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Single-domain antibodies, or VHH, nanobodies, are attractive tools in biotechnology and pharmaceuticals due to their favorable biophysical properties. Single-domain antibodies have potential for use in sensing materials to detect antigens, and in this paper, we propose a generic design strategy of single-domain antibodies for the highly efficient use of immobilized antibodies on a sensing substrate. Amine coupling was used to immobilize the single-domain antibodies on the substrate through a robust covalent bond. First, for two model single-domain antibodies with lysines at four highly conserved positions (K48, K72, K84, and K95), we mutated the lysines to alanine and measured the binding activity of the mutants (the percentage of immobilized antibodies that can bind antigen) using surface plasmon resonance. The two model single-domain antibodies tended to have higher binding activities when K72, which is close to the antigen binding site, was mutated. Adding a Lys-tag to the C-terminus of single-domain antibodies also increased the binding activity. We also mutated the lysine for another model single-domain antibodies with the lysine in a different position than the four residues mentioned above and measured the binding activity. Thus, single-domain antibodies immobilized in an orientation accessible to the antigen tended to have a high binding activity, provided that the physical properties of the single-domain antibodies themselves (affinity and structural stability) were not significantly reduced. Specifically, the design strategy of single-domain antibodies with high binding activity included mutating the lysine at or near the antigen binding site, adding a Lys-tag to the C-terminus, and mutating a residue away from the antigen binding site to lysine. It is noteworthy that mutating K72 close to the antigen binding site was more effective in increasing the binding activity than Lys-tag addition, and immobilization at the N-terminus close to the antigen binding site did not have such a negative effect on the binding activity compared to immobilization at the K72., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

A regenerable immunoaffinity layer comprising covalently immobilized orientation-controlled antibodies was developed for use in a surface plasmon resonance (SPR) biosensor. For antibody orientation control, antibody-binding Z-domain-autodisplaying Escherichia coli ( E. coli ) cells and their outer membrane (OM) were utilized, and a disuccinimidyl crosslinker was employed for covalent antibody binding. To fabricate the regenerable immunoaffinity layer, capture antibodies were bound to autodisplayed Z-domains, and then treated with the crosslinker for chemical fixation to the Z-domains. Various crosslinkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and poly (ethylene glycol)-ylated bis (sulfosuccinimidyl)suberate (BS(PEG) 5 ), were evaluated, and DSS at a concentration of 500 μM was confirmed to be optimal. The E. coli -cell-based regenerable HRP immunoassay was evaluated employing three sequential HRP treatment and regeneration steps. Then, the Oms of E. coli cells were isolated and layered on a microplate and regenerable OM-based HRP immunoassaying was evaluated. Five HRP immunoassays with four regeneration steps were found to be feasible. This regenerable, covalently immobilized, orientation-controlled OM-based immunoaffinity layer was applied to an SPR biosensor, which was capable of quantifying C-reactive protein (CRP). Five regeneration cycles were repeated using the demonstrated immunoaffinity layer with a signal difference of <10%.

We demonstrate a versatile nanoparticle with imaging-guided chemo-photothermal synergistic therapy and EpCAM-targeted delivery of liver tumor cells. EpCAM antibody (anti-EpCAM) and Pt(IV) were grafted onto the polydopamine carbon dots (PDA-CDs) by the amidation reaction. The EpCAM antibody of particles enables the targeted interaction with liver progenitor cells due to their overexpressed EpCAM protein. The tetravalent platinum prodrug [Pt(IV)] induces apoptosis with minimum toxic side effects through the interaction between cisplatin and tumor cell DNA. The nanoparticles displayed stable photothermal property and considerable anti-tumor therapeutic effect in vivo . Coupling with cellular imaging due to their fluorescence property, anti-EpCAM@PDA-CDs@Pt(IV) offers a convenient and effective platform for imaging-guided chemo-photothermal synergistic therapy toward liver cancers in the near future.

In this study, immobilization of single-chain Fv (scFv) antibodies on the surfaces of polystyrene (PS) latex beads via material-binding peptides was investigated for sensitive immuno-turbidimetric assay of C-reactive protein (CRP). Anti-CRP scFvs fused with polystyrene-binding peptide (PS-tag) and poly(methylmethacrylate)-binding peptide (PMMA-tag) were over-expressed in Escherichia coli cells and recovered in the active form following refolding. The beads with PMMA-tag-fused scFv (scFv-PM) were successfully suspended with sufficient dispersion at pH 8.0. Three types of alternative scFv-PMs with a penta-asparatic acid tag (D5-tag) introduced at different positions were then designed. All of the D5-tagged scFv-PMs were successfully immobilized on the surfaces of beads with no significant change in the diameter of the latex beads at pH levels ranging from 6.0 to 8.0. According to the results of turbidimetric assay for the detection of CRP, 13 ng/ml of CRP was detectable using beads with D5-tagged scFv-PMs at 400 ng/cm 3 , and no turbidity change was observed in the absence of antigen. When the density of scFv-PM was 250 ng/cm 2 , which was 63% of the maximum density, the beads were dispersed well and reactive with the antigen at a concentration range comparable to those with D5-tagged scFv-PMs. These results indicate that controlling charge density on the surface of beads after site-directed immobilization is definitely important in order to maintain high levels of dispersion and reactivity. Thus, the usefulness of the scFv-PM as well as D5-tagged scFv-PMs developed in the present study should be significant when used as ligand antibodies in the preparation of immuno-latex beads., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Sandwich-type electrochemical immunosensor was one of the main methods for detecting carcinoembryonic antigen (CEA). In this work, using Ce-MoF as the skeleton precursor, hyaluronic acid (HA) was coated on the surface of Ce-metal organic framework (Ce-MoF), which loaded with silver nanoparticles (Ag NPs) and horseradish peroxidase (HRP) to catalyze H 2 O 2 and double amplified the current signal. Thus, a sensitive sandwich-type electrochemical immunosensor (Ce-MoF@ HA/Ag-HRP) was designed to detect carcinoembryonic antigen (CEA). The designed immunosensor used Au NPs to enhance the ability of attach more the first antibody (Ab 1 ). This was due to Au NPs had good electrical conductivity and biocompatibility to accelerate electron transfer on the surface of the electrode. HA was riched in -COOH, -OH and had excellent biocompatibility, which can carry more Ag NPs to catalyze H 2 O 2 . Finally, the prepared sandwich-type electrochemical immunosensor had excellent biocompatibility and great catalytic performance. The immunosensor can be tested within 30 min and the logarithm of the current signal and CEA concentration showed a broad linear response range of 1 pg ml -1 -80 ng ml -1 , and the detection limit of CEA was 0.2 pg ml -1 . More importantly, the proposed immunosensor had good reproducibility, selectivity, stability and without matrix effect. This confirmed that the proposed immunosensor had broad prospects in early clinical trials.

