Chromatography affinity resin with photosynthetically-sourced protein A ligand.

Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.

Neuropeptidomics of Genetically Defined Cell Types in Mouse Brain.

Peptidomic techniques are powerful tools to identify peptides in a biological sample. In the case of brain, which contains a complex mixture of cell types, standard peptidomics procedures reveal the major peptides in a dissected brain region. It is difficult to obtain information on peptides within a specific cell type using standard approaches, unless that cell type can be isolated. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells. Many CPE products function as neuropeptides and/or peptide hormones, and therefore represent an important subset of the peptidome. Because CPE removes C-terminal Lys and Arg residues from peptide processing intermediates, organisms lacking CPE show a large decrease in the levels of the mature forms of most neuropeptides and peptide hormones, and a very large increase in the levels of the processing intermediates that contain C-terminal Lys and/or Arg (i.e., the CPE substrates). These CPE substrates can be purified on an anhydrotrypsin-agarose affinity resin, which specifically binds peptides with C-terminal basic residues. When this method is used with mice lacking CPE activity in genetically defined cell types, it allows the detection of peptides specifically produced in that cell type.

Rapid and sensitive quantitation of amitraz in orange, tomato, and eggplant samples using immunochromatographic assay.

Amitraz (AMT) is a broad-spectrum formamidine insecticide and acaricide. In this study, we produced an anti-AMT monoclonal antibody (mAb) with high performance. The half-maximal inhibitory concentration of the anti-AMT mAb was 4.418 ng/mL, the cross reactivity with other insecticides was negligible, and an affinity constant was 2.06 × 109 mmol/L. Additionally, we developed an immunochromatographic assay for the rapid detection of AMT residues in oranges, tomatoes, and eggplants. The cut-off values were 2000 µg/kg in oranges and tomato samples and 1000 µg/kg in eggplant samples and the calculated limits of detection were 14.521 µg/kg, 6.281 µg/kg, and 3.518 µg/kg in oranges, tomatoes, and eggplants, respectively, meeting the detection requirements for AMT in fruits and vegetables. The recovery rates ranged between 95.8 % and 105.2 %, consistent with the recovery rates obtained via LC-MS/MS. Our developed immunochromatographic assay can effectively, accurately, and rapidly determine AMT residues in oranges, tomatoes, and eggplants.

Polymer Microspheres Copolymerized with Deep Red Fluorescent Molecules as a Label for Lateral Flow Immunochromatography.

The development of fluorescently labeled microspheres is a critical aspect of advancing the technology of lateral flow immunochromatography (LFIA) for biological detection. Nevertheless, potential interference posed by the background fluorescence originating from the nitrocellulose (NC) membrane would significantly impact the sensitivity and accuracy of microsphere-based detection in LFIA. In this work, an attempt was made to extend the π-conjugated system and asymmetric structure of rhodamine fluorophore, resulting in the synthesis of dye molecules (RB2) incorporating double bonds, which can reach an absolute photoluminescence quantum yield (PLQY) of 30.01% in EtOH. Subsequently, carboxyl group functionalized fluorescent microspheres were prepared in a two-step copolymerization via soap-free emulsion polymerization. The obtained microspheres were characterized by scanning electron microscopy, transmission electron microscopy, DLS, Fourier transform infrared spectroscopy, ultraviolet spectrophotometry, and fluorescence spectrophotometry. The results showed that RB2 was successfully copolymerized into the microspheres, and the resulting microspheres had good dispersion and stability with high red fluorescence intensity (λabs ∼ 610 nm, λem ∼ 660 nm). Utilizing these microspheres, the resulting lateral flow immunoassay was successfully found to detect SARS-CoV-2 N protein with a detection limit of 2.5 pg/mL and the linear concentration spanning from 2.5 pg/mL to 10 ng/mL. The results confirm the effectiveness of the synthetic fluorescent microspheres as the label for LFIA.

Functional Green-Emitting Mn2+-doped Zinc Germanate Persistent Luminescent Nanoparticles for Dual-Mode Immunochromatographic Detection.

