration in the contaminated environment and no significant signal degradation.

[0118] Test 2: For H1N1 testing, two complex biological fluids were used -simulated saliva and artificial nasal fluid. These simulated fluids mimic the pH and composition of typical human saliva and nasal swabs. The artificial nasal fluid is quite viscous and was diluted 4 times with PBS before spiking with H1N1 live virus, and no such dilution was needed for simulated saliva. After spiking the fluids with H1N1, the calibration runs were seen in Fig. 5, Panel B. A linear increase in capacitance change with increasing virus concentration was observed, and the absence of significant signal degradation demonstrates the robust performance of the ECB in fouling-prone environments.

[0119] Example 2. Label -free capacitive biosensor for rapid multiplex detection of avian (H5N1) influenza and E. coli in aerosols, including to prevent zoonotic outbreaks.

[0120] In this example, a label-free capacitive biosensor was fabricated using an interlocked Prussian blue (PB, i.e., iron(III) hexacyanoferrate(II)) / graphene oxide (GO) network on a screen-printed carbon electrode (SPCE) for direct detection of avian H5N1 virus and E. coli bacteria. Presented is a simple, one-step co-electrodeposition of PB and GO to construct an interlocked hybrid network on screen-printed carbon electrodes (SPCEs) and to fabricate an ultrasensitive and label-free ECB.

[0121] Fig. 7 depicts a schematic of the capacitive biosensor platform in accordance with the present disclosure for pathogen aerosol monitoring. (A) Pathogen-laden aerosols from infected animals are collected using a wet-cyclone particle sampler. (B) The biosensing platform detects the collected pathogens. (C) Fabrication highlights of PB / GO based capacitive biosensor. The PB / GO co-deposition forms a redox capacitive layer (Cr) onSPCE. Capture probes (aptamers / antibodies) immobilize via glutaraldehyde crosslinking. The double-layer capacitance (Cai) is established at the biosensor / media interface, while diffusion capacitance (Car) represents the diffuse layer. Target cell binding to capture probes is detected through total capacitance (Ctot) changes.

[0122] A single-step electro-co-deposition process grows GO branches on the SPCE surface, while the PB nano-crystals simultaneously decorate around the GO branches, forming an ultrasensitive capacitive response at nanofarad levels. A simplified equivalent circuit representing this mechanism includes three capacitors in series (Fig. 7, Panel C). The total capacitance, Ctot, of the entire capacitive biosensing system was calculated using the following equation:1

[0123] where Crrepresents the redox capacitance of the modified PB / GO layer. Cai corresponds to the double-layer capacitance at the equilibrated electrode / solution interface. Car illustrates the diffusion layer capacitance. Ctot is primarily determined by the smallest capacitance of the three capacitors. Therefore, when Crof the dielectric PB / GO layer is sufficiently large, Cai becomes very sensitive to changes due to the bio-binding occurrences.

[0124] This is the first known report of a PB / GO interlock composite-based ECB.

[0125] Characterization of capacitive biosensor fabrication.

[0126] Fig. 8 depicts characterization of a capacitive biosensor in accordance with the present disclosure. (A) EDX element analysis of the cross-section of the PB / GO codeposited SPCE. (B) Raman spectroscopy on sole-PB deposition, sole-GO deposition, and PB / GO co-deposition. (C) The electrochemical capacitive spectroscopy to compare the different deposition strategies. (D) The complex C* of aptamer-functionalized PB / GO capacitive biosensor for different concentrations of H5N1.

[0127] A cross-section EDX element analysis revealed that the PB / GO layer is approximately 5 to 10 pm thick (Fig. 8, Panel A). The hybrid capacitive layer was observed to contain a large quantity of PB surrounding the GO, primarily composed of Fe moleculesaround a graphene frame, with a small percentage of oxygen atoms. From the top view of the modified layer, crosslinked PB nanocrystals with diameters around 50 nm as the dominant dimension were identified, while GO branches protrude from the film, serving as anchors for the further immobilization of aptamers or antibodies (not shown). It was also found that the modified layer formed a porous structure with a high surface-to-volume ratio, enabling rapid electron transfer and reducing response time. Raman Spectroscopy (Fig. 8, Panel B) revealed two bands at 1350 cm’1and 1610 cm’1, corresponding to the presence of GO, and a vibrational band at 2140 cm’1, likely caused by the CN group of PB. The PB / GO hybrid layer displayed characteristic peaks from both components, confirming successful codeposition on the SPCEs. The electro-co-deposition of PB / GO on the SPCEs significantly enhanced the electrochemical capacitive performance compared to either component alone (Fig. 8, Panel C). The cyclic voltammetry (CV) spectra of sole-GO deposition showed no obvious redox peak, while sole-PB deposition produced a pair of redox peaks at 0.25 V / 0.15 V from FeniFen(CN)6 (not shown). When PB / GO was co-deposited, the redox peaks of PB shifted slightly to 0.4 V / 0.05 V, indicating a change in the electron transfer pathway from PB — GO — SPCE instead of directly from PB — SPCE. The ECS spectra, fitted to a simple Randel equivalent circuit (R2> 0.99) showed that Ctot of the PB / GO co-deposition was 13.9 x and 20.3 x higher than sole-PB and sole-GO deposition, respectively. As higher concentrations of H5N1 virus were captured on the biosensor, the complex C* consistently decreased in the spectra (Fig. 8, Panel D).

[0128] Performance of the capacitive biosensors.

