An immobilized antibody-based affinity grid strategy for on-grid purification of target proteins enables high-resolution cryo-EM.

In cryo-electron microscopy (cryo-EM), sample preparation poses a critical bottleneck, particularly for rare or fragile macromolecular assemblies and those suffering from denaturation and particle orientation distribution issues related to air-water interface. In this study, we develop and characterize an immobilized antibody-based affinity grid (IAAG) strategy based on the high-affinity PA tag/NZ-1 antibody epitope tag system. We employ Pyr-NHS as a linker to immobilize NZ-1 Fab on the graphene oxide or carbon-covered grid surface. Our results demonstrate that the IAAG grid effectively enriches PA-tagged target proteins and overcomes preferred orientation issues. Furthermore, we demonstrate the utility of our IAAG strategy for on-grid purification of low-abundance target complexes from cell lysates, enabling atomic resolution cryo-EM. This approach greatly streamlines the purification process, reduces the need for large quantities of biological samples, and addresses common challenges encountered in cryo-EM sample preparation. Collectively, our IAAG strategy provides an efficient and robust means for combined sample purification and vitrification, feasible for high-resolution cryo-EM. This approach holds potential for broader applicability in both cryo-EM and cryo-electron tomography (cryo-ET).

Bilateral efforts to improve SERS detection efficiency of exosomes by Au/Na7PMo11O39 Combined with Phospholipid Epitope Imprinting.

Detection of cancer-related exosomes in body fluids has become a revolutionary strategy for early cancer diagnosis and prognosis prediction. We have developed a two-step targeting detection method, termed PS-MIPs-NELISA SERS, for rapid and highly sensitive exosomes detection. In the first step, a phospholipid polar site imprinting strategy was employed using magnetic PS-MIPs (phospholipids-molecularly imprinted polymers) to selectively isolate and enrich all exosomes from urine samples. In the second step, a nanozyme-linked immunosorbent assay (NELISA) technique was utilized. We constructed Au/Na7PMo11O39 nanoparticles (NPs) with both surface-enhanced Raman scattering (SERS) property and peroxidase catalytic activity, followed by the immobilization of CD9 antibodies on the surface of Au/Na7PMo11O39 NPs. The Au/Na7PMo11O39-CD9 antibody complexes were then used to recognize CD9 proteins on the surface of exosomes enriched by magnetic PS-MIPs. Lastly, the high sensitivity detection of exosomes was achieved indirectly via the SERS activity and peroxidase-like activity of Au/Na7PMo11O39 NPs. The quantity of exosomes in urine samples from pancreatic cancer patients obtained by the PS-MIPs-NELISA SERS technique showed a linear relationship with the SERS intensity in the range of 6.21 × 107-2.81 × 108 particles/mL, with a limit of detection (LOD) of 5.82 × 107 particles/mL. The SERS signal intensity of exosomes in urine samples from pancreatic cancer patients was higher than that of healthy volunteers. This bidirectional MIPs-NELISA-SERS approach enables noninvasive, highly sensitive, and rapid detection of cancer, facilitating the monitoring of disease progression during treatment and opening up a new avenue for rapid early cancer screening.

The Evaluation of a Lateral Flow Strip Based on the Covalently Fixed "End-On" Orientation of an Antibody for Listeria monocytogenes Detection.

In this study, the covalently fixed "end-on" orientation of a monoclonal Listeria monocytogenes antibody (mAb-Lis) to amino terminated oligo (ethylene glycol)-capped gold nanoparticles (NH2-TEG-AuNPs) was used to fabricate an in-house lateral flow strip (LFS), namely, the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS. The aim was to evaluate the performance of the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS in detecting L. monocytogenes. The proposed LFS enabled the sensitive detection of L. monocytogenes in 15 min with a visual limit of detection of 102 CFU/mL. Quantitative analysis indicated an LOD at 10 CFU/mL. The fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS showed no cross-reactivity with other pathogenic bacteria and practical performance across different food matrices, including human blood, milk, and mushroom samples. Furthermore, the clinical performance of the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS for detecting L. monocytogenes was evaluated by using 12 clinical samples validated by the hemoculture method. It demonstrated excellent concordance with the reference methods, with no false-positive or false-negative results observed. Therefore, the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS serves as a promising candidate for a point-of-care test (POCT), enabling the rapid, precise, and highly sensitive detection of L. monocytogenes in clinical samples and contaminated food.