Immunochromatography is a commonly used immediate detection technique, using signal labels to generate detection signals for rapid medical diagnosis. However, its detection sensitivity is affected by background fluorescence caused by the excitation light source. We have developed an immunochromatographic test strip using Zn2GeO4:Mn2+ (ZGM) persistent luminescent nanoparticles (PLNPs) for immediate fluorescence detection and highly sensitive persistent luminescence (PersL) detection without background fluorescence interference. ZGM emits a strong green light when exposed to ultraviolet (UV) excitation, and its green PersL can persist for over 30 min after the excitation light is turned off. We modified the surface of ZGM with heparin-binding protein (HBP) antibodies to create immunochromatographic test strips for the detection of HBP as the target analyte. Under UV excitation, the chromatography test paper can be visually observed at concentrations as low as 25 ng/mL. After the excitation light source is switched off, PersL can achieve a detection limit of 4.7 ng/mL without background interference. This dual-mode immunochromatographic detection, based on ZGM, shows great potential for in vitro diagnostic applications.

Generation of affinity maps for thiazolidinediones with human serum albumin using affinity microcolumns. I. Studies of effects by glycation on multisite drug binding.

Human serum albumin (HSA) is known to undergo modifications by glucose during diabetes. This process produces glycated HSA that can have altered binding to some drugs. In this study, high-performance affinity microcolumns and competition studies were used to see how glycation affects the binding by two thiazolidinedione-class drugs (i.e., pioglitazone and rosiglitazone) at specific regions of HSA. These regions included Sudlow sites I and II, the tamoxifen and digitoxin sites, and a drug-binding site located in subdomain IB. At Sudlow site II, the association equilibrium constants (or binding constants) for pioglitazone and rosiglitazone with normal HSA were 1.7 × 105 M-1 and 2.0 × 105 M-1 at pH 7.4 and 37 °C, with values that changed by up to 5.7-fold for glycated HSA. Sudlow site I of normal HSA had binding constants for pioglitazone and rosiglitazone of 3.4 × 105 M-1 and 4.6 × 105 M-1, with these values changing by up to 1.5-fold for glycated HSA. Rosiglitazone was found to also bind a second region that had a positive allosteric effect on Sudlow site I for all the tested preparations of HSA (binding affinity, 1.1-3.2 × 105 M-1; coupling constant for Sudlow site I, 1.20-1.34). Both drugs had a strong positive allosteric effect on the tamoxifen site of HSA (coupling constants, 13.7-19.9 for pioglitazone and 3.7-11.5 for rosiglitazone). Rosiglitazone also had weak interactions at a site in subdomain IB, with a binding constant of 1.4 × 103 M-1 for normal HSA and a value that was altered by up to 6.8-fold with glycated HSA. Neither of the tested drugs had any significant binding at the digitoxin site. The results were used to produce affinity maps that described binding by these thiazolidinediones with HSA and the effects of glycation on these interactions during diabetes.

Size-selective sampler combined with an immunochromatographic assay for the rapid detection of airborne Legionella pneumophila.

Airborne biological aerosols (also called bioaerosols) are found in various environmental and occupational settings. Among these, pathogenic bioaerosols can cause diseases such as legionellosis, influenza, measles, and tuberculosis. To prevent or minimize people's exposure to these pathogenic bioaerosols in the field, a rapid detection method is required. In this study, a size-selective bioaerosol (SSB) sampler was combined with the immunochromatographic assay (ICA). The SSB sampler can collect bioaerosols on the sampling swab and the lateral flow test kit used in ICA can rapidly detect the pathogens in bioaerosols collected on the swab. Before testing the combined method, the lower limit of detection (LOD) of the lateral flow test kit was determined. Legionella pneumophila (L. pneumophila) was used as a target pathogen. The results show that at least 1.3 × 103L. pneumophila cells are required to be detected by the lateral flow test kit. To test the developed method, L. pneumophila suspension was aerosolized in the sampling chamber and collected using two SSB samplers with different sampling times (10 and 20 min). The developed method could detect aerosolized L. pneumophila and also estimate the concentrations from the lower LOD, sampling time, and formation of a positive line on a test strip. When positive results were obtained from sampling for 10 min and 20 min, concentrations of respirable L. pneumophila were estimated ≥5.2 × 104 CFUresp/m3 and ≥2.6 × 104 CFUresp/m3, respectively. The conventional sampler Andersen impactor with colony counting was also used for comparison. In all cases, the estimated concentrations obtained by the developed method were higher than those obtained by the conventional method. These findings confirm that the developed method can overcome the limitations of conventional methods and eventually benefit environmental and occupational health by providing a better method for risk assessment.

