Wide Detection Research Articles

Portable Saliva Sensor Based on Dual Recognition Elements for Detection of Caries Pathogenic Bacteria.

Dental caries is one of the most common diseases affecting more than 2 billion people's health worldwide. In a clinical setting, it is challenging to predict and proactively guard against dental cavities prior to receiving a confirmed diagnosis. Streptococcus mutans (S. mutans) in saliva has been recognized as the main causative bacterial agent that causes dental caries. High sensitivity, good selectivity, and a wide detection range are incredibly important factors to affect S. mutans detection in practical applications. In this study, we present a portable saliva biosensor designed for the early detection of S. mutans with the potential to predict the occurrence of dental cavities. The biosensor was fabricated using a S. mutans-specific DNA aptamer and S. mutans-imprinted polymers. Methylene blue was utilized as a redox probe in the sensor to generate current signals for analysis. When S. mutans enters complementarily S. mutans cavities, it blocks electron transfer between methylene blue and the electrode, resulting in decreases in the reduction current signal. The signal variations are associated with S. mutans concentrations that are useful for quantitative analysis. The linear detection range of S. mutans is 102-109 cfu mL-1, which covers the critical concentration of high caries risk. The biosensor exhibited excellent selectivity toward S. mutans in the presence of other common oral bacteria. The biosensor's wide detection range, excellent selectivity, and low limit of detection (2.6 cfu mL-1) are attributed to the synergistic effect of aptamer and S. mutans-imprinted polymers. The sensor demonstrates the potential to prevent dental caries.

Multiwalled carbon nanotubes supported Fe nanostructured interfaces for electrochemical detection of uric acid

Uric acid (UA) levels in biological fluids are an important diagnostic and prognostic biomarker for various metabolic disorders. Therefore, a convenient, facile, one-step electroreduction method to develop Fe nanostructured (FeNS) interfaces for highly sensitive electrochemical detection of UA. The synthesis process was thoroughly investigated at various deposition potentials, pH of the electrochemical bath solutions, and growth durations to attain excellent electrochemical response towards UA detection. The morphology and elemental composition of the FeNS/MWCNT were analyzed using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDXS) techniques. We observed uniform deposition of FeNSs with sizes ranging from 100 − 120 nm on tubular nanostructures. The multiwalled carbon nanotubes supported Fe nanostructured interfaces (FeNS/MWCNT) generated at a potential of –1.1 V demonstrated excellent electrochemical response towards UA with a wide linear detection range of 5 to 500 µM with a sensitivity of 6.5605 µA µM−1 cm−2 and low detection limit of 3.26 µM. The excellent electrochemical response was attributed to the synergistic effect of MWCNTs, which facilitated FeNSs growth, leading to increased active sites for electrocatalysis and sensitive detection of UA. The convenience of the synthesis process, tunable electrochemical response, and reliability of the detection process make FeNS interfaces a potential option for developing electrochemical UA sensors.

Enhanced Label-Free Photoelectrochemical Strategy for Pollutant Detection: Using Surface Oxygen Vacancies-Enriched BiVO4 Photoanode.

Label-free photoelectrochemical sensors have the advantages of high sensitivity and a simple electrode structure. However, its performance is greatly limited due to the photoactive materials' weak photoactivity and poor stability. Herein, a robust homogeneous photoelectrochemical (PEC) aptasensor has been constructed for atrazine (ATZ) based on photoetching (PE) surface oxygen vacancies (Ov)-enriched Bismuth vanadate (BiVO4) (PE-BVO). The surface of the Ov improves the carrier separation ability of BiVO4, thus providing a superior signal substrate for the sensor. A thiol molecular layer self-assembled on PE-BVO acts as a blocker, while 2D graphene acts as a signal-on probe after release from the aptamer-graphene complex. The fabricated sensor has a wide linear detection range of 0.5 pM to 10.0 nM and a low detection limit of 0.34 pM (S/N = 3) for ATZ. In addition, it can efficiently work in a wide pH range (3-13) and high ionic strength (∼6 M Na+), which provides promising opportunities for detecting environmental pollutants under complex conditions.