Nitrocellulose/acrylic resin coated screen-printed carbon electrode to construct a capacitive immunosensor for anti-BSA.

The capacitive immunosensor, known for its label-free simplicity, has great potential for point-of-care diagnostics. However, the interaction between insulation and recognition layers on the sensing electrode greatly affects its performance. This study introduces a pioneering dual-layer strategy, implementing a novel combination of acrylic resin (AR) and nitrocellulose (NC) coatings on screen-printed carbon electrodes (SPCEs). This innovative approach not only enhances the dielectric properties of the capacitive sensor but also streamlines the immobilization of recognizing elements. Particularly noteworthy is the superior reliability and insulation offered by the AR coating, surpassing the limitations of traditional self-assembled monolayer (SAM) modifications. This dual-layer methodology establishes a robust foundation for constructing capacitive sensors optimized specifically for liquid medium-based biosensing applications. The NC coating in this study represents a breakthrough in effectively immobilizing BSA, unraveling the capacitive response intricately linked to the quantity of adsorbed recognizing elements. The results underscore the prowess of the proposed immunosensor, showcasing a meticulously defined linear calibration curve for anti-BSA (ranging from 0 to 25 µg/ml). Additionally, specific interactions with anti-HAS and anti-TNF-α further validate the versatility and efficacy of the developed immunosensor. This work presents a streamlined and highly efficient protocol for developing label-free immunosensors for antibody determination and introduces a paradigm shift by utilizing readily available electrodes and sensing systems. The findings are poised to catalyze a significant acceleration in the advancement of biosensor technology, opening new avenues for innovative applications in point-of-care diagnostics.

Versatile Metal-Organic Framework Incorporating Ag2S for Constructing a Photoelectrochemical Immunosensor for Two Breast Cancer Markers.

Breast cancer poses the significance of early diagnosis and treatment. Here, we developed an innovative photoelectrochemical (PEC) immunosensor characterized by high-level dual photocurrent signals and exceptional sensitivity. The PEC sensor, denoted as MIL&Ag2S, was constructed by incorporating Ag2S into a metal-organic framework of MIL-101(Cr). This composite not only enhanced electron-hole separation and conductivity but also yielded robust and stable dual photocurrent signals. Through the implementation of signal switching, we achieved the combined detection of cancer antigen 15-3 (CA15-3) and carcinoembryonic antigen (CEA) with outstanding stability, reproducibility, and specificity. The results revealed a linear range for CEA detection spanning 0.01-32 ng/mL, with a remarkably low detection limit of 0.0023 ng/mL. Similarly, for CA15-3 detection, the linear range extended from 0.1 to 320 U/mL, with a low detection limit of 0.014 U/mL. The proposed strategy introduces new avenues for the development of highly efficient, cost-effective, and user-friendly PEC sensors. Furthermore, it holds promising prospects for early clinical diagnosis, contributing to potential breakthroughs in medical detection and ultimately improving patient outcomes.

Preparation of immunomagnetic composite nanostructures with bifunctional four-arm PEG derivatives as linkers for the ultrafast enrichment of zearalenone and its metabolites.

It is challenging to prepare sample pretreatment materials with simple use, strong selectivity and satisfactory enrichment performance. In this study, the antibody (3D4) that can specifically recognize zearalenone (ZEN) and its metabolites was immobilized on the surface of gold-coated magnetic Fe3O4 nanoparticles (GMN) by streptavidin (SA)-biotin interaction using GMN as the substrate and our designed four-arm PEG derivative (HS-4ARMPEG10K-(CM)3) as the linker. The immunomagnetic nanoparticles (GMN-4ARMPEG10K-SA-3D4) prepared by this strategy can achieve rapid enrichment (only 5 min) of analytes directly in the matrix, and higher enrichment capacity compared with the previous immunomagnetic particles. The sensitive and accurate analysis of ZEN and its metabolites can be achieved coupled with HPLC-MS/MS. The LODs and LOQs were 0.02-0.05 µg/kg and 0.05-0.10 µg/kg, respectively. The recoveries were 84.13%-112.67%, and the RSDs were 1.09%-9.39%. The method can provide a powerful tool for highly sensitive and rapid monitoring of mycotoxins in complex matrices due to its' strong selectivity and resistance to matrix interference.

Exploring various carbon nanomaterials-based electrodes modified with polymelamine for the reagentless electrochemical immunosensing of Claudin18.2.