In this study, haptens were designed to produce highly sensitive and specific monoclonal antibodies (mAb) against carbamazepine (CBZ) and its metabolite carbamazepine-10, 11-epoxide (CBZ-EP). According to the results of our competitive enzyme-linked immunosorbent assay (ic-ELISA), the half-maximum inhibitory concentration values for anti-CBZ and anti-CBZ-EP mAb were 0.18 and 0.59 ng/mL, respectively. An immunochromatographic assay (ICA) was developed for the determination of CBZ and CBZ-EP concentrations. This method can provide visible limits of detection ranging from 0.25 to 1 ng/mL, and cut-off limits ranging from 5 to 10 ng/mL, and takes 10 min to evaluate with the naked eye. Importantly, these observations were consistent with those obtained by ic-ELISA and liquid chromatography-mass spectrometry. The ICA assay represented a reliable, fast, and high-throughput method for the determination of CBZ and CBZ-EP in serum samples., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We report cloning and expressing of recombinant human VEGF-A 165 , fused at the N-terminal with Hydrophobin II (HFBII) from Trichoderma reseei, in yeast Pichia pastoris. We validated the construct using SDS-PAGE and ELISA against VEGF-A 165 and efficiently performed protein purification and enrichment based on HFBII counterpart and using an aqueous two-phase system (ATPS) with nonionic surfactant X-114. We studied the effects of various culture medium additives and interaction effects of positive factors to increase the recombinant HFBII-VEGF-A 165 production. Supplementing the Pichia pastoris cell culture medium with Mg 2+ , Polysorbate 20 (PS 20), and 4-phenylbutyrate (PBA) improved the expression of the chimeric protein. Orthogonal experiments showed that the optimal condition to achieve maximal HFBII-VEGF-A 165 production was with the addition of PBA, PS 20, and MgSO 4 . Under this condition, the production of the target protein was 4.5 times more than that in the medium without the additives. Overall, our approach to produce chimeric HFBII-VEGF-A 165 and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A cohort of monoclonal antibodies (mAbs) were generated against Staphylococcal enterotoxin-B (SEB) and selected by double sandwich enzyme-linked immunosorbent assay (ELISA) for solution capture of the toxin. Clonal hybridoma cell lines were established and a pair of anti-SEB mAbs selected for the development of a sandwich ELISA. Immobilized 3D6 mAb (IgG1, kappa) when paired with 4C9 mAb (IgG1, kappa) conjugated to horseradish peroxidase generates a typical dose-response curve with an EC 50 of 24.8 ng/mL for purified SEB using chemiluminescent detection. These mAbs bind SEB by Western blot and ELISA binding to classical enterotoxin serotypes show that the 3D6 mAb binds both SEB and the SEC1 serotypes, whereas 4C9 binds only SEB. These mAbs effectively port onto lateral flow test strips with a visual detection sensitivity for SEB of 5 ng/mL in <10 minutes using a 4C9 conjugated to a 40 nm gold reporter.

We report a novel immunocapture (IC)-LC-MS/MS methodology to directly measure real time in vivo receptor occupancy (RO) for a covalent binding drug in blood lysate. A small molecule quencher was added immediately after sample collection to convert the free receptor to a quencher-bound receptor (QB-R) which was measured with the drug-bound receptor (DB-R) simultaneously by LC-MS/MS after immunocapture enrichment, followed by trypsin digestion. Addition of the quencher is necessary to prevent the free receptor from ex vivo binding with the drug. The real time RO was calculated based on the concentrations of DB-R and the free receptor (which is now QB-R) that were obtained from each sample. This strategy has been successfully applied to the measurement of the RO for Bruton's tyrosine kinase (BTK) in the blood lysate of monkeys after dosing with branebrutinib (BMS-986195), a covalent BTK inhibitor being evaluated to treat rheumatoid arthritis. A custom-made quencher, which is more reactive to BTK than branebrutinib, was added in excess amount to bind with all available free BTK to form quencher-bound BTK (QB-BTK) during blood sample collection. To measure a wide range of % BTK RO, including those of <5% or >95%, the required LLOQ at 0.125 nM for QB-BTK and 0.250 nM for drug-bound BTK (DB-BTK) in blood lysate were successfully achieved by using this IC-LC-MS/MS strategy. This proof-of-concept assay demonstrated its suitability with high throughput for real time in vivo BTK RO measurement as a pharmacodynamic (PD) biomarker for clinical drug development.