A label-free strategy for immobilization of GPCRs using site-specific encoded non-natural amino acids to develop a selectively chromatographic approach for pursuing potential ligands binding to 5-hydroxytryptamine 1A receptor.

G protein-coupled receptors (GPCRs) are one of the most prominent targets for drug discovery. Immobilizing GPCRs has proven to be an effective strategy for expanding the utility of GPCRs into nonbiological contexts. However, traditional strategies of immobilizing GPCRs have been severely challenged due to the loss of receptor function. Here, we reported a novel and general approach to realize the label-free and site-selective immobilization of 5-hydroxytryptamine 1A receptor (5-HT1AR) and the application in developing a chromatographic method with improved specificity for pursuing 5-HT1AR ligands from natural products. This method involved the use of a clickable non-natural amino acid, O-allyl-L-tyrosine (O-ALTyr) to immobilize the receptor onto thiol-functionalized silica gels through a 'thiol-ene' click chemistry, which allowed us to avoid the purification step and directly immobilize 5-HT1AR on silica gels. The immobilized receptor was characterized using immunofluorescence assay, and receptor-ligand interaction analysis was conducted through frontal analysis. To test the feasibility of the immobilized 5-HT1ARO-ALTyr in complex matrices, bioactive compounds in Ziziphi Spinosae Semen (ZSS) were screened and their interaction with the receptor was assessed using zonal elution. Our findings indicated that immobilizing the receptor through nnAAs effectively minimizes the chromatographic peak tailing and broadening of specific ligands, leading to a significant improvement in chromatographic performance. The association constants of the three ligands to 5-HT1AR were approximately one order of magnitude greater than those of Halo-tag attachment. These results demonstrated that the immobilized 5-HT1AR exhibits high specificity and the ability to recognize receptor ligands from complex matrices. This allowed us to identify magnoflorine (Mag) as a potential ligand of 5-HT1AR from ZSS extract. In vivo assay also proved that Mag presented a promising anxiolytic effect by promoting the expression of 5-HT1AR in mice brain. The above findings pointed to the fact that the immobilized 5-HT1AR affinity chromatographic strategy relying on the site-specific encoded non-natural amino acid is a powerful alternative for precisely determining the drug-protein interaction and discovering the specific ligand of GPCRs from complex matrixes.

Application of gold immunochromatographic assay strip combined with digital evaluation for early detection of Toxoplasma gondii infection in multiple species.

BACKGROUND: Timely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results. METHODS: Colloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera. RESULTS: AMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%-23.4% of these samples were found to be positive for T. gondii. CONCLUSIONS: This study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.

Scalable dual column cation exchange affinity chromatography based platform process for recombinant protein purification.

A novel tandem affinity tag is presented that enables the use of cation exchange resins for initial affinity purification, followed by an additional column step for enhanced purity and affinity tag self-removal. In this method, the highly charged heparin-binding tag binds strongly and selectively to either a strong or weak cation exchange resin based on electrostatic interactions, effectively acting as an initial affinity tag. Combining the heparin-binding tag (HB-tag) with the self-removing iCapTag™ provides a means for removing both tags in a subsequent self-cleaving step. The result is a convenient platform for the purification of diverse tagless proteins with a range of isoelectric points and molecular weights. In this work, we demonstrate a dual column process in which the tagged protein of interest is first captured from an E. coli cell lysate using a cation exchange column via a fused heparin-binding affinity tag. The partially purified protein is then diluted and loaded onto an iCapTag™ split-intein column, washed, and then incubated overnight to release the tagless target protein from the bound tag. Case studies are provided for enhanced green fluorescent protein (eGFP), beta galactosidase (ßgal), maltose binding protein (MBP) and beta lactamase (ßlac), where overall purity and host cell DNA clearance is provided. Overall, the proposed dual column process is shown to be a scalable platform technology capable of accessing both the high dynamic binding capacity of ion exchange resins and the high selectivity of affinity tags for the purification of recombinant proteins.

Novel melanin-derived stationary phase for immobilized metal ion affinity chromatography in recombinant His-tagged protein purification.