[0129] Fig. 9 depicts the performance of the capacitive biosensor in accordance with the present disclosure for H5N1 and / / . coli. The sensitivity of the capacitive biosensors was evaluated for (A) H5N1 and (B) E. coli using serial dilutions of the targets in PBS. Both biosensors showed well-defined linear response (R2> 0.95) to the log concentration of their respective targets. The capacitive response was statistically significant (t-distribution, p < 0.005) indicating that the biosensors reacted solely to different doses of the targets. The specificity was also assessed for (C) H5N1 and (D) E. coli. The signals for both targets, at concentration just above their LoDs, were significantly larger than those of interferences even at high concentrations, demonstrating that non-specific pathogens did not affect the capacitive biosensors.

[0130] The operating parameters were optimized for PB / GO electro-co-deposition to achieve the best capacitive performance. The ratio of PB / GO used for co-deposition and the deposition duration was studied in term of CV cycles (not shown). After optimizing the capacitive biosensor system (1 mg / ml of GO / 20 mM of PB through 20 cycles of CV deposition), its performance was evaluated in detecting different pathogens (Fig. 9, Panels A and B). Standard dilutions of H5N1 and A. coli in 1 * PBS were tested using the developed capacitive biosensors functionalized with anti-H5Nl aptamer and anti -A. coli antibody, respectively. The results from triplicate samples for each data point were analyzed. The capacitance responses decreased with higher concentration.

[0131] The normalized capacitive changes (AC%) were calculated using the equation 4C% =Ctar9et Cncx 100%, where Garget is the Got measured after target binding, and CNCCNCis the Got in PBS. The AC% showed a strong linear relationship with the logarithmic concentration of H5N1 (R2= 0.98), and E. coli (R2= 0.95) within the detection range of 2.0 to 1.6 x 105viral copies / ml and 2.0 to 1.8 * 104CFU / ml, respectively. The limits of detection (LoDs) were calculated based on a signal -to-noise ratio of three (S / N = 3), resulting in LoDs of 56 viral copies / ml for H5N1 and 5 CFU / ml for A. coli. The lower LoD for A. coli likely results from the biosensor’s distinct response to different species due to variations in physical dimensions, surface polarizability from shell proteins, and dielectric properties. This suggests that the capacitive biosensor has the potential to fingerprint different targets of interest based on their unique capacitive behaviors.

[0132] The capacitive biosensors in this disclosure provided highly sensitive responses with low LoDs, requiring only about 5 minutes for detection through a simple, label-free, and direct detection procedure. Specificity is crucial for accurate pathogen identification in complex biological samples, reducing the risk of false-positive results. Both the H5N1 (Fig. 9, Panel C) and E. coli (Fig. 9, Panel D) biosensors exhibited excellent specificity, as the normalized AC% values of the two targets at low concentrations remained significantly larger than those of non-specific interferences, even at high concentrations. This result demonstrates that the specificity of the biosensor is primarily determined by the bioreceptors used for functionalization, with negligible non-specific impacts on the sensor response.

[0133] Quasi -quantification assessment of airborne H5N1 and A. coli.

[0134] Fig. 10 depicts a quasi-quantitation of H5N1 and E. coli aerosols by the capacitive biosensor in accordance with the present disclosure integrated with a wet-cyclone bioaerosol sampler. (A) The flow chart of the process for quasi-quantification and (B) The quasi -quantification to screen H5N1 and E. coli aerosols collected by the wet-cyclone air sampler.The capacitive biosensor screened subdiluted samples, with results displayed as positive (O ) or negative (X) based on normalized AC% relative to the LoDs of each target.

[0135] To demonstrate the practical application of the biosensor for air quality monitoring, it was integrated with a custom wet-cyclone air sampler to detect H5N1 and E. coli aerosols. Inactivated H5N1 virus and E. coli particles were aerosolized using a medical nebulizer and the aerosols were collected in a fume hood over a five-minute period. The custom-built cyclone sampler, previously validated for capturing SARS-CoV-2 aerosols in both laboratory and field settings, successfully captured the aerosols, and the electrochemical biosensor accurately detected and quantified the particles (Fig. 10).

[0136] The wet-cyclone air sampler is beneficial in part because of its high collection efficiency and low noise. In addition, because noise is measured in decibels (which is a logarithmic scale), arranging multiple air pumps in series in single wet-cyclone air sampler can boost airflow while produces a non-linear increase in noise. The degree of sampling is therefore achieving higher sampling rates without greatly impacting noise. This property makes the wet-cyclone design highly tunable, enabling researchers to balance sensitivity, throughput, and user comfort in a flexible and scalable manner.

[0137] While exact quantification of pathogens is highly desirable, achieving it can be challenging due to various factors affecting the biosensor’s calibration curve, which often requires extensive data. Instead, a “quasi-quantification” method was developed, similar to methods used in polymerase chain reaction (PCR) analyses, offering a reliable risk assessment of pathogen transmission in the air through a binary (positive / negative) output based on the biosensor's limit of detection. The wet-cyclone sampler is used as an example, but the same method can be applied to other particle-to-liquid sampling systems, such as exhaled breath condensate (EBC) devices or condensation growth samplers.

[0138] After collecting pathogen aerosols with the wet-cyclone sampler, the samples were sub-diluted into no dilution (original sample), 10x dilution, and 100* dilution. Thesub-diluted samples were then screened using the capacitive biosensor, displaying the detection results as positive (O) or negative (X) based on the normalized AC% relative to the LoD for each target (Fig. 10, Panel B). The concentration range was estimated for each target in the air and validated the results using digital PCR (dPCR).

[0139] Both H5N1 and E. coli concentration estimates aligned well with dPCR validation, except for H5N1 sample #5, where the normalized AC% of its “original sample” was only slightly above the LoD threshold (-0.903% vs. -0.896%). Despite this, the overall accuracy of the quasi-quantification for target aerosols exceeded 90% (n=l 1). The infectious dose of H5N1 among animals can be as low as 100 virus copies / mL, and since the biosensor’s LoD is 56 viral copies / mL in PBS (equivalent to 93 viral copies / m3of air), the infection risk can be confidently classified as low if all sub-dilutions test negative.