A dual sites affinity protocol was developed for fluorescent analysis of Staphylococcus aureus (S. aureus) by employing daptomycin and immunoglobulin G (IgG) as the recognition elements. Pig IgG immobilized on microplate was employed as the first recognition element to capture S. aureus owing to the fact that the Fc segment of mammal IgG can selectively bind with protein A on the surface of the target bacteria. Meanwhile, fluorescein isothiocyanate-conjugated daptomycin was employed as the second recognition element as well as the signal tracer for the target bacteria utilizing the binding capability of daptomycin to Gram-positive bacteria. S. aureus can be analyzed within a concentration range of 5.0 × 10 3 -5.0 × 10 8  CFU mL -1 with a detection limit of 3.6 × 10 3  CFU mL -1 . The analytical process can be accomplished within 1.5 h by using a pre-coated microplate. The dual sites affinity protocol can exclude the interference led by Gram-negative bacteria and other common Gram-positive bacteria. We have successfully applied it to analyze S. aureus in spiked lake water and physiological saline injection samples, and the recovery values ranged from 88.0% to 120.0%. The results demonstrate its application potential for environmental sanitation and drug safety control., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Immunoaffinity chromatography has a huge interest in the biomedical and biotechnological fields, in particular for one-step isolation, purification and removal of analyte compounds. In this study, uniform-sized microcryogels, a new type of cryogels, were synthesized using 2-hydroxyetyhl methacrylate and epoxy-group-containing monomer, glycidyl methacrylate for purification of a plasma protein, transferrin. Immunoaffinity microcryogels containing anti-Tf antibodies were characterized by Raman spectroscopy, Fourier transform infrared spectroscopy, optical microscopy, scanning electron microscopy, density measurements and swelling tests. Adsorption studies in aqueous media were carried out in order to examine the effects of medium pH, initial concentration of analyte and contact time. It was found that the optimum pH was 6.0 and the maximum adsorption capacity of immunoaffinity microcryogels at this pH value found to be 9.82 mg/g. The K L constant for Langmuir isotherm was calculated as 2.65 mL/mg. The maximum adsorption capacity obtained from experimental studies is also very close to the calculated Langmuir adsorption capacity (11.27 mg/g). Langmuir adsorption isotherms and pseudo-second-order kinetic models are consistent with the adsorption process, which means that the adsorption is single layered and chemically controlled. The purity of the eluted hsTf from plasma was about 84% with yield about 82%. After the tenth use of the same microcryogels, the maximum hsTf adsorption capacity decreased by about 20%. The results indicated that the immunoaffinity microcryogels having anti-Tf antibody ligands could be a safe and cost-friendly method for purification of transferrin., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Transport of the majority of therapeutic molecules to the brain is precluded by the presence of the blood-brain barrier (BBB) rendering efficient treatment of many neurological disorders impossible. This BBB, nonetheless, may be circumvented by targeting receptors and transport proteins expressed on the luminal surface of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) has remained a popular target since its original description for this purpose, although clinical progression of TfR-targeted drug constructs or nanomedicines remains unsuccessful. One proposed issue pertaining to the use of TfR-targeting in nanomedicines is the efficient tuning of the ligand density on the nanoparticle surface. We studied the impact of TfR antibody density on the uptake and transport of nanoparticles into the brain, taking a parallel approach to investigate the impact on both antibody-functionalized gold nanoparticles (AuNPs) and cargo-loaded liposomes. We report that among three different low-range mean ligand densities (0.15, 0.3, and 0.6 ∗ 10 3 antibodies/μm 2 ), the highest density yielded the highest ability towards both targeting of the BCECs and subsequent transport across the BBB in vivo, and in vitro using primary cultures of the murine BBB. We also find that TfR-targeting on liposomes in the mouse may induce severe adverse effects after intravenous administration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A MEMS-based impedance biosensor was designed, fabricated, and tested to effectively detect the presence of bacterial cells including E. coli O157:H7 and Salmonella typhimurium in raw chicken products using detection region made of multiple interdigitated electrode arrays. A positive dielectrophoresis based focusing electrode was used in order to focus and concentrate the bacterial cells at the centerline of the fluidic microchannel and direct them toward the detection microchannel. The biosensor was fabricated using surface micromachining technology on a glass substrate. The results demonstrate that the device can detect Salmonella with concentrations as low as 10 cells/mL in less than 1 h. The device sensitivity was improved by the addition of the focusing electrodes, which increased the signal response by a factor between 6 and 18 times higher than without the use of the focusing electrodes. The biosensor is selective and can detect other types of pathogen by changing the type of the antibody immobilized on the detection electrodes. The device was able to differentiate live from dead bacteria., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we fabricated a label-free electrochemical immunosensor for sensitive and selective detection of tumor marker p53. This immunosensor was based on chitosan/carbon black composite (Chitosan-CB) layer coated ITO electrode. This composite was utilized for enhancement of the conductivity of the immunosensor. Anti-p53 antibodies were captured on the modified ITO electrode through the cross-linking of chitosan and glutaraldehyde. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques were utilized for electrochemical characterization of the proposed immunosensor. Moreover, the biosensor construction steps were monitored by using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The immobilization of anti-p53 antibodies on the electrode surface was investigated by using Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy. The change in impedance which formed during the specific interaction between anti-p53 antibody and p53 antigen was used to detect p53. Under optimized experimental conditions, the fabricated immunosensor had a wide linear range of 0.01-2 pg/mL and low detection limit of 3 fg/mL. The fabricated immunosensor had good sensitivity, stability and repeatability. Furthermore, it was successfully applied to analyze p53 in human serum., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An approach based on multifunctional fluorescent magnetic nanoparticles was proposed for the enrichment and identification of Alicyclobacillus spp. in apple juice simultaneously. The prepared Fe 3 O 4 magnetic particles (MNPs) were modified by the sol-gel process and a silica shell was formed to improve the reactivity, and then the obtained MNPs@SiO 2 -SH nanoparticles were conjugated with Thioglycolic acid functionalized CdTe/CdS QDs via thiols chemistry. The characteristic evaluation results indicated that the MNPs-QD nanocomposites exhibited good magnetic properties and optical characterization. The polyclonal anti-Alicyclobacillus IgG antibody was immobilized onto the surface of MNPs-QD materials via esterification reactions. The maximum antibody immobilization capacity was 119.62 μg/mg and the adsorption reaction could be accomplished in 60 min. The adsorption process could be represented by Langmuir model and pseudo-second order kinetics equation, respectively. Based on the high immunocapture efficiency and sensitive fluorescence characteristics, the obtained MNPs-QDs-antibody conjugates could be applied to recognize the contamination of Alicyclobacillus spp. and a quantitative analysis method was established for target cells detection. The minimum quantitative limit was 10 4  CFU (colony forming unit)/mL and the testing process could be completed in 90 min. The results indicated that the MNPs-QDs-antibody conjugates can be successfully applied for immunocapture and detection of Alicyclobacillus spp. in apple juice. That is to say, the developed MNPs-QDs-antibody conjugates have exhibit more attractive and great potential for the immunocapture and recognition of target bacteria, fully demonstrated a new method for enrichment and rapid detection of Alicyclobacillus spp. in fruit juices., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The accurate and rapid detection of cardiac troponin I (cTnI) at an early stage can prevent people from getting acute myocardial infarction to a great extent. Here, an ultrasensitive sandwich-type electrochemical immunosensor for the quantitative detection of cTnI is designed based on a novel signal amplification strategy. Gold nanoparticles (Au NPs) doped covalent organic frameworks (COFs) and electron mediator toluidine blue (TB-Au-COFs) behave as labels for achieving signal amplification. Note that polypyrrole modified titanium dioxide nanoparticles (TiO 2 -PPy) with a high electronic conductivity and a large specific surface area can promote the performance of the immunosensor. For the immobilization of primary antibodies, Au NPs decorated TiO 2 -PPy (TiO 2 -PPy-Au) is used as the substrate material to improve electron transfer. The proposed method exhibits a linear range from 0.5 pg mL -1 to 10.0 ng mL -1 and a low detection limit of 0.17 pg mL -1 (S/N = 3). The immunosensor exhibits good stability, acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A new approach to enhance the electrochemical performance of biosensor was attempted by using Super P© carbon black/Star polymer composite material. In this study, we developed an electrochemical IL 8 biosensor by modification with a conductive composite including Super P, polyvinylidene fluoride (PVDF) and star polymer (SPGMA) of disposable ITO electrode surface. The Super P carbon black as carbonaceous material had a high conductivity and was used for the enhancement of electron transfer between electrode surface and electrolyte. Anti-IL 8 antibodies were utilized as biorecognition molecules and bound to epoxy groups of star polymer covalently. The chemical characterization of antibody immobilization on this composite was performed by using Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy. The characterizations of stepwise modification of this immunosensor were performed by electrochemical techniques such as Electrochemical Impedance Spectroscopy (EIS), Cyclic Voltammetry (CV) and Single Frequency Impedance (SFI); and morphological techniques such as Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Several variables that affect the immunosensor performance were optimized. Under optimum conditions, a wide linear range 0.01-3 pg/mL and low detection limit 3.3 fg/mL were obtained. Super P-star polymer composite modified immunosensor was easy, sensitive, cheap and reliable analytical method for IL 8 detection. The applicability of the proposed immunosensor to determine IL 8 in saliva and serum samples were examined. The results of biosensor and Enzyme-linked Immunosorbent Assay (ELISA) kit were in compatible. Consequently, it was concluded that the electrochemical immunosensor offers a potential approach for IL 8 detection in clinical applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Gap junctions, transmembrane protein channels that directly connect the cytoplasm of neighboring cells and enable the exchange of molecules between cells, are a promising new frontier for therapeutic delivery. Specifically, cell-derived lipid vesicles that contain functional gap junction channels, termed Connectosomes, have recently been demonstrated to substantially increase the effectiveness of small molecule chemotherapeutics. However, because gap junctions are present in nearly all tissues, Connectosomes have no intrinsic ability to target specific cell types, which potentially limits their therapeutic effectiveness. To address this challenge, here we display targeting ligands consisting of single-domain antibodies on the surfaces of Connectosomes. We demonstrate that these targeted Connectosomes selectively interact with cells that express a model receptor, promoting the selective delivery of the chemotherapeutic doxorubicin to this target cell population. More generally, our approach has the potential to boost cytoplasmic delivery of diverse therapeutic molecules to specific cell populations while protecting off-target cells, a critical step toward realizing the therapeutic potential of gap junctions.