The matrix of the stationary phase is a crucial element in affinity chromatography for protein purification. Various materials, including polymer or magnetic materials, have been employed as the matrix in the purification of His-tagged protein. Here, for the first time, we utilized a combination of melanin and alginate, both natural polymer materials, to synthesize Ni-melanin/alginate (Ni-M/A) beads for His-tagged protein purification. We investigated the binding of His-tagged Mpro on the Ni-M/A beads, referred to as Ni-M/A-Mpro, and assessed the elution efficiency of Mpro from the beads. Our examination involved FTIR, EDS, XRD, SDS-PAGE, and Western blotting methods. FTIR spectra revealed notable changes in the stretching patterns and intensities of hydroxyl, amine, carbonyl, imine and amide chemical groups, when Mpro protein was present in the Ni-M/A sample. XRD spectra demonstrated the occurrence of two Nickel peaks at 35-40 deg and 40-45 deg in Ni-M/A, but only one nickel peak at 35-40 deg in Ni-M/A-Mpro, indicating the binding of Mpro on the Nickel ions. EDS analysis reported a decrease in the concentration of Nickel on the surface of Ni-M/A from 16% to 7% when Mpro protein was loaded into the stationary phase. Importantly, our data indicated that the purity of the His-tagged protein Mpro after purification reached 97% after just one-step purification using the Ni-M/A stationary phase. Moreover, the binding capacity of Ni-M/A for Mpro was approximately 5.2 mg/g with recovery efficiency of 40%. Our results suggested Ni-M/A as a highly potential solid phase for affinity chromatography in the purification of His-tagged protein.

Rapid determination of furazolidone residues in animal foods by time-resolved fluorescence immunochromatography.

Although furazolidone (FZD) was completely banned from livestock production in many countries many years ago due to its mutagenicity and carcinogenicity, the abuse of FZD is still common today. Accurate and rapid detection of FZD residues in animal-derived food products is highly important for human health. Here, a time-resolved fluorescence immunochromatography (TRFI) test strip for rapid and quantitative detection of 3-amino-2-oxazolidinone (AOZ) residues in animal foods was developed and validated. Its limit of detection and limit of quantification were 0.05 and 0.14 µg/kg, respectively. The typical recovery rates were 95-105 % in chicken breast samples spiked with the AOZ standard substance at concentrations of 0.05-2 µg/kg, with a coefficient of variation value ≤8.5 %. The cross-reaction rates of the TRFI-AOZ test strips with 3-amino-5-morpholinomethyl-2-oxazolidone, semicarbazide, and 1-amino-imidazolidin-2,4-dione were less than 1 %. The newly developed TRFI test strip has high sensitivity, high specificity, cost effectiveness and user-friendly control, and is suitable for the rapid and large-scale screening of AOZ residues in animal foods.

Enhancing hyaluronidase enzyme activity: Insights from advancement in bovine and ovine testicular hyaluronidase purification.

This essay investigates the use of an affinity resin named Capto lentil lectin for the purification of bovine and ovine testicular hyaluronidase. Hyaluronidase, an enzyme that degrades hyaluronic acid, is used widely in medical fields like dermatology, orthopedics, and ophthalmology. The research highlights the importance of optimizing the purification process to increase enzyme activity and purity. A new purification method is proposed, which begins with ammonium sulfate precipitation, followed by Blue Sepharose and Capto Lentil Lectin chromatography. This novel approach significantly increases the yield, purity, and activity of the enzyme. This study paves the way for further research into improving the purification process. The study further discusses challenges in identifying hyaluronidase bands using SDS-PAGE and highlights the necessity of using Western blotting for precise results.

Combination of click chemistry and Schiff base reaction: Post-synthesis of covalent organic frameworks as an immobilized metal ion affinity chromatography platform for efficient capture of global phosphopeptides in serum with chronic obstructive pulmonary disease.

Reasonable design and construction of functionalized materials are of great importance for the enrichment of global phosphopeptides. In this work, Ti4+ functionalized hydrophilic covalent organic frameworks by introducing glutathione (GSH) and 2,3,4-trihydroxy benzaldehyde (THBA) via click chemistry and Schiff base reaction (COF-V@GSH-THBA-Ti4+ ) was constructed and applied for selective enrichment of phosphopeptides in serum. Benefit from the high surface area, excellent hydrophilicity as well as regular mesoporous structure, COF-V@GSH-THBA-Ti4+ displayed high selectivity (molar ratio of 2000:1), low limit of detection (0.5 fmol), high load capacity (100.0 mg/g) and excellent size-exclusion effect (1:10000) for enrichment of phosphopeptides. For actual bio-sample analysis, 15 phosphopeptides assigned to 10 phosphoproteins with 16 phosphorylated sites and 33 phosphopeptides assigned to 25 phosphoproteins with 34 phosphorylated sites were detected from the serum of patients with chronic obstructive pulmonary disease (COPD), and normal controls. Biological processes and molecular functions analysis further disclosed the difference of serums with phosphoproteomics between COPD and normal controls.