A self-heating gas sensor for online monitoring of endogenous ethylene of post-harvest cut chrysanthemums

The decay of post-harvest cut chrysanthemums (PHCCs) during storage and shipment results in enormous loss, and the accumulated endogenous ethylene is one of the main reasons. However, it is challenging for current technologies to onsite monitoring of endogenous ethylene of PHCCs whose concentration is very low. Here, we propose a ethylene sensor with high performances enabled by self-heating capability. Specifically, laser-induced graphene (LIG) patterns are in-situ fabricated on the upper and lower sides of a polyimide substrate. The upper LIG is coated with chemo-resistive SnO2 for ethylene sensing and the lower LIG serves as heater for self-heating. The working temperature is well tuned by the heater and effectively transferred to SnO2. Owing to the self-heating capability, the sensor displays outstanding ethylene detection performances, such as ultra-low limit of detection (1.654 ppb), wide detection range (0.05–100 ppm), high sensitivity (0.2249 ppm−1), high detection linearity (0.9925), and long-term stability (>14 days). Moreover, the sensor is integrated with a data acquisition circuit and connected to a mobile application, realizing online monitoring of endogenous ethylene of PHCCs. The results demonstrate that the decay of PHCCs is accelerated as the endogenous ethylene accumulates. This work would promote the online monitoring of endogenous ethylene of plants.

Electrochemical sensor based on reduced graphene oxide paste electrode for detection of gemcitabine as a chemotherapy drug in breast cancer

The development of a precise and sensitive electrochemical sensor is essential in the field of medical diagnostics in order to identify gemcitabine, a chemotherapy drug that is commonly used to treat breast cancer. This work presents a novel electrochemical sensor based on a reduced graphene oxide (rGO) nanocomposite and a zirconium-based metal-organic framework (Zr-MOF) with terephthalic acid as a ligand. Gemcitabine (GEM), a chemotherapy medication used to treat breast cancer, is detected with remarkable sensitivity and selectivity by the Zr-MOF-rGO modified graphene paste electrode (GrPE) sensor. By combining the strong structure and active sites of Zr-MOF with the large surface area and electrical conductivity of rGO, the sensor's design maximizes electron transfer rates and GEM adsorption capacity. The sensor outperforms current GEM sensors with its wide linear detection range (0.5–490 μM) and low detection limit (0.008 μM). The sensor's long-term analytical applicability is confirmed by stability and reusability studies, which produced accuracy rates surpassing 94 % and RSD values below 4.21 % in recovery trials using spiked human blood serum and urine samples. Overall, the results of this investigation show that the Zr-MOF-rGO/GrPE electrode has the potential to be a useful platform for the detection of gemcitabine in patients with breast cancer.

A CHA amplifier assisted DNA logical circuits for accurate diagnosis of acute myocardial infarction

Developing sensitive and reliable strategies for the simultaneous detection of multiple miRNAs is crucial for the accurate diagnosis of acute myocardial infraction (AMI). Herein, we have rationally designed a signal amplifier-assisted DNA “AND” logic gate for simultaneously detecting three AMI-related miRNAs. To achieve amplified signal output, hairpins labeled with FAM and BHQ1 are employed, which remain in the signal-off state. Upon the input of all three targets, the “AND” logic gate is activated, resulting in the release of a trigger molecule. This trigger molecule then initiates the catalytic hairpin assembly (CHA) reaction, leading to amplified fluorescence signal enhancement. Notably, the system demonstrates excellent sensing performance in serum, exhibiting a wide detection range spanning from 0.05 to 20 nM and low limits of detection (31.34, 39.07, and 23.86 pM) for the three miRNAs. The AND-CHA system ensures that fluorescence signal output is obtained only when all three inputs are present simultaneously. It provides a direct result without the need for further signal analysis, thereby improving diagnostic accuracy and convenience.