Platelets can be found on the surface of inflamed and ruptured atherosclerotic plaques. Thus, targeting of activated platelets may allow for molecular imaging of vulnerable atherosclerotic lesions. We here investigated microbubbles (MB) functionalized with the selectin ligand sialyl Lewis a individually (MB sLea ) or dually with sLe a and an antibody targeting ligand-induced binding sites of the activated GPIIb/IIIa receptor (MB Dual ). Assessed by in vitro flow chamber, targeted MB exhibited increased adhesion to platelets as compared to MB Control . While MB sLea rolled slowly on the platelets' surface, MB Dual enhanced the percentage of firm adhesion. In vivo, MB were investigated by ultrasound in a model of ferric chloride induced non-occlusive carotid artery thrombosis. MB sLea and MB Dual revealed a higher ultrasound mean acoustic intensity than MB Control (p < 0.05), however MB Dual demonstrated no additional increase in mean signal intensity as compared to MB sLea . The degree of carotid artery stenosis on histology correlated well with the ultrasound acoustic intensity of targeted MB (p < 0.05). While dual targeting of MB using fast binding carbohydrate polymers and specific antibodies is a promising strategy to support adhesion to activated platelets under arterial shear stress, these advantages seem not readily translatable to in vivo models.

Fibrinogen (FIB) is a secretory glycoprotein synthesized by hepatocytes that has a key role in blood clotting. Its glycosylation has not been studied in detail and little is known about the biological variability of FIB N-glycosylation, mainly due to the lack of fast, simple, and robust approaches to purify FIB from blood plasma samples. In recent years, customised chromatographic monoliths have been used for a variety of biological applications due to their unique characteristics. Here we describe development and optimisation of monolithic supports bearing monoclonal anti-human fibrinogen antibodies in a single column as well as in multi-well plate formats with high FIB specificity and binding capacity for fast immunoaffinity purification of FIB from human blood samples. The developed semi-high-throughput workflow has been successfully applied for FIB immunoaffinity isolation and subsequent ultra performance liquid chromatography N-glycosylation analysis in ten healthy human individuals, demonstrating the potential of monolithic supports in glycomics studies., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

15α-Hydroxyestrogens (15α-OHEs) are metabolites of the female hormone estradiol. In this study, to discover physiological markers that can be utilized for monitoring fetal conditions and estrogen-induced cancers, we established a method for quantifying 15α-OHEs in rat urine via immunoaffinity column extraction and HPLC-electrochemical detection, and detected 15α-OHEs in urine obtained male rats treated with estradiol. Notably, the standard curves for quantification obtained using the column were linear over a range of 0.5-50ng 15α-OHEs. The accuracy of the analytical method with cleanup was 97-109% for the three kinds of 15α-OHEs examined, and the intra-assay precision of the measured values had a coefficient of variation of ≤20.6%. Therefore, the theoretical limit of quantification was 0.5ng. However, the actual measured values obtained from the urine of male rats indicated that the detection limits were 0.425, 0.103, and 0.047ng for estetrol, 15α-hydroxyestradiol, and 15α-hydroxyestrone, respectively. Our newly established method for measuring 15α-OHE concentrations in urine could facilitate characterization of the in vivo metabolic profile of 15α-OHEs in mammals under various physiological conditions, which could comprise clinical markers for monitoring human fetal health conditions in mammals., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Immunosensors are used to detect the presence of certain bio-reagents mostly targeted at the diagnosis of a condition or a disease. Here, a general purpose electrical immunosensor has been fabricated for the quantitative detection of multiple bio-reagents through the formation of an antibody-antigen pair. The sensors were fabricated using all printing approaches. 2D transition metal dichalcogenide (TMDC) MoS 2 thin film was deposited using Electrohydrodynamic atomization (EHDA) on top of an interdigitated transducer (IDT) electrode fabricated by reverse offset printing. The sensors were then treated with three different types of antibodies that were immobilized by physisorption into the highly porous multi-layered structure of MoS 2 active layer. BSA was used as blocking agent to prevent non-specific absorption (NSA). The sensors were then employed for the targeted detection of the specific antigens including prostate specific antigen (PSA), mouse immunoglobulin-G (IgG), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). IgG was then selected to test the sensors for point of care (POC) diagnosis through a specially designed electronic readout system for sensors and interfacing it with a smartphone using Bluetooth connection. The sensors showed promising performance in terms of stability, specificity, repeatability, sensitivity, limit of detection (LoD), and range of detection (RoD).

Objective: To develop a high-sensitivity immunochromatographic test for fumonisin B1 in plant extracts., Results: Unlike conventional immunochromatographic tests, this assay is performed in two stages: competitive reaction with free specific antibodies and identifying immune complexes by their interaction with the anti-species antibody-conjugated gold nanoparticles. The use of a new geometry for the test strip membranes and a novel reagent application method ensures the proper order of these stages without additional manipulations. The contact of the ready-to-use test strip with the liquid sample suffices in initiating all stages of the assay and obtaining test results. The developed test was used on corn extracts; its instrumental limit of fumonisin B1 detection was 0.6 ng ml -1 at 15 min of assay duration., Conclusions: The proposed approach is flexible and can be used for a wide range of low molecular compounds. The use of anti-species antibody-conjugated gold nanoparticles in immunochromatography significantly facilitates the development of test systems by eliminating the need to synthesize and characterize the conjugates with specific antibodies for each new compound to be detected.

The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to bind to the generic structure of amyloid deposits and possibly to protect them against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca 2+ concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins, of which 24 were direct or indirect interaction partners not previously reported. The SAP interactome can be divided into categories that include apolipoproteins, the complement system, coagulation, and proteolytic regulation.

Background: Biosensor-based detection provides a rapid and low-cost alternative to conventional analytical methods for revealing the presence of the contaminants in water as well as solid matrices. Although important to be detected, small analytes (few hundreds of Daltons) are an issue in biosensing since the signal they induce in the transducer, and specifically in a Quartz-Crystal Microbalance, is undetectable. A pesticide like parathion (M = 292 Da) is a typical example of contaminant for which a signal amplification procedure is desirable., Methods/findings: The ballasting of the analyte by gold nanoparticles has been already applied to heavy target as proteins or bacteria to improve the limit of detection. In this paper, we extend the application of such a method to small analytes by showing that once the working surface of a Quartz-Crystal Microbalance (QCM) has been properly functionalized, a limit of detection lower than 1 ppb is reached for parathion. The effective surface functionalization is achieved by immobilizing antibodies upright oriented on the QCM gold surface by a simple photochemical technique (Photonic Immobilization Technique, PIT) based on the UV irradiation of the antibodies, whereas a simple protocol provided by the manufacturer is applied to functionalize the gold nanoparticles. Thus, in a non-competitive approach, the small analyte is made detectable by weighing it down through a "sandwich protocol" with a second antibody tethered to heavy gold nanoparticles. The immunosensor has been proved to be effective against the parathion while showing no cross reaction when a mixture of compounds very similar to parathion is analyzed., Conclusion/significance: The immunosensor described in this paper can be easily applied to any small molecule for which polyclonal antibodies are available since both the functionalization procedure of the QCM probe surface and gold nanoparticle can be applied to any IgG, thereby making our device of general application in terms of target analyte., Competing Interests: The authors have declared that no competing interests exist.