Claudin18.2 (CLDN18.2) is a tight junction protein often overexpressed in various solid tumors, including gastrointestinal and esophageal cancers, serving as a promising target and potential biomarker for tumor diagnosis, treatment assessment, and prognosis. Despite its significance, no biosensor has been reported to date for the detection of CLDN18.2. Here, we present the inaugural immunosensor for CLDN18.2. In this study, an amine-rich conducting polymer of polymelamine (PM) was electrografted onto different carbon nanomaterial-based screen-printed electrodes (SPEs), including carbon (C), graphene (Gr), graphene oxide (GO), carbon nanotube (CNT), and carbon nanofiber (CNF) via cyclic voltammetry. A comparative study was performed to explore the best material for the preparation of the PM-modified electrodes to be used as in-situ redox substrate for the immunosensor fabrication. The surface chemistry and structural features of pristine and PM-deposited electrodes were analyzed using Raman and scanning electron microscopy (SEM) techniques. Our results showed that the PM deposited on Gr and CNT/SPEs exhibited the most significant and stable redox behavior in PBS buffer. The terminal amine moieties on the PM-modified electrode surfaces were utilized for immobilizing anti-CLDN18.2 monoclonal antibodies via N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide chemistry to construct the electrochemical immunosensor platform. Differential pulse voltammetry-based immunosensing of CLDN18.2 protein on BSA/anti-CLDN18.2/PM-Gr/SPE and BSA/anti-CLDN18.2/PM-CNT/SPE exhibited excellent selectivity against other proteins such as CD1, PDCD1, and ErBb2. The limits of detection of these two immunosensor platforms were calculated to be 7.9 pg/mL and 0.104 ng/mL for the CNT and Gr immunosensors, respectively. This study demonstrated that the PM-modified Gr and CNT electrodes offer promising platforms not only for the reagentless signaling but also for covalent immobilization of biomolecules. Moreover, these platforms offer excellent sensitivity and selectivity for the detection of CLDN18.2 due to its enhanced stable redox activity. The immunosensor demonstrated promising results for the sensitive detection of CLDN18.2 in biological samples, addressing the critical need for early gastric cancer diagnosis.

Laser-printed paper ELISA and hydroxyapatite immobilization for colorimetric congenital anomalies screening in saliva.

BACKGROUND: Alpha-fetoprotein (AFP) is a fetal protein that can indicate congenital anomalies such as Down syndrome and spinal canal blockage when detected at abnormal levels in pregnant women. Current AFP detection methods rely on invasive blood or serum samples, which require sophisticated equipment. From the many solutions proposed, colorimetric paper-based assays excel in point-of-care settings. The concept of paper-based ELISA (p-ELISA) enhances traditional methods, aligning with the ASSURED criteria for diagnostics in resource-limited regions. Despite success in microfluidic paper-based assay devices, laser printing remains underexplored for p-ELISA. Additionally, modifying the paper surface provides an additional layer of sensitivity enhancement. RESULTS: In this study, we developed a novel laser-printed paper-based ELISA (LP-pELISA) for rapid, sensitive, and noninvasive detection of AFP in saliva samples. The LP-pELISA platform was fabricated by printing hydrophobic barriers on filter paper using a laser printer, followed by depositing hydroxyapatite (HAp) as an immobilization material for the antibodies. The colorimetric detection was achieved using AuNPs functionalized with anti-AFP antibodies and silver nitrate enhancement. The LP-pELISA exhibited a linear response for AFP detection in both buffer and saliva samples over a range of 1.0-800 ng mL-1, with a limit of detection (LOD) reaching 1.0 ng mL-1. The assay also demonstrated good selectivity, repeatability, reproducibility, and stability. The LP-pELISA was further validated by testing spiked human saliva samples, showing its potential for point-of-care diagnosis of congenital disabilities. SIGNIFICANCE: The LP-pELISA is a noninvasive platform showcasing simplicity, cost-effectiveness, and user-friendliness, utilizing laser printing, hydroxyapatite modification, and saliva samples to efficiently detect AFP. Beyond its application for AFP, this method's versatility extends to other biomarkers, positioning it as a catalyst for the evolution of paper-based biosensors. The LP-pELISA holds promise as a transformative tool for point-of-care diagnostics, fostering advancements in healthcare with its innovative technology.