Integrated surface microstructure and enhanced dielectric constant for constructing simple, low-cost, and high-performance flexible capacitive pressure sensor

Designing microstructure surface or enhancing the dielectric constant of functional layer is a common useful strategy to improve the pressure sensing performances of flexible capacitive pressure sensors, but it usually involves complex preparation processes (such as artificial microstructure and preparation of composite materials). Considering the limitations of a single strategy in improving the pressure sensing performance of capacitive pressure sensor, herein, we propose a simple strategy that integrates surface microstructure and enhanced dielectric constant of functional layer for constructing a high-performance flexible capacitive pressure sensor. Specifically, the polyester fiber conductive tape and filter paper with intrinsic rough surfaces are selected as microstructure electrodes and dielectric layer, respectively. Meanwhile, by introducing the daily carbon ink into filter paper, the moderate conductivity of the carbon ink is utilized to enhance the dielectric constant of the filter paper dielectric layer. The results show that the proposed the pressure sensor has wide pressure detection range of 0.1−100 kPa, high sensitivity (1.47 kPa−1, 0.1−10 kPa) and good durability (4000 cycles at 20 kPa). Taking the pressure range of 10−100 kPa as an example, the sensitivity of carbon ink paper-based sensor is 60 times higher than that of sensor without carbon ink. The enhanced working mechanism of the sensor is clarified by combining testing and characterization. In addition, the proposed pressure sensor has potential applications in wrist pulse, respiratory rate, walking record, morse code transmission，and stoma bag detection. This work provides a useful reference for constructing simple, low-cost and high-performance flexible capacitive pressure sensor.

A PEDOT: PSS/GO fiber microelectrode fabricated by microfluidic spinning for dopamine detection in human serum and PC12 cells.

Rapid and accurate in situ determinationof dopamine is of great significance in the study of neurological diseases. In this work, poly (3,4-ethylenedioxythiophene): poly (styrenesulfonic acid) (PEDOT: PSS)/graphene oxide (GO) fibers were fabricated by an effective method based on microfluidic wet spinning technology. The composite microfibers with stratified and dense arrangement were continuously prepared by injecting PEDOT: PSS and GO dispersion solutions into a microfluidic chip. PEDOT: PSS/GO fiber microelectrodes with high electrochemical activity and enhanced electrochemical oxidation activity of dopamine were constructed by controlling the structure composition of the microfibers with varying flow rate. The fabricated fiber microelectrode had a low detection limit (4.56 nM) and wide detection range (0.01-8.0 µM) for dopamine detection with excellent stability, repeatability, and reproducibility. In addition, the PEDOT: PSS/GO fiber microelectrode prepared was successfully used for the detection of dopamine in human serum and PC12 cells. The strategy for the fabrication of multi-component fiber microelectrodes isa new and effective approach for monitoring the intercellular neurotransmitter dopamine and has highpotential as an implantable neural microelectrode.

A novel AgNFs/MOF/CFC substrate-based flexible SERS sensor for high-sensitive detection of dye molecules

Metal–organic framework (MOF) materials have been increasingly used owing to their high porosity, chemical stability, and semiconducting properties. With the help of MOF materials, MOF-based SERS substrates can exhibit high sensitivity. Herein, we proposed a flower-like Ag nanoparticles (AgNFs)/metal–organic framework (MOF)/ carbon fiber cloth (CFC) substrate-based surface-enhanced Raman scattering (SERS) sensor for the high-sensitive detection of dye molecules. MOF is used as a signal amplifier to accelerate the approach of dye molecules near AgNFs on the MOF surface and anchor AgNFs on the substrate surface. The detection occurred on the AgNFs/MOF/CFC SERS substrate, providing an enhancement factor (EF) of 5.53 × 107. Benefitting from the signal amplification function of AgNFs/MOF/CFC and the superiority of SERS, this sensor represented high sensitivity performance and a wide detection range. Importantly, the combination of Co elements with silver in the AgNFs/MOF/CFC composite substrate enhances the resistance of Ag to oxidation by atmospheric moisture resulting in excellent stability (the substrate maintained good SERS activity after 45 days of exposure to air) of the SERS substrate. The basic performance of the substrate was evaluated using rhodamine 6 G (R6G). (Detection limit reached 10−12 M) The detection limit of TC was 0.1 ppm. This method provides a potential and cost-effective strategy to develop advanced SERS sensing devices to effectively monitor pollutants in environmental waters.