Nanoparticles have become a powerful tool for cell imaging and biomolecule, cell and protein interaction studies, but are difficult to rapidly and accurately measure in most assays. Dark-field microscope (DFM) image analysis approaches used to quantify nanoparticles require high-magnification near-field (HN) images that are labor intensive due to a requirement for manual image selection and focal adjustments needed when identifying and capturing new regions of interest. Low-magnification far-field (LF) DFM imagery is technically simpler to perform but cannot be used as an alternate to HN-DFM quantification, since it is highly sensitive to surface artifacts and debris that can easily mask nanoparticle signal. We now describe a new noise reduction approach that markedly reduces LF-DFM image artifacts to allow sensitive and accurate nanoparticle signal quantification from LF-DFM images. We have used this approach to develop a "Dark Scatter Master" (DSM) algorithm for the popular NIH image analysis program ImageJ, which can be readily adapted for use with automated high-throughput assay analyses. This method demonstrated robust performance quantifying nanoparticles in different assay formats, including a novel method that quantified extracellular vesicles in patient blood sample to detect pancreatic cancer cases. Based on these results, we believe our LF-DFM quantification method can markedly decrease the analysis time of most nanoparticle-based assays to impact both basic research and clinical analyses.

Background: The field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with not only insights into basic cell biology but may also enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role. The nanoneedle technology is a powerful tool allowing for intracellular investigations, as it can be directly inserted into live cells by penetrating through the plasma membrane causing minimal damage to cells, under the precise manipulation using atomic force microscope. Modifications of the nanoneedles using antibodies have allowed for accurate mechanical detection of various cytoskeletal components, including actin, microtubules and intermediate filaments. However, successful penetration of the nanoneedle through the plasma membrane has been shown to vary greatly between different cell types and conditions. In an effort to overcome this problem and improve the success rate of nanoneedle insertion into the live cells, we have focused here on the fluidity of the membrane lipid bilayer, which may hinder nanoneedle penetration into the cytosolic environment., Results: We aimed to reduce apparent fluidity of the membrane by either increasing the approach velocity or reducing experimental temperatures. Although changes in approach velocity did not have much effect, lowering the temperature was found to greatly improve the detection of unbinding forces, suggesting that alteration in the plasma membrane fluidity led to increase in nanoneedle penetration., Conclusions: Operation at a lower temperature of 4 °C greatly improved the success rate of nanoneedle insertion to live cells at an optimized approach velocity, while it did not affect the binding of antibodies immobilized on the nanoneedle to vimentins for mechanical detection. As these experimental parameters can be applied to various cell types, these results may improve the versatility of the nanoneedle technology to other cell lines and platforms.

Here we developed an integrated volumetric bar-chart chip (IV-Chip) technology by integration of our previous V-Chip with a fluid handling design to generate an instrument-free POC device and greatly reduce the detection time and effort. The IV-Chip test requires only 1 μL of serum separated from finger-prick blood. The serum sample and ELISA reagents are directly loaded into the device using a pipette, and a shift of the two layers of the device generates homogeneous liquid segments in the microfluidic channel. Under vacuum pressure generated by a regular syringe, the segments flow into the ELISA wells in sequence and a sandwich ELISA reaction takes place. As a result of the automated washing and reacting strategy, the IV-Chip allows rapid tests for myocardial infarction biomarkers, and turnaround time is greatly reduced to 15 min. The specificity and accuracy of quantitative multiplex detection of MI biomarkers CK-MB, troponin I and myoglobin, are 87.5% and 95.8%, respectively.

Microfluidic technologies have miniaturized a variety of biomedical applications, and these chip-based systems have several significant advantages over their large-scale counterparts. Recently, this technology has been used for automating labor-intensive and time-consuming screening processes, whereby affinity reagents, including aptamers, peptides, antibodies, polysaccharides, glycoproteins, and a variety of small molecules, are used to probe for molecular biomarkers. When compared to conventional methods, the microfluidic approaches are faster, more compact, require considerably smaller quantities of samples and reagents, and can be automated. Furthermore, they allow for more precise control of reaction conditions (e.g., pH, temperature, and shearing forces) such that more efficient screening can be performed. A variety of affinity reagents for targeting cancer cells or cancer biomarkers are now available and will likely replace conventional antibodies. In this review article, the selection of affinity reagents for cancer cells or cancer biomarkers on microfluidic platforms is reviewed with the aim of highlighting the utility of such approaches in cancer diagnostics.

Antibody fragments, such as the single-chain variable fragment (scFv), have much potential in research and diagnostics because of their antigen-binding ability similar to a full-sized antibody and their ease of production in microorganisms. Some applications of antibody fragments require immobilization on a surface, and we have established a simple immobilization method that is based on the biotin-streptavidin interaction and does not require a separate purification step. We genetically fused two biotinylation tags-the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequence-to six different scFvs (scFv13R4, scFvD10, scFv26-10, scFv3, scFv5, and scFv12) for site-specific biotinylation in vivo by endogenous biotin ligases produced by Escherichia coli. The biotinylated scFvs were immobilized onto streptavidin-coated plates directly from cell lysates, and immobilization was detected through enzyme-linked immunosorbent assays. All scFvs fusions were successfully immobilized, and scFvs biotinylated via the BCCP tag tended to immobilize better than those biotinylated via the AviTag, even when biotinylation efficiency was improved with the biotin ligase BirA. The ability of immobilized scFvs to bind antigens was confirmed using scFv13R4 and scFvD10 with their respective targets β-galactosidase and bacteriophage lambda head protein D (gpD). The immobilized scFv13R4 bound to β-galactosidase at the same level for both biotinylation tags when the surface was saturated with the scFv, and immobilized scFvs retained their functionality for at least 100days after immobilization. The simplicity and robustness of our method make it a promising approach for future applications that require antibody fragment immobilization., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The purpose of this study was the measurement of enzyme activity within a single-drop of biological fluid after micropurification. Esterase and lactate dehydrogenase (LDH) retained their enzymatic activities after being captured by membrane-immobilized antibodies, which were prepared by non-denaturing two-dimensional electrophoresis, transferred to polyvinylidene difluoride and then stained by Ponceau S. The activities of both enzymes were also measured after being captured by antibodies and biotinylated antibodies bound to membrane-immobilized protein A or avidin, respectively. After esterase and LDH were captured from biological samples by membrane-immobilized protein A or avidin, their activities were semi-quantitatively measured on the surface of the membrane using fluorescence determination. More than 51% of enzyme activities were retained even after the enzymes were captured by biotinylated antibody bound to membrane-immobilized avidin and eluted by rinsing with 5μL of 1% Triton X-100, compared with the activities of the enzyme on the immunoaffinity membrane., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A colloidal gold-based immunochromatographic (IC) strip test was developed and validated for the detection of HIV-1 protease (HIV-PR) activity and inhibitory effect of HIV-PR inhibitors (PIs). It is a unique 'two-step' process requiring the combination of proteolysis of HIV-PR and an immunochromatographic reaction. Monoclonal antibodies to the free C-terminus of HIV matrix protein (HIV-MA) conjugated to gold particles and a monoclonal antibody against intact and cleaved forms of the HIV-MA are immobilized on the 'Test'-line of the IC strip. Using lopinavir, a potent HIV protease inhibitor, the IC-strip was optimized to detect inhibitory activity against HIV-protease. At a lopinavir concentration of 1000ng/mL (its suggested minimum effective concentration), a HIV-PRH6 concentration of 6mg/mL and incubation period of 60min were the optimal conditions. A preliminary comparison between a validated high-performance liquid chromatography assay and the IC-strip to semi-quantify HIV protease inhibitor concentrations (lopinavir and atazanavir) demonstrated good agreement. This simplified method is suitable for the rapid screening of novel protease inhibitors for future therapeutic use. Moreover, the IC strip could also be optimized to semi-quantify PIs concentrations in plasma samples., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first application of immunoaffinity chromatography for the analysis of MTX., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 μm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL.