Precursor carboxy-silica for functionalization with interactive ligands. IV. Carbodiimide assisted preparation of immobilized antibody stationary phases for high performance immuno-affinity chromatography of human serum.

In this Part IV of the article series dealing with the functionalization of the precursor carboxy silica with various chromatographic ligands, immuno affinity (IA) columns were prepared with immobilized anti-apolipoprotein B (AAP B) and anti-haptoglobin (AHP) antibodies for use in immuno affinity chromatography (IAC) in the aim of selectivily capturing their corresponding antigens from healthy and cancer human sera. Diseased human serum with adenocarcinoma cancer was selected as a typical diseased biological fluid. Besides preferentially capturing their corresponding antigens, the AAP B column captured from disease-free and cancer sera, 34 proteins and 33 proteins, respectively, while the AHP column enriched 38 and 47 proteins, respectively. This nonspecific binding can be attributed to the many proteins human serum have, which could mediate protein-protein interactions thus leading to the so-called "sponge effect". This kind of behavior can be exploited positively in the determination of differentially expressed proteins (DEPs) for diseased serum with respect to healthy serum and in turn allow the identification of an array of potential biomarkers for cancer. In fact, For AHP column, 13 upregulated and 22 downregulated proteins were identified whereas for AAP B column the numbers were 23 and 10, respectively. The DEPs identified with both columns match those reported in the literature for other types of cancers. The different expression of proteins in each IAC column can be related to the variability of protein-protein interactions. In addition, an array of a few biomarkers is more indicative of a certain disease than a single biomarker.

Functionalized gold nanoparticle enhanced nanorod hyperbolic metamaterial biosensor for highly sensitive detection of carcinoembryonic antigen.

Hyperbolic metamaterial (HMM) biosensors based on metals have superior performance in comparison with conventional plasmonic biosensors in the detection of low concentrations of molecules. In this study, a nanorod HMM (NHMM) biosensor based on refractive index changes for carcinoembryonic antigen (CEA) detection is developed using secondary antibody modified gold nanoparticle (AuNP-Ab2) nanocomposites as signal amplification element for the first time. Numerical analysis based on finite element method is conducted to simulate the perturbation of the electric field of bulk plasmon polariton (BPP) supported by a NHMM in the presence of a AuNP. The simulation reveals an enhancement of the localized electric field, which arises from the resonant coupling of BPP to the localized surface plasmon resonance supported by AuNPs and is beneficial for the detection of changes of the refractive index. Furthermore, the AuNP-Ab2 nanocomposites-based NHMM (AuNP/Ab2-NHMM) biosensor enables CEA detection in the visible and near-infrared regions simultaneously. The highly sensitive detection of CEA with a wide linear range of 1-500 ng/mL is achieved in the near-infrared region. The detectable concentration of the AuNP/Ab2-NHMM biosensor has a 50-fold decrease in comparison with a NHMM biosensor. A low detection limit of 0.25 ng/mL (1.25 pM) is estimated when considering a noise level of 0.05 nm as the minimum detectable wavelength shift. The proposed method achieves high sensitivity and good reproducibility for CEA detection, which makes it a novel and viable approach for biomedical research and early clinical diagnostics.

Construction of AlGaN/GaN high-electron-mobility transistor-based biosensor for ultrasensitive detection of SARS-CoV-2 spike proteins and virions.

The COVID-19 pandemic has highlighted the need for rapid and sensitive detection of SARS-CoV-2. Here, we report an ultrasensitive SARS-CoV-2 immunosensor by integration of an AlGaN/GaN high-electron-mobility transistor (HEMT) and anti-SARS-CoV-2 spike protein antibody. The AlGaN/GaN HEMT immunosensor has demonstrated the capability to detect SARS-CoV-2 spike proteins at an impressively low concentration of 10-22 M. The sensor was also applied to pseudoviruses and SARS-CoV-2 ΔN virions that display the Spike proteins with a single virion particle sensitivity. These features validate the potential of AlGaN/GaN HEMT biosensors for point of care tests targeting SARS-CoV-2. This research not only provides the first HEMT biosensing platform for ultrasensitive and label-free detection of SARS-CoV-2.

Sandwich-type electrochemical immunosensing of CA125 by using nanoribbon-like Ti3C2Tx MXenes and toluidine blue/UIO-66-NH2.