Enzymes encapsulated in organic–inorganic hybrid nanoflower with spatial localization for sensitive and colorimetric detection of formate

Formate is an important environmental pollutant, and meanwhile its concentration change is associated with a variety of diseases. Thus, rapid and sensitive detection of formate is critical for the biochemical analysis of complex samples and clinical diagnosis of multiple diseases. Herein, a colorimetric biosensor was constructed based on the cascade catalysis of formate oxidase (FOx) and horseradish peroxidase (HRP). These two enzymes were co-immobilized in Cu3(PO4)2-based hybrid nanoflower with spatial localization, in which FOx and HRP were located in the shell and core of nanoflower, respectively (FOx@HRP). In this system, FOx could catalyze the oxidation of formate to generate H2O2, which was then utilized by HRP to oxidize 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid to yield blue product. Ideal linear correlation could be obtained between the absorbance at 420 nm and formate concentration. Meanwhile, FOx@HRP exhibited excellent detection performance with low limit of detection (6 μM), wide linear detection range (10–900 μM), and favorable specificity, stability and reusability. Moreover, it could be applied in the detection of formate in environmental, food and biological samples with high accuracy. Collectively, FOx@HRP provides a useful strategy for the simple and sensitive detection of formate and is potentially to be used in biochemical analysis and clinical diagnosis.

A Ni-doped coral-like three-dimensional Mo2C nanorod for sensitive electrochemical monitoring of environmentally polluting nitrobenzene

This paper reports the synthesis of a Ni-doped coral-like three-dimensional Mo2C nanorod via the pyrolysis of polypyrrole (PPy)-loaded NiMoO4·xH2O nanorods for nitrobenzene (NB) detection. The incorporation of Ni species into Mo2C enhances dual-atom synergistic catalysis, while the inclusion of PPy as a preferred carbon source improves the composite's active area. The coral-like three-dimensional rod structure not only provides various types of sites for adsorbing reactants but also offers more channels for ion migration and electron transfer, promoting charge transfer and further enhancing its electrical conductivity. The resulting composite demonstrates a wide linear detection range (2.11–1053.61 μM), high sensitivity, low limit of detection (0.64 μM, S/N = 3), and excellent stability and anti-interference performance in NB detection. This novel composite preparation approach offers promising prospects for practical NB detection applications.

A Low-cost Two-in-one Fluorescence/ Colorimetry Ammonium Detector: Design and Application

In this study, a compact and low-cost ammonium detector integrated spectrophotometric and fluorescence detection was designed based on the reaction of ammonium and o-phthalaldehyde in the presence of sodium sulfite solution to form substances with both photometric properties and fluorescence. This two-in-one detector detects ammonium in spectrophotometric mode at high concentrations and in fluorescence mode at low concentrations. The detector with a total size of 5×5×5 cm2 and integrates the detector skeleton box, light source, attenuator, filter, photoelectric sensor, data acquisition card, constant current source circuit, and LabVIEW upper computer acquisition program. Also, the detector was applied to the determination of water samples with a wide range of ammonium concentrations, showing good performance. The detector has the advantages of low cost, miniaturization, easy operation, and wide detection concentration range. This two-in-one detector has good potential for ammonium detection in the field, especially where ammonium concentration varies widely.

Bioinspired composite fiber aerogel pressure sensor for machine-learning-assisted human activity and gesture recognition

Machine-learning-assisted human activity and gesture recognition are valuable for human-computer interaction, and data acquisition often necessitates high-performance sensors. Here, inspired by spider web and bat wing airflow sensing system, polyimide fiber (PIF)/carbon black (CB) composite fiber aerogel (CFA) pressure sensor with biomimetic hair-Merkel cell sensitive unit was developed, exhibiting ultralow detection limit (2 Pa), high pressure sensitivity (S=23.1 kPa-1), wide linear detection capacity up to 67.61 kPa, and fast response/recovery time (140/100 ms). Thanks to the excellent mechanical property and environmental tolerance of CFA, it also possesses excellent low fatigue over >4000 cycles and good durability even at extreme high-temperature (200 °C) and underwater conditions. The superior signal data of the sensor, combined with the Convolutional Neural Network machine learning algorithm, achieves ultra-high prediction accuracies of 96.73% and 98.26% for human activity and gesture recognition, respectively. Additionally, CFA also has amazing thermal management properties, making it to be an ideal candidate for wearable electronics with excellent wearing comfort and safety.