Studying protein interactions is of vital importance both to fundamental biology research and to medical applications. Here, we report on the experimental proof of a universally applicable label-free homogeneous platform for rapid protein analysis. It is based on optically detecting changes in the rotational dynamics of magnetically agitated core-shell nanorods upon their specific interaction with proteins. By adjusting the excitation frequency, we are able to optimize the measurement signal for each analyte protein size. In addition, due to the locking of the optical signal to the magnetic excitation frequency, background signals are suppressed, thus allowing exclusive studies of processes at the nanoprobe surface only. We study target proteins (soluble domain of the human epidermal growth factor receptor 2 - sHER2) specifically binding to antibodies (trastuzumab) immobilized on the surface of our nanoprobes and demonstrate direct deduction of their respective sizes. Additionally, we examine the dependence of our measurement signal on the concentration of the analyte protein, and deduce a minimally detectable sHER2 concentration of 440 pM. For our homogeneous measurement platform, good dispersion stability of the applied nanoprobes under physiological conditions is of vital importance. To that end, we support our measurement data by theoretical modeling of the total particle-particle interaction energies. The successful implementation of our platform offers scope for applications in biomarker-based diagnostics as well as for answering basic biology questions.

Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes., (Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the current study we have investigated the protein content of blood plasma deoxyribonucleoprotein complexes. The complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies. Proteins were separated by SDS PAAGE and identified by MALDI-TOF mass-spectrometry. 111 and 56 proteins (excluding histones), respectively, were identified with a good score in deoxyribonucleoprotein complexes of healthy females and breast cancer patients. However, only four of these proteins were found in 30 % of all samples. Fourteen proteins previously described as tumor specific proteins were found in cancer patients whereas not one of them was found in healthy individuals. The data obtained demonstrate the involvement of different cellular and extracellular proteins in circulating cell-free DNA.

An antibody-modified reduced graphene oxide (rGO) film with unexpected -extreme sensitivity to circulating tumor cells (CTCs) is reported. The antibody--modified rGO films efficiently capture CTCs from billions of blood cells and minimize the background of white blood cells, without complex microfluidic operations., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Targeting nanoparticles to desired intracellular compartments is a major challenge. Integrin-type adhesion receptors are connected to different endocytosis routes in a receptor-specific manner. According to our previous observations, the internalization of an α2β1-integrin-echovirus-1 complex takes place via a macropinocytosis-like mechanism, suggesting that the receptor could be used to target nanoparticles to this specific entry route. Here, silica-based nanoparticles, carrying monoclonal antibodies against the α2β1 integrin as address labels, were synthesized. Studies with flow cytometry, atomic force microscopy and confocal microscopy showed the particles to attach to the cell surface via the α2β1 integrin. Furthermore, quantitative analysis of nanoparticle trafficking inside the cell performed with the BioImageXD software indicated that the particles enter cells via a macropinocytosis-like process and end up in caveolin-1 positive structures. Thus, we suggest that different integrins can guide particles to distinct endocytosis routes and, subsequently, also to specific intracellular compartments. In addition, we show that with the BioImageXD software it is possible to conduct sensitive and complex analyses of the behavior of small fluorescent particles inside cells, using basic confocal microscopy images.

A nonenzymatic electrochemical immunosensor based on palladium nanoparticles conjugated reduced graphene nanosheets (Pd-GS) for sensitive detection of cancer biomarker a-fetoprotein (AFP) is described. Primary antibody-AFP (Ab1) was immobilized onto the surface of reduced graphene nanosheets (rGO) through an amidation reaction between the carboxylic acid group of the rGO and the available amine groups of Ab1. Pd-GS which was prepared by one-spot synthesis is employed to immobilize secondary antibody (Ab2). The resulting Pd-GS-Ab2 conjugate was used as a label for the immunosensor to detect AFP. The amplified immunoassay exhibits high sensitivity, wide linear rang (0.01-10 ng/mL), low detection limit (3.0 pg/mL), acceptable stability and reproducibility. And such immunosensor also shows good recovery in the assay results for AFP in human serum samples.