[0140] The E. coli aerosols were also used as a positive control to assess general air hygiene. E. coli becomes aerosolized from animal feces and can account for 2-6% of airborne bacteria in poultry houses, with concentrations ranging from dozens to millions of cells per cubic meter. The H5N1 and coli biosensors showed no interference between the two targets, confirming they can be used in parallel to examine the same sample in future applications. Since E. coli concentrations vary depending on barn type, animal size, and ventilation, multiple biosensing systems integrated with the capacitive biosensor and wetcyclone sampler can be deployed at different ventilation locations to provide comprehensive air quality monitoring. Dilutions can also be adjusted for quasi-quantification based on the specific location.

[0141] The platform’s flexibility allows integration with other particle-to-liquid samplers, such as those designed to capture human respiratory aerosols. For example, combining the biosensor with exhaled breath condensate (EBC) devices or condensation growth samplers enables real-time detection of pathogens like influenza or SARS-CoV-2 in exhaled breath. This setup offers a promising tool for non-invasive diagnostics and real-time health monitoring in healthcare settings, where tracking respiratory pathogen transmission is crucial.

[0142] Example 3. Hybrid Composite based Electrochemical Capacitive Biosensors (HC-ECBs).

[0143] Properties of biosensors in accordance with the present disclosure were analyzed by the above-described methods and then compared. The biosensors of these examples are electrochemical capacitive biosensors that utilize hybrid composites (PB / GO or PEDOT :PSS / rGO) and demonstrate improved capacitive properties. The detected targets are representative of all major types of pathogens (viruses, bacteria, allergens, etc.).

[0144] Figure 12 shows the capacitive property comparison with the addition of GO or rGO for a HC-ECBs in accordance with the present disclosure, namely: (A) The Ctot when different materials are electrodeposited on the electrode (first protocol). The hybrid composite of PB / GO showed more than 10-times higher capacitance compared to that of single redox material. (B) The hybrid composite of PEDOT:PSS / rGO showed improved capacitive value almost doubled that of a PEDOT:PSS-only film (second protocol).

[0145] In the first protocol, PB / GO was deposited via electrochemical co-deposition. PB / GO was thereby grown intertwined. GO provides anchoring sites for various receptors, such as aptamers, antibodies, genes, or other probes. This protocol resulted in a Prussian Blue (Fe2+Fe3+(CN)) / GO based capacitive biosensor.

[0146] In the second protocol, GO / PEDOT:PSS was deposited first via drop-casting, and then an electrochemical reduction was performed. This protocol resulted in a PEDOT:PSS / rGO based capacitive biosensor, which may be particularly useful to avoid biofouling and to analyze complex biological samples.

[0147] These capacitive composites are merely illustrative. The composites can contain a variety of capacitive materials (e.g., PPy, PANi, polyimide, metal-organic framework (MOF), metal oxides, metal nanoparticles, etc.) and GO replacements (e.g., carbon nanotubes, metal nanowires, metal oxides, etc.). They can also contain more than 2 elements.

[0148] In addition, the composites can be applied to a variety of electrode materials (e.g., gold, carbon, metal, indium tin oxide, etc.) in a variety of electrode layouts (e.g., rectangular, interdigitated, circular, multi-electrode array, etc.).

[0149] Figure 11 shows the performance of the capacitive composite biosensor to detect dust mite allergen Der fl. The LoD is calculated to be 0.012 ng / ml (S / N=3).

[0150] The biosensor can be used to detect a variety of targets of interest, including viruses (e.g., influenza A virus (IAV) (Hl, H3, H5, etc.), influenza B virus (IBV), respiratory syncytial virus (RSV), coronavirus disease (CoV), etc.), bacteria (e.g., E. coli, Pseudomonas, Staphylococcus, etc.), allergens (e.g., dust mite allergens), and antimicrobial resistant agents (e.g., genes, pathogens, etc.).

[0151] Figure 13 shows the performance of HC-ECBs for different pathogens at a fixed frequency (10 Hz). The Ctot was evaluated at different frequencies and was determined to depict the largest resolution at 10 Hz, which is in a low frequency range that primarily shows the behavior of the double layer capacitance. The capacitive biosensor still shows strong correlation between the normalized AC% and the log concentration of H5N1 (R2> 0.92), E. coli (R2> 0.99) and Der fl (R2>0.92). The LoDs were calculated for H5N1 (16 RNA copies / ml), E. coli (5 bacterial cells / ml) and Der fl (0.99 ng / ml), which are close to that obtained from the whole frequency range ECS analysis (H5N1 at 56 RNA copies / ml and E. coli at 4 bacterial cells / ml, respectively).

[0152] It is demonstrated that the biosensors in accordance with the present disclosure are capable of single-frequency capacitance measurements. Without being bound to any particular theory, it is believed that such measurements in the low frequency range represent the double-layer capacitance.

[0153] The performance analysis (sensitivity, LoD, R2) shows close values to the whole frequency range scan. It is therefore feasible to use single-frequency measurements of the biosensors for the detection of targets. Single-frequency measurements have significant value for multiplex biosensors, for commercial prototypes and for in-field applications.

[0154] The current disclosure relates to the development of an ECB for rapid (~5 min detection time) and sensitive detection of respiratory pathogens, including but not limited to, influenza strains, namely H5N1 and H1N1. A reduced graphene oxide / PEDOT:PSS (PP / rGO) nanocomposite-modified electrode was integrated with electrochemical capacitance spectroscopy (ECS) for real-time pathogen detection. The presence ofPEDOT:PSS minimizes biofouling and enables signal stability even in complex and contaminated environments. This anti-fouling property is critical for achieving reliable, realtime pathogen detection in challenging settings such as poultry farm environments and clinical biological fluids, addressing a significant research gap in biosensor performance under interference-prone conditions.