Copper oxide nanoparticles modified electrodes for high-sensitivity detection of uric acid in athletes

This study introduces an innovative electrochemical sensor leveraging copper oxide nanoparticles for detecting uric acid levels in athletes with enhanced sensitivity and selectivity. Employing a novel synthesis protocol, uniform copper oxide nanoparticles were integrated into a chitosan-glutathione biogel matrix, creating a high-performance sensing electrode. The nanoparticles were extensively characterized using XRD, SEM, FTIR, and XPS techniques, confirming their crystalline structure, morphology, and purity. The average crystallite size was determined to be 18 nm. Electrochemical evaluation of the sensor using cyclic voltammetry and electrochemical impedance spectroscopy revealed significantly improved electron transfer kinetics and catalytic activity towards uric acid oxidation. The sensor exhibited a wide linear detection range from 1 μM to 1.2 mM, encompassing both physiological and pathological uric acid levels. Notably, the limit of detection was found to be 0.27 μM, surpassing the performance of most existing metal oxide based uric acid sensors. The sensor also demonstrated excellent anti-interference capability against commonly coexisting species in biological fluids. Furthermore, the practical applicability of the sensor was validated by successfully determining uric acid concentrations in human serum, plasma, and sweat samples, with impressive recovery rates ranging from 96% to 102%. The stability, reproducibility, and cost-effectiveness of the developed sensor make it a promising tool for routine health monitoring and early intervention in athletes, facilitating personalized training and preventing complications associated with uric acid imbalances. The present work offers a significant advancement in non-invasive, reliable, and efficient assessment of uric acid, with potential for widespread implementation in sports medicine and general healthcare.

Electrochemiluminescence quenching effect of Cu2O towards flower-like ferric ion-doped g-C3N4 and its application for Cyfra21-1 immunosensing

In this article, ferric ion-doped floral graphite carbon nitride (Fe–CN-3, energy donor) was used to construct the substrate of the immunosensor and copper oxide nanocubes (Cu2O, energy acceptor) were taken as an efficient ECL quenching probe. A sandwich quench electrochemiluminescence (ECL) immunosensor for soluble cytokeratin 19 fragment (Cyfra21-1) detection was preliminarily developed based on a novel resonant energy transfer donor-acceptor pair. Fe–CN-3, a carbon nitride that combines the advantages of metal ion doping as well as morphology modulation, is used in ECL luminophores to provide more excellent ECL performance, which makes a significant contribution to the application and development of carbon nitride in the field of ECL biosensors. The regular shape, high specific surface area and excellent biocompatibility of the quencher Cu2O nanocubes facilitate the labeling of secondary antibodies and the construction of sensors. Meanwhile, as an energy acceptor, the UV absorption spectrum of Cu2O can overlap efficiently with the energy donor's ECL emission spectrum, making it prone to the occurrence of ECL-RET and thus obtaining an excellent quenching effect. These merits of the donor-acceptor pair enable the sensor to have a wide detection range of 0.00005–100 ng/mL and a low detection limit of 17.4 fg/mL (S/N = 3), which provides a new approach and theoretical basis for the clinical detection of lung cancer.