A PMMA-binding peptide (PMMA-tag) was genetically fused with the C-terminal region of an anti-human chorionic gonadotropin (hCG) single-domain antibody (VHH). It was over-expressed in an insoluble fraction of E. coli cells, and recovered in the presence of 8 M urea via one-step IMAC purification. Monomeric and denatured PMMA-tag-fused VHH (VHH-PM) was successfully prepared via the reduction and oxidation of VHH-PM at a concentration less than 1 mg/mL in the presence of 8 M of urea. Furthermore, the VHH-PM was refolded with a recovery of more than 95% by dialysis against 50 mM TAPS at pH 8.5, because the genetic fusion of PMMA-tag resulted in a decrease in the apparent isoelectric point (pI) of the fusion protein, and its solubility at weak alkaline pH was considerably increased. The antigen-binding activities of VHH-PM in the adsorptive state were 10-fold higher than that of VHH without a PMMA-tag. The density of VHH-PM on a PMMA plate was twice that of VHH, indicating that the site-directed attachment of a PMMA-tag resulted in positive effects to the adsorption amount as well as to the orientation of VHH-PM in its adsorptive state. The preparation and immobilization methods for VHH-PM against hCG developed in the present study were further applied to VHH-PMs against four different antigens, and consequently, those antigens with the concentrations lower than 1 ng/mL were detected by the sandwich ELISA. Thus, the VHH-PMs developed in the present study are useful for preparation of high-performance and economical immunosorbent for detection of biomarkers., (© 2015 American Institute of Chemical Engineers.)

Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 °C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10(-9) M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal., Competing Interests: Statement The authors have no competing interests., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Protein A from Staphylococcus aureus specifically binds to the Fc region of immunoglobulin G (IgG) and is widely used as a scaffold for the immobilization of IgG antibodies on solid supports. It is known that the oriented immobilization of Protein A on solid supports enhances its antibody-binding capability in comparison with immobilization in a random manner. In the current work, we developed a novel method for the oriented immobilization of the IgG-binding domain of Protein A based on the biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property in that the enzyme, biotin protein ligase (BPL), forms a stable complex with its biotinylated substrate protein, biotin carboxyl carrier protein (BCCP). Here, BCCP was fused to the IgG-binding domain of Protein A, and the resulting fusion protein was immobilized on the BPL-modified gold surface of the sensor chip for quartz crystal microbalance through complexation between BCCP and BPL. The layer of the IgG-binding domain prepared in this way successfully captured the antibody, and the captured antibody retained high antigen-binding capability., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 μL/min). In stationary arrangement it is possible to detect MT concentrations up to 30 ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5 ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 μg/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Flagellated bacteria have been utilized as potential swimming micro-robotic bodies for propulsion of spherical liposome by attaching several bacteria on their surface. Liposome as a drug delivery vehicle can contain biologically active compounds. In this work, the antibody binding technique is developed to attach bacteria to liposome's surface. Consequently, the stochastic effect of bacterial propulsion of liposome is investigated analytically and experimentally. It is shown that the mobility of liposome with bacteria was higher than that of liposome without bacteria. Experimental data matches well with statistical calculation.

We describe the use of a computational neural network platform to optimize the fluorescence upon binding 5-carboxyfluorescein-d-Ala-d-Ala-d-Ala (5-FAM(DA)3 ) (1) to the antibiotic teicoplanin covalently attached to a glass slide. A three-level response surface experimental design was used as the first stage of investigation. Subsequently, three defined experimental parameters were examined by the neural network approach: (i) the concentration of teicoplanin used to derivatize a glass platform on the microfluidic device, (ii) the time required for the immobilization of teicoplanin on the platform, and (iii) the length of time 1 is allowed to equilibrate with teicoplanin in the microfluidic channel. Optimal neural structure provided a best fit model, both for the training set (r(2) = 0.961) and test set (r(2) = 0.934) data. Model simulated results were experimentally validated with excellent agreement (% difference) between experimental and predicted fluorescence shown, thus demonstrating efficiency of the neural network approach., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Reusability of a biosensor has recently received considerable attention, and it is closely related with the effective desorption of probe molecules. We present a novel mechanical desorption technique to reuse biosensors by using periodic jets of carbon dioxide (CO2) aerosols (a mixture of solid and gaseous CO2), and demonstrate its feasibility by removing physically adsorbed and covalently bonded fluorescent proteins i.e., Escherichia coli fluorescein isothiocyanate antibody and bovine serum albumin (E. coli FITC-Ab and FITC-BSA) from silicon chips. The proteins on the chip surfaces were measured by fluorescent images before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured for various concentrations (1-20 μg mL(-1)) of E. coli FITC-Ab and FITC-BSA with two different removal cycles (5 and 11 cycles; each cycle: 8s). We observed high removal efficiencies (>93.5% for physically adsorbed Ab and >84.6% for covalently bonded Ab) at 11 cycle aerosol treatment. This CO2 aerosol treatment did not undermine re-functionalization, which was confirmed by the fluorescent images of FITC-Abs for fresh and reused chips. Desorption of the immobilized layers was validated by Fourier transform infrared and X-ray photoelectron spectroscopic analyses. We also conducted an experiment on the regeneration of E. coli sensing chips using this aerosol treatment, and the chips were re-used 5 times successfully. This mechanical desorption technique is a highly effective and novel strategy for reusable biosensors., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A tumor-targeted and pH-responsive drug release system based on superparamagnetic iron oxide nanoparticles (IONPs) coated by poly(ethylene glycol) (PEG) and dodecylamine (DDA)-modified polyitaconic acid (PIA) connecting with bortezomib (BTZ) (PIA-PEG-DDA-BTZ@IOs) has been constructed and characterized. The anticancer drug BTZ was first conjugated using dopamine as the linker via catechol borate ester bond, which is acid cleavable and used as an ideal pH-responsive drug release system. The IONPs were then coated by PIA-PEG-DDA-BTZ to form micelles with good biocompatibility. The conjugates were further designed to target liver cancer cells overexpressing vascular endothelial growth factor (VEGF) by the targeting molecule anti-vascular endothelial growth factor (anti-VEGF). The magnetic resonance imaging showed that the targeting capability of IONPs-anti-VEGF conjugates to Hep G2 cells was more significant than that of non-anti-VEGF IONPs. From the above, this kind of novel dual-functional targeting probe could provide a new idea for the diagnosis and treatment of cancer.