[0155] The results with H5N1 and H1N1 detection indicate that the ECB is highly sensitive and specific, with detection limits of 39 copies / mL for H5N1 and 10 copies / mL for H1N1 respectively. The ECB is extensively validated in complex matrices, including simulated human saliva and nasal fluids spiked with H1N1, and contaminated real -world poultry farm samples spiked with H5N1 — confirming its robust stability and effective resistance to interference in complex / dirty environments. Overall, the system provides an affordable, real-time diagnostic solution with excellent performance in diverse environments, offering significant advantages over conventional biosensing systems.

[0156] In addition, the label-free capacitive biosensor in accordance with the present disclosure leverages the dielectric properties of PB and GO to form an interlocked nanonetwork on the surface of SPCEs. This PB / GO layer greatly enhanced the sensor’ s capacitive properties, leading to significant improvements in detection performance. The biosensor demonstrated impressive versatility, targeting a variety of pathogens with wide detection ranges and excellent linear responses to concentrations of H5N1 and E. coli. With low detection limits achieved in under 5 minutes and high specificity, the biosensor performed exceptionally well even in complex samples.

[0157] When paired with a wet-cyclone bioaerosol sampler, the biosensor provided an effective platform for airborne pathogen detection, using the “quasi-quantification” method to offer reliable risk assessments. The biosensor is highly adaptable, allowing easy functionalization for the detection of additional pathogens. Its single-step electrochemical co-deposition process also ensures cost-effective mass production. Its potential has been demonstrated for multiplex pathogen detection, including its application for H1N1 detection. When integrated with aerosol samplers and automated sample delivery systems, this platform holds great promise for near real-time surveillance of multiple airborne pathogens.

[0158] Embodiments.

[0159] Further aspects of the invention are provided by the embodiments of the following clauses. These clauses may be combined in any permutation or combination.

[0160] 1. An electrochemical capacitive biosensor (ECB), comprising:an electrode; anda capacitive composite disposed on the electrode;wherein the capacitive composite comprises:a capacitive material; anda dielectric matrix; andwherein the ECB is configured for electrochemical capacitance spectroscopy.

[0161] 2. The ECB of embodiment 1, wherein the electrode is selected from the group consisting of carbon electrodes, carbon paste electrodes, graphite electrodes, glassy carbon electrodes, carbon nanotube electrodes, graphene electrodes, graphene oxide electrodes, screen-printed carbon electrodes, metal electrodes, platinum electrodes, gold electrodes, silver electrodes, copper electrodes, nickel electrodes, stainless steel electrodes, metal oxide electrodes, indium tin oxide (ITO) electrodes, TiO2electrodes, SnO2electrodes, RUO2electrodes, MnO2electrodes, conductive polymer electrodes, PEDOT:PSS electrodes, polyaniline electrodes, polypyrrole electrodes, and combinations thereof.

[0162] 3. The ECB of embodiment 1, wherein the dielectric matrix is selected from the group consisting of graphene oxide (GO), carbon nanotubes, metal nanowires, metal oxides, nanocellulose, hexagonal boron nitride, transition metal dichalcogenides, polymer dielectrics (polyvinyl alcohol, polyethylene glycol, Nafion, zwitterionic polymers, and combinations thereof.

[0163] 4. The ECB of embodiment 1, wherein the capacitive material comprises a conducting metal salt selected from the group consisting of iron(III) hexacyanoferrate(II), iron(III) hexacyanoferrate(II), Prussian Blue (PB) analogues (e.g., cobalt hexacyanoferrate, nickel hexacyanoferrate, manganese hexacyanoferrate, copper hexacyanoferrate, zinc hexacyanoferrate, iron hexacyanomanganate, vanadium hexacyanoferrate, mixed-metal PBanalogues, etc.), transition metal oxides (e.g., MnCh, RuCh, TiCh, NiO, CO3O4, Fe20s, FesO4, ZnO, V2O5, WO3, MoOs, CuO, Q12O, etc.), conductive metal-organic frameworks (e.g., MIL-100(Fe), MIL-lOl(Cr), MIL-53(A1), ZIF-8 (Zn-based), ZIF-67 (Co-based), HKUST-1 (CU3(BTC)2), UiO-66 (Zr-based), MOF-74 (Mg, Ni, Co variants), Co-MOF-74, Ni-MOF-74 (conductive variants), Cu3(HHTP)2 (2D conductive MOF), Fe-TCPP MOF (porphyrinbased, redox-active), MOF-derived TMOs (e.g., pyrolyzed MOFs forming porous CO3O4 or Fe20s), etc.), and combinations thereof.

[0164] 5. The ECB of embodiment 1, wherein the capacitive material comprises a conducting polymer selected from the group consisting of poly(3,4 ethylenedioxythiophene):poly-styrene-sulfonic acid (PEDOT:PSS), polyaniline (PANI), polypyrrole (Ppy), polyimide, poly(thiophene), poly(3 -hexylthiophene), poly(phenylene vinylene), poly(acetylene), poly(fluorene) derivatives, poly(benzimidazole), poly(anthracene), poly (3, 4-porpylenedi oxythiophene), poly(3 -thiopheneacetic acid), poly(ethyleneimine), zwitterionic polymers (PEDOT-sulfobetaine, PEDOT-carboxybetaine, PEDOT-phosphorylcholine, PPy-sulfobetaine, PPy-phosphorylcholine, Poly(sulfobetaine methacrylate) hybrids, poly(carboxybetaine methacrylate) hybrids, zwitterionic poly(thiophene) derivatives), and combinations thereof.