Multistage Microstructured Ionic Skin for Real‐Time Vital Signs Monitoring and Human‐Machine Interaction

Skin‐like electronics research aiming to mimic even surpass human‐like specific tactile cognition by operating perception‐to‐cognition‐to‐feedback of stimulus to build intelligent cognition systems for certain imperceptible or inappreciable signals was so attractive. Herein, we constructed an all‐in‐one tri‐modal pressure sensing wearable device to address the issue of power supply by integrating multistage microstructured ionic skin (MM i‐skin) and thermoelectric self‐power staffs, which exhibits high sensitivity simultaneously. The MM i‐skin with multi‐stage “interlocked” configurations achieved precise recognition of subtle signals, where the sensitivity reached up to 3.95 kPa−1, as well as response time of 46 ms, cyclic stability (over 1500 cycles), a wide detection range of 0–200 kPa. Furthermore, we developed the thermoelectricity nanogenerator, piezoelectricity nanogenerator, and piezocapacitive sensing as an integrated tri‐modal pressure sensing, denoted as P‐iskin, T‐iskin, and C‐iskin, respectively. This multifunctional ionic skin enables real‐time monitoring of weak body signals, rehab guidance, and robotic motion recognition, demonstrating potential for Internet of things (IoT) applications involving the artificial intelligence‐motivated sapiential healthcare Internet (SHI) and widely distributed human‐machine interaction (HMI).

Portable electrochemical sensor for adrenaline detection using CoNi-MOF-based CS-PAM hydrogel

The development of a portable smartphone-based electrochemical sensor for analyzing adrenaline levels in real samples can make a great contribution to the research community worldwide. In order to achieve this goal, the key challenge is to build sensing interfaces with excellent electrocatalytic properties. In this work, microspherical bimetallic metal–organic frameworks (CoNi-MOF) consisting of nanoclusters were first synthesized using a hydrothermal method. On this basis, the catalytic activity of pure chitosan-polyacrylamide hydrogel (CS-PAM) was modulated by adding different amounts of CoNi-MOF during the in-situ synthesis of CS-PAM. Finally, a portable electrochemical detection system based on CS-PAM was established for the detection of adrenaline. A series of resulting composite hydrogels with a large specific surface area, abundant active sites, and unique network structure facilitate the enrichment and catalysis of adrenaline molecules. Under optimal conditions, the analytical platform constructed by using CoNi-MOF-based CS-PAM has the advantages of a wide detection range (0.5–10 and 10–2500 μM), a low detection limit (0.167 μM), and high sensitivity (0.182 and 0.133 μA·μM·cm−2). In addition, the sensor maintains selective detection of the target in the presence of many different types of interferences, and the current response is not significantly reduced even after 60 cycles of testing. We strongly believe that the designed smart portable sensing can realize the accurate determination of adrenaline in complex systems, and this study can provide new ideas for the research of MOFs-based hydrogels in electrochemical analysis.

Electrochemical aptasensor based on CRISPR/Cas12a-mediated and DNAzyme-assisted cascade dual-enzyme transformation strategy for zearalenone detection

The combination of the CRISPR/Cas system and biosensing is of profound significance in small molecule diagnostics. Herein, we report a cascade dual-enzyme transformation strategy for ultrasensitive analysis of zearalenone (ZEN) by utilizing the CRISPR/Cas12a trans-cleavage activity and DNAzyme amplification. NH2-MnO2/Pd@Au NBs nanocomposites are greatly promising electrode modification material for improving electrode sensing performance. PtPd@Fe3O4 nanocomposites are synthesized as signal label materials to load the electroactive molecule methylene blue (MB). The recognition of ZEN target and aptamer is converted into DNA signal to regulate the trans-cleavage activity of the CRISPR/Cas12a on Mg2+-DNAzyme probes. After that, in the presence of DNAzyme probes and Mg2+, the MB signal changes caused by Mg2+dependent DNAzyme-assisted catalytic cleavage of signal labels on the electrode surface are evaluated. The quantitative detection of ZEN is ultimately achieved. Under optimal conditions, the as-prepared electrochemical aptasensor shows a wide linear detection range of 1 × 10-5 to 10 ng·mL−1, and the detection limit is 6.27 × 10-6 ng·mL−1. Moreover, the constructed aptasensor exhibits satisfactory selectivity, stability and repeatability, which also has a wide application prospect in the real sample analysis.