Affinity Resins for the Isolation of Immunoglobulins G Obtained Using Biocatalytic Technology.

Affinity chromatography resins that are obtained by conjugation of matrices with proteins of bacterial origin, like protein A, are frequently used for the purification of numerous therapeutic monoclonal antibodies. This article presents the development of a biocatalytic method for the production of novel affinity resins with an immobilized mutant form of protein A via sortase A mediated reaction. The conditions for activation of the agarose Seplife 6FF matrix, selection of different types of linkers with free amino groups and conditions for immobilization of recombinant protein A on the surface of the activated matrix were studied. Finally, the basic operational properties, like dynamic binding capacity (DBC), temperature dependance of DBC and stability during the cleaning-in-place process of the affinity resin with the Gly-Gly-EDA-Gly-Gly linker, were assessed using recombinant hyperchimeric monoclonal antibodies. The main characteristics show comparable results with the widely used commercial samples.

Improved mRNA affinity chromatography binding capacity and throughput using an oligo-dT immobilized electrospun polymer nanofiber adsorbent.

Increased demand for mRNA-based therapeutics and improved in vitro transcription (IVT) yields have challenged the mRNA purification platform. Hybridization-affinity chromatography with an immobilized oligo-deoxythymidilic acid (oligodT) ligand is often used to capture mRNA through base pairing with the polyadenylated tail. Commercially available oligodT matrices include perfusive cross-linked poly(styrene-divinylbenzene) 50 µm POROS™ chromatography resin beads and convective polymethacrylate CIMmultus® monolithic columns consisting of 2 µm interconnected channels. POROS™ columns may be limited by poor mass transfer for larger mRNAs and slow flowrates, while monoliths can operate at higher flowrates but are limited by modest binding capacity. To enable both high flowrates and binding capacity for mRNA of all lengths, prototype chromatography media was developed by Cytiva using oligodT immobilized electrospun cellulose nanofibers (Fibro™) with a 0.3-0.4 µm pore size. In this work, four polyadenylated mRNAs ranging from ∼1900-4300 nucleotides were used to compare the dynamic binding capacity (DBC) of Fibro™, POROS® and CIMmultus® columns as a function of residence time and binding buffer compositions. Fibro™ improved the DBC ∼2-4-fold higher than CIMmultus® and ∼2-13-fold higher than POROS™ across all residence times, mRNA length, and binding matrix compositions tested. CIMmultus® DBC was least dependent on residence time and mRNA size, while both Fibro™ and POROS™ DBC increased at slower flowrates and with shorter mRNA. Surprisingly, inverse size exclusion (ISE) experiments showed that POROS™ was not limited by diffusion and POROS™ along with CIMmultus® demonstrate higher mRNA permeation however the Fibro™ prototype is not in the final configuration. Lastly, IVT reaction products were subjected to purification and oligodT elution product yield, quality, and purity were consistent across the three matrices investigated. These results highlight the benefits of high DBC and equivalent product profiles offered by the oligodT Fibro™ prototype compared to commercially available oligodT media.

Purification of pineapple bromelain by IMAC chromatography using chlorophyll-activated macroporous matrices.

This study investigated the purification of bromelain obtained from pineapple fruit using a new adsorbent for immobilized metal ion affinity chromatography (IMAC), with chlorophyll obtained from plant leaves as a chelating agent. The purification of bromelain was evaluated in batches from the crude extract of pineapple pulp (EXT), and the extract precipitated with 50 % ammonium sulfate (EXT.PR), the imidazole buffer (200 mM, pH 7.2) being analyzed and sodium acetate buffer, pH 5.0 + 1.0 NaCl as elution solutions. All methods tested could separate forms of bromelain with molecular weights between ±21 to 25 kDa. Although the technique using EXT.PR stood out in terms of purity, presenting a purification factor of around 3.09 ± 0.31 for elution with imidazole and 4.23 ± 0.12 for acetate buffer solution. In contrast, the EXT methods obtained values between 2.44 ± 0.23 and 3.21 ± 0.74 for elution with imidazole and acetate buffer, respectively, for purification from EXT.PR has lower yield values (around 5 %) than EXT (around 15 %). The number of steps tends to reduce yield and increase process costs, so the purification process in a monolithic bed coupled to the chromatographic system using the crude extract was evaluated. The final product obtained had a purification factor of 6, with a specific enzymatic activity of 59.61 ± 0.00 U·mg-1 and a yield of around 39 %, with only one band observed in the SDS-PAGE electrophoresis analysis, indicating that the matrix produced can separate specific proteins from the total fraction in the raw material. The IMAC matrix immobilized with chlorophyll proved promising and viable for application in protease purification processes.