[0165] 6. The ECB of embodiment 1, wherein the capacitive material comprises a surface area enhancer selected from the group consisting of nanocomposites, graphene oxide, metal organic frameworks (MOFs), metal nanoparticles, metal oxides, gold nanoparticles (AuNP), nickel nanoparticles (NiNP), titanium / cerium oxide (TiO2 / CeO2), and combinations thereof.

[0166] 7. The ECB of embodiment 1, wherein the capacitive material is in the form of a film.

[0167] 8. The ECB of embodiment 1, wherein the capacitive material has antifouling properties.

[0168] 9. The ECB of embodiment 1, wherein the ECB does not comprise a chemical label or a biochemical label.

[0169] 10. The ECB of embodiment 1, wherein the ECB further comprises a chemical label or a biochemical label selected from the group consisting of aptamers, antibodies, nanobodies, enzymes, oligonucleotides, peptides, cells, tissues, organelles, lectins, molecularly imprinted polymers, and combinations thereof.

[0170] 11. The ECB of embodiment 1, wherein the ECB is configured for detection of a target selected from the group consisting of viruses, bacteria, allergens, fungi, parasites, biomarkers (e.g., inflammatory, cardiac, cancer, coagulation, enzymatic, etc.), nucleic acid (e.g., DNA or RNA, etc.), small molecules and metabolites (e.g., glucose, lactate, urea, creatinine, neurotransmitters, hormones, drugs, etc.), pollutants (e.g., heavy metals, toxins, pesticides, herbicides, etc.), whole cells (e.g., tumor cells, immune cells, exosomes, extracellular vesicles, etc.), antimicrobial resistant (AMR) agents (e.g., AMR genes, AMR pathogens, etc.), and combinations thereof.

[0171] 12. The ECB of embodiment 1, wherein the ECB comprises a limit of detection of less than 50 copies / mL or less than 10 cells / mL.

[0172] 13. A method of making an electrochemical capacitive biosensor (ECB), comprising:an electrode; anda capacitive composite disposed on the electrode;wherein the capacitive composite comprises:a capacitive material; anda dielectric matrix; andwherein the ECB is configured for electrochemical capacitance spectroscopy,the method comprising:disposing the capacitive composite on the electrode.

[0173] 14. The method of embodiment 13, wherein the disposing comprises a technique selected from the group consisting of drop-casting, spin-coating, spray-coating,dip-coating, electrodeposition, electrograft, electropolymerization, screen-printing, inkjet printing, 3D printing, electrophoretic deposition, layer-by-layer assembly, vacuum and physical deposition, self-assembly, sol-gel deposition, and combinations thereof.

[0174] 15. The method of embodiment 13, further comprising disposing on the capacitive composite a chemical label or a biochemical label selected from the group consisting of aptamers, antibodies, nanobodies, enzymes, oligonucleotides, peptides, cells, tissues, organelles, lectins, molecularly imprinted polymers, and combinations thereof.

[0175] 16. A method of using an electrochemical capacitive biosensor (ECB), comprising:an electrode; anda capacitive composite disposed on the electrode;wherein the capacitive composite comprises:a capacitive material; anda dielectric matrix; andwherein the ECB is configured for electrochemical capacitance spectroscopy,the method comprising:exposing the ECB to a sample; andanalyzing the sample with the ECB using electrochemical capacitance spectroscopy to detect a target.

[0176] 17. The method of embodiment 16, wherein the target is selected from the group consisting of viruses, bacteria, allergens, fungi, parasites, biomarkers (e.g., inflammatory, cardiac, cancer, coagulation, enzymatic, etc.), nucleic acid (e.g., DNA or RNA, etc.), small molecules and metabolites (e.g., glucose, lactate, urea, creatinine, neurotransmitters, hormones, drugs, etc.), pollutants (e.g., heavy metals, toxins, pesticides, herbicides, etc.), whole cells (e.g., tumor cells, immune cells, exosomes, extracellularvesicles, etc.), antimicrobial resistant (AMR) agents (e.g., AMR genes, AMR pathogens, etc.), and combinations thereof.

[0177] 18. The method of embodiment 16, wherein the sample is selected from the group consisting of bodily fluids, saliva, nasal fluid, blood, poultry farm samples, air samples, breath samples, wastewater, environmental samples, and combinations thereof.

[0178] 19. The method of embodiment 16, wherein the exposing the ECB to a sample is achieved via a sampler selected from the group consisting of particle-to-liquid sampling systems, wet-cyclone particle samplers, exhaled breath condensate (EBC) devices, condensation growth samplers, clinical and diagnostic sampling (e.g., swabs, sputum, blood / serum / plasma, urine, etc.), filter membranes, precipitators, surface swabs / wipes, microfluidic devices, lab-on-a-chip devices, sweat patches, tear fluid sampling, wearable sampler, breath masks / filters, and combinations thereof.

[0179] 20. The method of embodiment 16, wherein the ECB further comprises a chemical label or a biochemical label selected from the group consisting of enzymes (e.g., horseradish peroxidase, alkaline phosphatase, glucose oxidase, urease, etc.), redox-active labels (e.g., ferrocene and derivatives, methylene blue, anthraquinone, etc.), nanoparticles (e.g., gold, silver, magnetic, etc.), quantum dots (e.g., CdSe, CdTe, ZnS, etc.), polystyrene beads, polymer / conductive agents, and combinations thereof.

[0180] 21. An electrochemical capacitive biosensor (ECB), comprising:an electrode; anda conducting polymer matrix disposed on the electrode;wherein the ECB is configured for electrochemical capacitance spectroscopy.

[0181] 22. The ECB of embodiment 21, wherein the electrode is selected from the group consisting of carbon electrodes (e.g., carbon paste, graphite, glassy carbon, carbon nanotubes, graphene, graphene oxide, screen printed carbon, etc.), metal electrodes (e.g., platinum, gold, silver, copper, nickel, stainless steel, etc.) , metal oxide electrodes (e.g., indium tin oxide (ITO), TiO?, SnO2, RuO?, MnO?, etc.), conductive polymer electrodes (e.g., PEDOT:PSS, polyaniline, polypyrrole, etc.), and combinations thereof.