CA125 (carbohydrate antigen 125) is an important biomarker of ovarian cancer, so developing effective method for its detection is of great significance. In the present work, a novel sandwich-like electrochemical immunosensor (STEM) of CA125 was constructed by preparing nanoribbon-like Ti3C2Tx MXenes (Ti3C2TxNR) to immobilize primary antibody (PAb) of CA125 and UIO-66-NH2 MOFs structure to immobilize second antibody (SAb) and electroactive toluidine blue (Tb) probe. In this designed STEM assay, the as-prepared Ti3C2TxNR nanohybrid offers the advantages in large surface area and conductivity as carrier, and UIO-66-NH2 provided an ideal platform to accommodate SAb and a large number of Tb molecules as signal amplifier. In the presence of CA125, the peak currents of Tb from the formed STEM structure increase with the increase of CA125 level. After optimizing the related control conditions, a wide linear range (0.2-150.0 U mL-1) and a very low detection limit (0.05 U mL-1) of CA125 were achieved. It's thus expected the developed STEM strategy has important applications for the detection of CA125.

Dual sensitivity-enhanced microring resonance-based integrated microfluidic biosensor for Aß42 detection.

Sensitive, accurate, and straightforward biosensors are pivotal in the battle against Alzheimer's disease, particularly in light of the escalating patient population. These biosensors enable early adjunctive diagnosis, thereby facilitating prompt intervention, alleviating socioeconomic burdens, and preserving individual well-being. In this study, we introduce the development of a highly sensitive add-drop dual-microring resonant microfluidic sensing chip boasting a sensitivity of 188.11 nm/RIU, marking a significant 20.7% enhancement over single microring systems. Leveraging ultra-thin Parylene C for streamlined antibody immobilization and non-destructive removal, this platform facilitates the precise quantification of the Alzheimer's disease biomarker Aß42. Employing an immune sensing strategy that amplifies and captures antigen signals using Au-labeled antibodies, we achieve an exceptional limit of detection of 9.02 pg/mL. The designed microring-based microfluidic biosensor chip exhibits outstanding specificity and sensitivity for Aß42 in serum samples, offering a promising avenue for the early adjunctive diagnosis of Alzheimer's disease.

A Co-Reactive Immunosensor Based on Ti3C2Tx MXene@TiO2-MoS2 Hybrids Promoting luminol@Au@Ni-Co NCs Electrochemiluminescence for CYFRA 21-1 Detection.

The construction of assays is capable of accurately detecting cytokeratin-19 (CYFRA 21-1), which is critical for the rapid diagnosis of nonsmall cell lung cancer. In this work, a novel electrochemiluminescence (ECL) immunosensor based on the co-reaction promotion of luminol@Au@Ni-Co nanocages (NCs) as ECL probe by Ti3C2Tx MXene@TiO2-MoS2 hybrids as co-reaction accelerator was proposed to detect CYFRA 21-1. Ni-Co NCs, as a derivative of Prussian blue analogs, can be loaded with large quantities of Au NPs, luminol, and CYFRA 21-1 secondary antibodies due to their high specific surface area. To further improve the sensitivity of the developed ECL immunosensor, Ti3C2Tx MXene@TiO2-MoS2 hybrids were prepared by in situ growth of TiO2 nanosheets on highly conductive Ti3C2Tx MXene, and MoS2 was homogeneously grown on Ti3C2Tx MXene@TiO2 surfaces by the hydrothermal method. Ti3C2Tx MXene@TiO2-MoS2 hybrids possess excellent catalytic performance on the electro-redox of H2O2 generating more O2·- and obtaining optimal ECL intensity of the luminol/H2O2 system. Under the appropriate experimental conditions, the quantitative detection range of CYFRA 21-1 was from 0.1 pg mL-1 to 100 ng mL-1, and the limit of detection (LOD) was 0.046 pg mL-1. The present sensor has a lower LOD with a wider linear range, which provides a new analytical assay for the early diagnosis of small-cell-type lung cancer labels.

Detection of tetanus toxoid with iron magnetic nanobioprobe.

