Autologous Adipose-Derived Tissue Stromal Vascular Fraction (AD-tSVF) for Knee Osteoarthritis.

Adipose tissue contains adult mesenchymal stem cells that may modulate the metabolism when applied to other tissues. Stromal vascular fraction (SVF) can be isolated from adipose tissue mechanically and/or enzymatically. SVF was recently used to decrease the pain and improve the function of knee osteoarthritis (OA) patients. Primary and/or secondary OA causes inflammation and degeneration in joints, and regenerative approaches that may modify the natural course of the disease are limited. SVF may modulate inflammation and initiate regeneration in joint tissues by initiating a paracrine effect. Chemokines released from SVF may slow down degeneration and stimulate regeneration in joints. In this review, we overviewed articular joint cartilage structures and functions, OA, and macro-, micro-, and nano-fat isolation techniques. Mechanic and enzymatic SVF processing techniques were summarized. Clinical outcomes of adipose tissue derived tissue SVF (AD-tSVF) were evaluated. Medical devices that can mechanically isolate AD-tSVF were listed, and publications referring to such devices were summarized. Recent review manuscripts were also systematically evaluated and included. Transferring adipose tissues and cells has its roots in plastic, reconstructive, and aesthetic surgery. Micro- and nano-fat is also transferred to other organs and tissues to stimulate regeneration as it contains regenerative cells. Minimal manipulation of the adipose tissue is recently preferred to isolate the regenerative cells without disrupting them from their natural environment. The number of patients in the follow-up studies are recently increasing. The duration of follow up is also increasing with favorable outcomes from the short- to mid-term. There are however variations for mean age and the severity of knee OA patients between studies. Positive outcomes are related to the higher number of cells in the AD-tSVF. Repetition of injections and concomitant treatments such as combining the AD-tSVF with platelet rich plasma or hyaluronan are not solidified. Good results were obtained when combined with arthroscopic debridement and micro- or nano-fracture techniques for small-sized cartilage defects. The optimum pressure applied to the tissues and cells during filtration and purification of the AD-tSVF is not specified yet. Quantitative monitoring of articular joint cartilage regeneration by ultrasound, MR, and synovial fluid analysis as well as with second-look arthroscopy could improve our current knowledge on AD-tSVF treatment in knee OA. AD-tSVF isolation techniques and technologies have the potential to improve knee OA treatment. The duration of centrifugation, filtration, washing, and purification should however be standardized. Using gravity-only for isolation and filtration could be a reasonable approach to avoid possible complications of other methodologies.

Delineating the clinical spectrum of isolated methylmalonic acidurias: cblA and mut.

INTRODUCTION: Long-term outcome is postulated to be different in isolated methylmalonic aciduria caused by mutations in the MMAA gene (cblA type) compared with methylmalonyl-CoA mutase deficiency (mut), but case definition was previously difficult. METHOD: Cross-sectional analysis of data from the European Registry and Network for Intoxication type Metabolic Diseases (Chafea no. December 1, 2010). RESULTS: Data from 28 cblA and 95 mut patients in most cases confirmed by mutation analysis (including 4 new mutations for cblA and 19 new mutations for mut). Metabolic crisis is the predominant symptom leading to diagnosis in both groups. Biochemical disturbances during the first crisis were similar in both groups, as well as the age at diagnosis. Z scores of body height and body weight were similar in both groups at birth, but were significantly lower in the mut group at the time of last visit. Glomerular filtration rate was significantly higher in cblA; and as a consequence, chronic renal failure and related complications were significantly less frequent and renal function could be preserved even in older patients. Neurological complications were predominantly found in the mut subgroup. Methylmalonic acidemia (MMA) levels in urine and plasma were significantly lower in cblA. 27/28 cblA patients were reported to be responsive to cobalamin, only 86% of cblA patients were treated with i.m. hydroxocobalamin. In total, 73% of cblA and 98% of mut patients followed a calculated diet with amino acid supplements in 27% (cblA) and 69% (mut). During the study interval, six patients from the mut group died, while all cblA patients survived. CONCLUSION: Although similar at first, cblA patients respond to hydroxocobalamin treatment, subsequently show significantly lower levels of MMA and a milder course than mut patients.

Recent trends in sorbents for bioaffinity chromatography.

Isolation or enrichment of biological molecules from complex biological samples is mostly a prerequisite in proteomics, genomics, and glycomics. Different techniques have been used to advance the efficiency of the purification of biological molecules. Bioaffinity chromatography is one of the most powerful technique that plays an important role in the isolation of target biological molecules by the specific interactions with ligands that are immobilized on different support materials. This review examines the recent developments in bioaffinity chromatography particularly over the past 5 years in the literature. Also properties of supports, immobilization techniques, types of binding agents, and methods used in bioaffinity chromatography applications are summarized.

Silica microspheres functionalized with the iminodiacetic acid/copper(II) complex as a peroxidase mimic for use in metal affinity chromatography-based colorimetric determination of histidine-tagged proteins.