[0182] 23. The ECB of embodiment 21, wherein the conducting polymer matrix is in the form of a film.

[0183] 24. The ECB of embodiment 21, wherein the conducting polymer matrix has anti-fouling properties.

[0184] 25. The ECB of embodiment 21, wherein the conducting polymer matrix comprises a conducting polymer and a surface area enhancer.

[0185] 26. The ECB of embodiment 25, wherein the conducting polymer is selected from the group consisting of poly(3,4 ethylenedioxythiophene):poly-styrene-sulfonic acid (PEDOT:PSS), polyaniline (PANI), polypyrrole (Ppy), zwitterionic polymers (PEDOT-sulfobetaine, PEDOT-carboxybetaine, PEDOT-phosphorylcholine, PPy-sulfobetaine, PPy-phosphorylcholine, poly(sulfobetaine methacrylate) hybrids, poly(carboxybetaine methacrylate) hybrids, zwitterionic poly(thiophene) derivatives), and combinations thereof.

[0186] 27. The ECB of embodiment 25, wherein the surface area enhancer is selected from the group consisting of nanocomposites, graphene oxide, metal organic frameworks (MOFs), metal nanoparticles, metal oxides, gold nanoparticles (AuNP), nickel nanoparticles (NiNP), titanium / cerium oxide (TiO2 / CeO2), and combinations thereof.

[0187] 28. The ECB of embodiment 21, wherein the ECB does not comprise a chemical label or a biochemical label.

[0188] 29. The ECB of embodiment 21, wherein the ECB further comprises a chemical label or a biochemical label selected from the group consisting of aptamers, antibodies, nanobodies, enzymes, oligonucleotides, peptides, cells, tissues, organelles, lectins, molecularly imprinted polymers, and combinations thereof.

[0189] 30. The ECB of embodiment 21, wherein the ECB is configured for detection of respiratory pathogens.

[0190] 31. The ECB of embodiment 30, wherein the respiratory pathogens are selected from strains of viruses (Influenza A strains (H1N1, H3N2, H5N1, H5N2, H7N2, H7N9), Influenza B strains (Yamagata, Victoria lineages), Coronavirus stains (229E, NL63, OC43, HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2), Paramyxoviridae (respiratorysyncytial virus (RSV), Human Metapneumovirus (hMPV), Parainfluenza viruses (HPIV)), Picornaviridae (rhinoviruses, enteroviruses), Adenoviridae, Parvoviridae, Measles, Varicella-zoster virus, Cytomegalovirus), bacteria (Streptococus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Moraxella catarrhalis, Neisseria meningitidis, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Legionella pneumophila, Bordetella pertussis, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Chlamydophila psittaci, Mycobacterium tuberculosis, Non-tuberculous mycobacteria, Corynebacterium diphtheriae, Bacillus anthracis, Nocardia species), fungi (Aspergillus fumigatus, Histoplasma capsulatum, Coccidioides immitis / posadasii, Blastomyces dermatitidis, Cryptococcus neoformans, Pneumocystis jirovecii), parasites (Strongyloides stercoralis, Paragonimus westermani, Toxoplasma gondii, Echinococcus granulosus), other strains identified now or in the future, and combinations thereof.

[0191] 32. The ECB of embodiment 21, wherein the ECB comprises a limit of detection of less than 50 copies / mL or less than 10 cells / mL.

[0192] 33. A method of making an electrochemical capacitive biosensor (ECB), comprising:an electrode; anda conducting polymer matrix disposed on the electrode;wherein the ECB is configured for electrochemical capacitance spectroscopy,the method comprising:disposing the conducting polymer matrix on the electrode.

[0193] 34. The method of embodiment 33, wherein the disposing comprises a technique selected from the group consisting of drop-casting, spin-coating, spray-coating, dip-coating, electrodeposition, electrograft, electropolymerization, screen-printing, inkjet printing, 3D printing, electrophoretic deposition, layer-by-layer assembly, vacuum and physical deposition, self-assembly, sol-gel deposition, and combinations thereof.

[0194] 35. The method of embodiment 33, further comprising disposing on the conducting polymer matrix a chemical label or a biochemical label selected from the group consisting of aptamers, antibodies, nanobodies, enzymes, oligonucleotides, peptides, cells, tissues, organelles, lectins, molecularly imprinted polymers, and combinations thereof.

[0195] 36. A method of using an electrochemical capacitive biosensor (ECB), comprising:an electrode; anda conducting polymer matrix disposed on the electrode;the method comprising:exposing the ECB to a sample; andanalyzing the sample with the ECB using electrochemical capacitance spectroscopy to detect respiratory pathogens.

[0196] 37. The method of embodiment 36, wherein the respiratory pathogens are selected from strains of viruses (Influenza A strains (H1N1, H3N2, H5N1, H5N2, H7N2, H7N9), Influenza B strains (Yamagata, Victoria lineages), Coronavirus stains (229E, NL63, OC43, HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2), Paramyxoviridae (respiratory syncytial virus (RSV), Human Metapneumovirus (hMPV), Parainfluenza viruses (HPIV)), Picornaviridae (rhinoviruses, enteroviruses), Adenoviridae, Parvoviridae, Measles, Varicella-zoster virus, Cytomegalovirus), bacteria (Streptococus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Moraxella catarrhalis, Neisseria meningitidis, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Legionella pneumophila, Bordetella pertussis, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Chlamydophila psittaci, Mycobacterium tuberculosis, Non-tuberculous mycobacteria, Corynebacterium diphtheriae, Bacillus anthracis, Nocardia species), fungi (Aspergillus fumigatus, Histoplasma capsulatum, Coccidioides immitis / posadasii, Blastomyces dermatitidis, Cryptococcus neoformans, Pneumocystis jirovecii), parasites (Strongyloides stercoralis, Paragonimus westermani, Toxoplasma gondii, Echinococcus granulosus), other strains identified now or in the future, and combinations thereof.