Optimization of a bovine cytokine multiplex assay using a new bovine and cross-reactive equine monoclonal antibodies

Cytokines are important markers for immune activation, regulation, and homeostasis. The lack of monoclonal antibodies (mAbs) and sensitive assays to evaluate cytokine secretion has hindered research of bovine inflammation and immune regulation. We recently developed a fluorescent bead-based multiplex assay (multiplex assay) for bovine IL-10, TNF-α, and IFN-γ. Although the original assay covers a broad concentration range for the 3 targets, analytical sensitivity for IL-10 and IFN-γ could be improved to facilitate detection of these cytokines in their physiological low pg/mL range. To optimize the multiplex assay, we generated a new bovine IL-10 mAb and explored its use for the detection of intracellular and secreted bovine IL-10. The new bovine IL-10 mAb 130 recognized recombinant bovine IL-10 fusion protein and did not react with the fusion protein tag, or the TNF-α and IFN-γ standards in the multiplex assay. For improving IFN-γ detection, we explored cross-reactivity of anti-equine IFN-γ mAbs by intracellular staining of bovine stimulated peripheral blood mononuclear cells (PBMC). Equine IFN-γ mAb 3 showed excellent cross-reactivity with bovine IFN-γ by intracellular detection. Adding IL-10 mAb 130 and IFN-γ mAb 3 to the bovine multiplex assay substantially improved the analytical sensitivity with lower limits of detection in the low pg/mL range for all analytes. The detection ranges for the optimized multiplex assay were determined as 2 – 134,000 pg/mL for IL-10, 8 – 127,000 pg/mL for IFN-γ, and 12 – 193,000 pg/mL for TNF-α. The assay was next used to measure cytokine concentrations in cell culture supernatants from PBMC stimulated in plasma from whole blood stimulation to confirm native IL-10, TNF-α, and IFN-γ recognition and to explore the upper detection limits of the assay. In PBMC stimulation with a mix of phorbol myristate acetate (PMA) and ionomycin resulted in highest cytokine concentrations, while in plasma from whole blood stimulation, highest concentrations were observed in samples stimulated with a mix of lipopolysaccharide (LPS), phytohemagglutinin (PHA), and the TLR-2/6 agonist Pam2Csk4. PBMC and whole blood stimulation protocols showed that the optimized multiplex assay covers a wide linear detection range for measuring cytokine concentrations in bovine samples. For whole blood stimulation, a cocktail of pathogen associated molecular patterns elicited a stronger cytokine response than a mix of PMA and ionomycin, but response varied considerably between individual cattle. In conclusion, optimizing the bovine cytokine assay with new reagents improved the lower detection limits and widened the linear detection ranges while lowering the background of the multiplex assay.

A Simplistic Label‐Free Electrochemical Immunosensing Approach for Rapid and Sensitive Detection of Anti‐SARS‐COV‐2 Nucleocapsid Antibodies

AbstractRapid and precise detection of SARS‐CoV‐2 antibodies is paramount for effective outbreak monitoring and vaccine efficacy assessment. While existing approaches for antibody detection often rely on complex electrochemical immunosensing with nanomaterial functionalization targeting S‐protein antibodies, their limitations in sensitivity and complexity have hindered widespread application. Here, we present a simplistic immunosensing platform designed for the rapid, and precise detection of SARS‐CoV‐2 specific IgG and Nucleocapsid antibodies. Notably, this study marks only the second exploration of SARS‐CoV‐2 N‐protein antibody detection. The platform utilizes traditional self‐assembled monolayers to establish selective bio‐affinity between SARS‐CoV‐2 specific Nucleocapsid antibodies and a gold electrode functionalized with the N‐protein antigen. Interestingly, despite the absence of nanomaterial functionalization, the developed platform achieves sensitivity comparable to existing sensors across a wide detection range (0.025 to 1 ng/mL) with an impressive limit of detection (0.019 ng/mL). The simplicity of the approach, relying solely on immunocomplex reactions, underscores that effective binding efficiency may be achieved in the absence of complex functionalization and determines its affordability, specificity, and high sensitivity. By eliminating the need for additional functionalization steps, the platform offers a streamlined solution for SARS‐CoV‐2 antibody detection and demonstrates the possibility of N‐protein antibody detection as a promising avenue for widespread application in SARS‐CoV‐2 outbreak monitoring and vaccine efficacy assessment particularly in underdeveloped regions.