Diagnosis of diseases with low facilities, speed, accuracy and sensitivity is an important matter in treatment. Bioprobes based on iron oxide nanoparticles are a good candidate for early detection of deadly and infectious diseases such as tetanus due to their high reactivity, biocompatibility, low production cost and sample separation under a magnetic field. In this study, silane groups were coated on surface of iron oxide nanoparticles using tetraethoxysilane (TEOS) hydrolysis. Also, NH2groups were generated on the surface of silanized nanoparticles using 3-aminopropyl triethoxy silane (APTES). Antibody was immobilized on the surface of silanized nanoparticles using TCT trichlorothriazine as activator. Silanization and stabilized antibody were investigated by using of FT-IR, EDX, VSM, SRB technique. UV/vis spectroscopy, fluorescence, agglutination test and ELISA were used for biosensor performance and specificity. The results of FT-IR spectroscopy showed that Si-O-Si and Si-O-Fe bonds and TCT chlorine and amine groups of tetanus anti-toxoid antibodies were formed on the surface of iron oxide nanoparticles. The presence of Si, N and C elements in EDX analysis confirms the silanization of iron oxide nanoparticles. VSM results showed that the amount of magnetic nanoparticles after conjugation is sufficient for biological applications. Antibody stabilization on nanoparticles increased the adsorption intensity in the uv/vis spectrometer. The fluorescence intensity of nano bioprobe increased in the presence of 10 ng ml-1. Nanobio probes were observed as agglomerates in the presence of tetanus toxoid antigen. The presence of tetanus antigen caused the formation of antigen-nanobioprobe antigen complex. Identification of this complex by HRP-bound antibody confirmed the specificity of nanobioprobe. Tetanus magnetic nanobioprobe with a diagnostic limit of 10 ng ml-1of tetanus antigen in a short time can be a good tool in LOC devices and microfluidic chips.

An ultrasensitive electrochemical immunosensor based on meso-PdN NCs and Au NPs/N-CNTs for quantitative cTnI detection.

Electrochemical immunosensors have gained considerable attention in detecting human disease markers due to their excellent specificity, high sensitivity, and facile operation. Herein, a rational-designed sandwich-type electrochemical immunosensor is constructed for the sensitive detection of cardiac troponin I (cTnI) using nitrogen-doped carbon nanotubes loaded with gold nanoparticles (Au NPs/N-CNTs) as substrate and highly active mesoporous palladium-nitrogen nanocubes (meso-PdN NCs) as secondary antibody markers. Benefitting from its large specific surface area (638.04 m2 g-1) and high nitrogen content, novel polydopamine (PDA)/ halloysite nanotubes (HNTs) hybrid derived one-dimensional (1D) N-CNTs can provide more binding sites for the in-situ growth of Au NPs to connect Ab1. Furthermore, as an ideal substrate material, Au NPs/N-CNTs exhibit finely tuned mesoporous structures and outstanding conductivity, which facilitate the mass and electron transfer during the electrocatalysis process. Besides, highly concave surfaces and crystalline mesopores of meso-PdN NCs expose more surfaces and crevices, providing abundant reactive sites for H2O2 reduction. Remarkably, the as-obtained immunosensor presented a wide linear range (from 10 fg mL-1 to 100 ng mL-1) and an excellent low detection limit (9.85 fg mL-1). This study may offer new insights into the precise fabrication of efficient electrochemical immunosensors for various clinical diagnosis applications.

Surface chemical modification of poly(dimethylsiloxane) for stabilizing antibody immobilization and T cell cultures.

Advances in cell immunotherapy underscore the need for effective methods to produce large populations of effector T cells, driving growing interest in T-cell bioprocessing and immunoengineering. Research suggests that T cells demonstrate enhanced expansion and differentiation on soft matrices in contrast to rigid ones. Nevertheless, the influence of antibody conjugation chemistry on these processes remains largely unexplored. In this study, we examined the effect of antibody conjugation chemistry on T cell activation, expansion and differentiation using a soft and biocompatible polydimethylsiloxane (PDMS) platform. We rigorously evaluated three distinct immobilization methods, beginning with the use of amino-silane (PDMS-NH2-Ab), followed by glutaraldehyde (PDMS-CHO-Ab) or succinic acid anhydride (PDMS-COOH-Ab) activation, in addition to the conventional physical adsorption (PDMS-Ab). By employing both stable amide bonds and reducible Schiff bases, antibody conjugation significantly enhanced antibody loading and density compared to physical adsorption. Furthermore, we discovered that the PDMS-COOH-Ab surface significantly promoted IL-2 secretion, CD69 expression, and T cell expansion compared to the other groups. Moreover, we observed that both PDMS-COOH-Ab and PDMS-NH2-Ab surfaces exhibited a tendency to induce the differentiation of naïve CD4+ T cells into Th1 cells, whereas the PDMS-Ab surface elicited a Th2-biased immunological response. These findings highlight the importance of antibody conjugation chemistry in the design and development of T cell culture biomaterials. They also indicate that PDMS holds promise as a material for constructing culture platforms to modulate T cell activation, proliferation, and differentiation.