[0197] 38. The method of embodiment 36, wherein the sample is selected from the group consisting of bodily fluids, saliva, nasal fluid, blood, poultry farm samples, air samples, breath samples, wastewater, environmental samples, and combinations thereof.

[0198] 39. The method of embodiment 36, wherein the ECB does not comprise a chemical label or a biochemical label.

[0199] 40. The method of embodiment 36, wherein the ECB further comprises a chemical label or a biochemical label selected from the group consisting of aptamers, antibodies, nanobodies, enzymes, oligonucleotides, peptides, cells, tissues, organelles, lectins, molecularly imprinted polymers, and combinations thereof.

[0200] 41. A biosensor comprising:Prussian blue (PB) nano-crystals;graphene oxide (GO); anda screen-printed carbon electrode (SPCE).

[0201] 42. The biosensor of embodiment 41, wherein the biosensor is label-free.

[0202] 43. A method of detecting airborne pathogens, the method comprising:exposing a biosensor to an air sample, wherein the biosensor is a label-free biosensor comprising:Prussian blue (PB) nano-crystals;graphene oxide (GO); anda screen-printed carbon electrode (SPCE); anddetecting a level of at least one airborne pathogen (or 10 RNA copies) in the air sample.

[0203] 44. The method of embodiment 43, wherein the at least one airborne pathogen comprises at least one of bacteria and viruses.

[0204] 45. The method of embodiment 44, wherein the at least one airborne pathogen comprises E. coli.

[0205] 46. The method of embodiment 44, wherein the at least one airborne pathogen comprises influenza virus.

[0206] 47. The method of embodiment 46, wherein the influenza virus comprises at least one ofH5Nl and H INI.

[0207] 48. A system for detecting airborne pathogens, the system comprising:a label-free biosensor comprising:Prussian blue (PB) nano-crystals;graphene oxide (GO); anda screen-printed carbon electrode (SPCE); anda wet-cyclone bioaerosol sampler.

[0208] Definitions.

[0209] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0210] Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0211] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. In some embodiments, the term “about” means plus or minus 10% of the value.

[0212] Unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). As used herein, “and / or” refers to andencompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0213] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any limitation explicitly indicated. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process or method.

[0214] The transitional phrase “consisting of’ excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0215] The transitional phrase “consisting essentially of’ is used to define a composition or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0216] Where an invention or a portion thereof is defined with an open-ended term such as “comprising,” it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms “consisting essentially of’ or “consisting of.”

[0217] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0218] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0219] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0220] As used herein, references to “example embodiment” or “one embodiment” or “some embodiments” of the present disclosure are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features.

[0221] Unless otherwise indicated, approximating language, such as “generally,” “substantially,” and “about,” as used herein indicates that the term so modified may apply to only an approximate degree, as would be recognized by one of ordinary skill in the art, rather than to an absolute or perfect degree. Accordingly, a value modified by a term or terms such as “about,” “approximately,” and “substantially” is not to be limited to the precise value specified. In at least some instances, the approximating language may correspond to the precision of an instrument for measuring the value. Additionally, unless otherwise indicated, the terms “first,” “second,” etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to, for example, a “second” item does not require or preclude the existence of, for example, a “first” or lower-numbered item or a “third” or higher-numbered item.

[0222] Although specific features of various embodiments of the invention may be shown in some drawings and not in others, this is for convenience only. In accordance with the principles of the invention, any feature of a drawing may be referenced and / or claimed in combination with any feature of any other drawing.

[0223] While the invention has been described in terms of various specific embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the claims.

[0224] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Claims

WHAT IS CLAIMED IS:

1. An electrochemical capacitive biosensor (ECB), comprising:an electrode; anda capacitive composite disposed on the electrode;wherein the capacitive composite comprises:a capacitive material; anda dielectric matrix; andwherein the ECB is configured for electrochemical capacitance spectroscopy.

2. The ECB of claim 1, wherein the electrode is selected from the group consisting of carbon electrodes, carbon paste electrodes, graphite electrodes, glassy carbon electrodes, carbon nanotube electrodes, graphene electrodes, graphene oxide electrodes, screen-printed carbon electrodes, metal electrodes, platinum electrodes, gold electrodes, silver electrodes, copper electrodes, nickel electrodes, stainless steel electrodes, metal oxide electrodes, indium tin oxide (ITO) electrodes, TiCh electrodes, SnO? electrodes, RuCh electrodes, MnCh electrodes, conductive polymer electrodes, PEDOT:PSS electrodes, polyaniline electrodes, polypyrrole electrodes, and combinations thereof.

3. The ECB of claim 1, wherein the dielectric matrix is selected from the group consisting of graphene oxide (GO), carbon nanotubes, metal nanowires, metal oxides, nanocellulose, hexagonal boron nitride, transition metal dichalcogenides, polymer dielectrics (polyvinyl alcohol, polyethylene glycol, Nafion, zwitterionic polymers, and combinations thereof.