Facile synthesis of Fe3+ immobilized magnetic polydopamine-polyethyleneimine composites for phosphopeptide enrichment.

As one of the most common post-translational modification of proteins, protein phosphorylation plays a vital role in many physiological processes. The enrichment of phosphopeptides is highly important before the mass spectrometry detection since phosphopeptides are susceptible to interferences from high-abundance non-phosphopeptides. In this study, we designed a novel magnetic composite (Fe3O4@PDA-PEI-Fe3+) for phosphopeptide enrichment with a facile protocol. The developed Fe3O4@PDA-PEI-Fe3+ is a marvelous material with multiple functional groups, and can effectively enrich phosphopeptides through the synergistic effect of three mechanisms, i.e., immobilized metal ion affinity chromatography raised form Fe3+, electrostatic interaction between amine and phosphate groups, and hydrogen bond between the hydrogen atoms of amine groups and oxygen atoms of phosphate groups. Combined with mass spectrometry, the material shows excellent enrichment performance, high sensitivity (0.4 fmol), good selectivity (ß-casein:BSA= 1:500, w:w), and stable reusability (at least 5 cycles). In addition, the material was successfully applied to enrich phosphopeptides from skim milk and human saliva samples, implying that it is an ideal adsorbent for the phosphopeptide enrichment in complex biological samples and provides valuable insights into the field of phosphopeptide analysis.

Immunochromatographic visualization detection platform for bitertanol in foods.

As a widely used fungicide in agriculture, bitertanol (BIT) significantly affects hormone regulation leading to imbalance of homeostasis in vivo, which makes it necessary to monitor BIT residues in foods. In this research, a novel hapten derivation scheme was designed by analyzing the chemical structure of BIT to prepare an anti-BIT monoclonal antibody with high affinity, specificity and sensitivity (half inhibitory concentration of 4.78 ng/mL). Subsequently, a visualized gold immunochromatographic assay (GICA) platform was established based on antigen-antibody specific recognition, with a limit of detection of 0.06 mg/kg and 0.18 mg/kg in cucumber and tomato, respectively. GICA has spiked recoveries of 84.3 %-114.1 %, determines results are not significantly different from those of LC-MS/MS, and the complex purification treatments can be reduced during the detection process. Therefore, the developed GICA is a reliable, rapid, and sensitive method for on-site rapid monitoring of BIT in foods.

Ultrasensitive detection of imatinib in human serum using a gold-based paper sensor.

Imatinib is the tyrosine kinase inhibitor of choice for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. However, imatinib has drawbacks such as drug resistance and significant differences in pharmacokinetics within patients. Therefore, a colloidal gold-based immunochromatographic assay (CG-IA) was developed for measuring and monitoring imatinib in human serum. An imatinib derivative containing carboxyl groups was used for the synthesis of the immunogen, and 4-(4-methyl-1-piperazinylmethyl) benzoic acid was selected as the hapten for the heterologous coating antigen. Next, a highly sensitive and specific monoclonal antibody (mAb), 2F7 was screened for the construction of a CG-IA, with an IC50 value of 0.091 ng/mL. For the qualification of imatinib in human serum, the visual limit of detection (vLOD) and cut-off values of the CG-IA were 2 and 20 ng/mL, respectively. For quantitative detection, the calculated LOD value of the CG-IA was 0.068 ng/mL, with a linearity range of 1.004 and 23.087 ng/mL. The recovery rate of spiked serum samples was between 88.24 % and 104.75 %. In addition, the concentration of imatinib in the serum samples from 10 patients was detected by CG-IA and revealed a good correlation with those from LC-MS/MS. These results indicated that the developed gold-based paper sensor could become an effective tool for the rapid monitoring of imatinib in human serum samples.