Label-free electrochemical biosensor for direct detection of Oncostatin M (OSM) inflammatory bowel diseases (IBD) biomarker in human serum.

Oncostatin M (OSM) is an interleukin-6 (IL-6) member family cytokine implicated in the pathogenesis of chronic diseases including inflammatory bowel disease (IBD). OSM is a novel diagnostic biomarker over-expressed in the serum of IBD patients. This paper reports on the first electrochemical OSM immunosensor, developed using a multistep fabrication process aimed at covalently immobilizing OSM antibodies on a mixed self-assembled monolayer coated gold working electrode. Cyclic voltammetry, atomic force microscopy (AFM), IR spectroscopy and optical characterizations were used to validate the sensor functionalization protocol. Electrochemical impedance spectroscopy (EIS) measurements were performed to assess the reliability of the immunosensor preparation and to verify the antibody-antigen complexes formation. The label-free immunosensor showed high sensitivity identifying OSM at clinically relevant concentrations (37-1000 pg mL-1) with low detection limit of 2.86 pg mL-1. Both sensitivity and selectivity of the proposed immunosensor were also demonstrated in human serum in the presence of interfering biomarkers, making it an innovative potential platform for the OSM biomarker detection in IBD patients' serum.

A sensitive sandwich-type electrochemical immunosensor using nitrogen-doped graphene/metal-organic framework-derived CuMnCoOx and Au/MXene for the detection of breast cancer biomarker.

In terms of cancer-related deaths among women, breast cancer (BC) is the most common. Clinically, human epidermal growth receptor 2 (HER2) is one of the most commonly used diagnostic biomarkers for facilitating BC cell proliferation and malignant growth. In this study, a disposable gold electrode (DGE) modified with gold nanoparticle-decorated Ti3C2Tx (Au/MXene) was utilized as a sensing platform to immobilize the capturing antibody (Ab1/Au/MXene). Subsequently, nitrogen-doped graphene (NG) with a metal-organic framework (MOF)-derived copper-manganese-cobalt oxide, tagged as NG/CuMnCoOx, was used as a probe to label the detection antibody (Ab2). A sandwich-type immunosensor (NG/CuMnCoOx/Ab2/HER2-ECD /Ab1/Au/MXene/DGE) was developed to quantify HER2-ECD. NG/CuMnCoOx enhances the conductivity, electrocatalytic active sites, and surface area to immobilize Ab2. In addition, Au/MXene facilitates electron transport and captures more Ab1 on its surface. Under optimal conditions, the resultant immunosensor displayed an excellent linear range of 0.0001 to 50.0 ng. mL-1. The detection limit was 0.757 pg·mL-1 with excellent selectivity, appreciable reproducibility, and high stability. Moreover, the applicability for determining HER2-ECD in human serum samples indicates its ability to monitor tumor markers clinically.

Significance of the antibody orientation for the lateral flow immunoassays: A mini-review.

Lateral flow immunoassays (LFIAs) are well-established and broadly commercialized tools in the field of point-of-care testing due to their simplicity, rapidity, cost-effectiveness, and low requirements for users and equipment. However, the insensitivity and the possibility of producing inaccurate results associated with conventional LFIAs have impeded their wide-ranging implementation, especially for monitoring ultra-trace level of analytes. Moreover, the heterogeneous distribution of amino acids on the surface of antibody (Ab) results in a lack of precise control over their orientation, which ultimately leads to unsatisfactory detection performance. To address those concerns, herein we provide an overview of the emerging efforts to prepare well-established LFIAs from the perspective of orientation manipulation of immobilized Abs on the nanoprobes or membranes. The preparation of excellent nanoprobes with Abs being oriented immobilized, consisting of the nanoprobe types, Ab types, and their conjugation chemistries, are reviewed. Followed by the introduction of efforts highlight the importance of directionally immobilized Ab on the membrane. The effects of Ab orientation on the analytical performance of LFIA platforms in terms of sensitivity, specificity, rapidity, reliability, cost-effectiveness, and stability are also summarized. Finally, the future development and challenges of Ab-oriented immobilization-assisted LFIAs are also discussed.