4. The ECB of claim 1, wherein the capacitive material comprises a conducting metal salt selected from the group consisting of iron(III) hexacyanoferrate(II), iron(III) hexacyanoferrate(II), Prussian Blue (PB) analogues (e.g., cobalt hexacyanoferrate, nickel hexacyanoferrate, manganese hexacyanoferrate, copper hexacyanoferrate, zinc hexacyanoferrate, iron hexacyanomanganate, vanadium hexacyanoferrate,mixed-metal PB analogues, etc.), transition metal oxides (e.g., MnCh, RuCh, TiCh, NiO, CO3O4, Fe2C>3, FesO4, ZnO, V2O5, WO3, MoOs, CuO, C O, etc ), conductive metal-organic frameworks (e.g., MIL-lOO(Fe), MIL-lOl(Cr), MIL-53(A1), ZIF-8 (Zn-based), ZIF-67 (Co-based), HKUST-1 (Cu3(BTC)2), UiO-66 (Zr-based), MOF- 74 (Mg, Ni, Co variants), Co-MOF-74, Ni-MOF-74 (conductive variants), CU3(HHTP)2 (2D conductive MOF), Fe-TCPPMOF (porphyrin-based, redox-active), MOF-derived TMOs (e.g., pyrolyzed MOFs forming porous CO3O4 or Fe20s), etc.), and combinations thereof.

5. The ECB of claim 1 , wherein the capacitive material comprises a conducting polymer selected from the group consisting of poly(3,4 ethylenedioxythiophene):poly- styrene-sulfonic acid (PEDOT:PSS), polyaniline (PANI), polypyrrole (Ppy), polyimide, poly(thiophene), poly(3 -hexylthiophene), poly(phenylene vinylene), poly(acetylene), poly(fluorene) derivatives, poly(benzimidazole), poly(anthracene), poly(3,4-porpylenedioxythiophene), poly (3 -thiopheneacetic acid), poly(ethyleneimine), zwitterionic polymers (PEDOT-sulfobetaine, PEDOT- carboxybetaine, PEDOT-phosphorylcholine, PPy-sulfobetaine, PPy- phosphorylcholine, Poly(sulfobetaine methacrylate) hybrids, poly(carboxybetaine methacrylate) hybrids, zwitterionic poly(thiophene) derivatives), and combinations thereof.

6. The ECB of claim 1, wherein the capacitive material comprises a surface area enhancer selected from the group consisting of nanocomposites, graphene oxide, metal organic frameworks (MOFs), metal nanoparticles, metal oxides, gold nanoparticles (AuNP), nickel nanoparticles (NiNP), titanium / cerium oxide (TiO2 / CeO2), and combinations thereof.

7. The ECB of claim 1, wherein the capacitive material is in the form of a film.

8. The ECB of claim 1, wherein the capacitive material has anti-fouling properties.

9. The ECB of claim 1, wherein the ECB does not comprise a chemical label or a biochemical label.

10. The ECB of claim 1, wherein the ECB further comprises a chemical label or a biochemical label selected from the group consisting of aptamers, antibodies, nanobodies, enzymes, oligonucleotides, peptides, cells, tissues, organelles, lectins, molecularly imprinted polymers and combinations thereof.

11. The ECB of claim 1, wherein the ECB is configured for detection of a target selected from the group consisting of viruses, bacteria, allergens, fungi, parasites, biomarkers, nucleic acid, small molecules and metabolites, pollutants, whole cells, antimicrobial resistant (AMR) agents, and combinations thereof.

12. The ECB of claim 1, wherein the ECB comprises a limit of detection of less than 50 copies / mL or less than 10 cells / mL.

13. A method of making an electrochemical capacitive biosensor (ECB), comprising:an electrode; anda capacitive composite disposed on the electrode;wherein the capacitive composite comprises:a capacitive material; anda dielectric matrix; andwherein the ECB is configured for electrochemical capacitance spectroscopy,the method comprising:disposing the capacitive composite on the electrode.

14. The method of claim 13, wherein the disposing comprises a technique selected from the group consisting of drop-casting, spin-coating, spray-coating, dip-coating, electrodeposition, electrograft, electropolymerization, screen-printing, inkjet printing, 3D printing, electrophoretic deposition, layer-by-layer assembly, vacuum and physical deposition, self-assembly, sol-gel deposition, and combinations thereof.

15. The method of claim 13, further comprising disposing on the capacitive composite a chemical label or a biochemical label selected from the group consisting of aptamers, antibodies, nanobodies, enzymes, oligonucleotides, peptides, cells, tissues, organelles, lectins, molecularly imprinted polymers, and combinations thereof.

16. A method of using an electrochemical capacitive biosensor (ECB), comprising:an electrode; anda capacitive composite disposed on the electrode;wherein the capacitive composite comprises:a capacitive material; anda dielectric matrix; andwherein the ECB is configured for electrochemical capacitance spectroscopy,the method comprising:exposing the ECB to a sample; andanalyzing the sample with the ECB using electrochemical capacitance spectroscopy to detect a target.

17. The method of claim 16, wherein the target is selected from the group consisting of viruses, bacteria, allergens, fungi, parasites, biomarkers, nucleic acid, small molecules and metabolites, pollutants, whole cells, antimicrobial resistant (AMR) agents, and combinations thereof.

18. The method of claim 16, wherein the sample is selected from the group consisting of bodily fluids, saliva, nasal fluid, blood, poultry farm samples, air samples, breath samples, wastewater, environmental samples, and combinations thereof.

19. The method of claim 16, wherein the exposing the ECB to a sample is achieved via a sampler selected from the group consisting of particle-to-liquid sampling systems, wet-cyclone particle samplers, exhaled breath condensate (EBC) devices,condensation growth samplers, clinical and diagnostic sampling, filter membranes, precipitators, surface swabs / wipes, microfluidic devices, lab-on-a-chip devices, sweat patches, tear fluid sampling, wearable sampler, breath masks / filters, and combinations thereof.

20. The method of claim 16, wherein the ECB further comprises a chemical label or a biochemical label selected from the group consisting of aptamers, antibodies, nanobodies, enzymes, oligonucleotides, peptides, cells, tissues, organelles, lectins, molecularly imprinted polymers, and combinations thereof.

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