Monodisperse porous silica microspheres were functionalized with the iminodiacetic acid/copper(II) complex and then evaluated as a group-specific peroxidase-mimicking nanozyme for colorimetric determination of histidine-tagged (His-tagged) proteins. The green fluorescent protein (GFP) was selected as a typical His-tagged protein. The specificity for GFP and the peroxidase-like activity for the selected substrate were obtained by immobilizing the complex on the porous microspheres. The modified microspheres were also evaluated as a group specific immobilized metal affinity chromatography (IMAC) sorbent for the purification of GFP from Escherichia coli extract. The peroxidase-like activity of the microspheres was inhibited by the GFP adsorbed onto the microspheres due to the interaction of His-tagged protein with the immobilized Cu(II) complex. Ortho-phenylenediamine is used as a substrate for the enzyme mimic. The photometric response (measured at 416 nm) is linear in the 9.0-92 µg·mL-1 GFP concentration range in E. coli lysate. The limit of detection is 6.9 µg·mL-1. Graphical abstractSchematic representation of metal affinity chromatography-based colorimetric determination of histidine-tagged proteins using silica microspheres functionalized with iminodiacteic acid/copper (II) complex as a peroxidase mimic.

Molecularly imprinted polymeric shell coated monodisperse-porous silica microspheres as a stationary phase for microfluidic boronate affinity chromatography.

Molecular imprinting of cis-diol functionalized agents via boronate affinity interaction has been usually performed using nanoparticles as a support which cannot be utilized as a stationary phase in continuous microcolumn applications. In this study, monodisperse-porous, spherical silica particles in the micron-size range, with bimodal pore diameter distribution were selected as a new support for the synthesis of a molecularly imprinted boronate affinity sorbent, using a cis-diol functionalized agent as the template. A specific surface area of 158 m2 /g was achieved with the imprinted sorbent by using monodisperse-porous silica microspheres containing both mesoporous and macroporous compartments as the support. High porosity originating from the macroporous compartment and sufficiently high particle size provided good column permeability to the imprinted sorbent in microcolumn applications. The mesoporous compartment provided a large surface area for the parking of imprinted molecules while the macroporous compartment facilitated the intraparticular diffusion of imprinted target within the microsphere interior. A microfluidic boronate affinity system was first constructed by using molecularly imprinted polymeric shell coated monodisperse-porous silica microspheres as a stationary phase. The synthetic route for the imprinting process, the reversible adsorption/ desorption behavior of selected target and the selectivity of imprinted sorbent in both batch and microfluidic boronate affinity chromatography systems are reported.

Ti (IV) attached-phosphonic acid functionalized capillary monolith as a stationary phase for in-syringe-type fast and robust enrichment of phosphopeptides.

In this study, poly(vinylphosphonic acid-co-ethylene dimethacrylate), poly(VPA-co-EDMA) capillary monolith was synthesized as a starting material for obtaining a stationary phase for microscale enrichment of phosphopeptides. The chelation of active phosphonate groups with Ti (IV) ions gave a macroporous monolithic column with a mean pore size of 5.4 µm. The phosphopeptides from different sources were enriched on Ti (IV)-attached poly(VPA-co-EDMA) monolith using a syringe-pump. The monolithic capillary columns exhibited highly sensitive/selective enrichment performance with phosphoprotein concentrations as low as 1.0 fmol/mL. Six different phosphopeptides were detected with high intensity by the treatment of ß-casein digest with the concentration of 1.0 fmol/mL, using Ti (IV)@poly(VPA-co-EDMA) monolith. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts, in a complex mixture of digested proteins (molar ratio of ß-casein to bovine serum albumin, 1:1500) and three phosphopeptides were successfully detected. Four highly intense signals of phosphopeptides in human serum were also observed with high signal-to-noise ratio and a clear background after enrichment with Ti (IV)@poly(VPA-co-EDMA) monolith. It was concluded that the capillary microextraction system enabled fast, efficient and robust enrichment of phosphopeptides from microscale complex samples. The whole enrichment process was completed within 20 min, which was shorter than in the previously reported studies.

Alkanethiol-functionalized organosilicon monoliths for nano-reversed-phase liquid chromatography.

Organosilicon monoliths carrying chromatographic ligands with different alkyl chain lengths were obtained by thiol-methacrylate photopolymerization. The use of thiol-ene chemistry in the presence of a main monomer with a series of methacrylate functionality (i.e., methacrylate substituted polyhedral oligomeric silsesquioxane) allowed the synthesis of organosilicon monoliths with high cross-linking density and carrying hydrophobic alkyl-chain ligands by a one-pot process. In the synthesis runs, 1-butanethiol, 1-octanethiol, and 1-octadecanethiol were used as the hydrophobic thiol ligands with the number of methylene units between 4 and 18. The selectivity analysis performed using cytosine/uracil retention ratio showed that alkanethiol-attached organosilicon monoliths exhibited hydrophobicity close to octadecyl-attached silica-based RP columns. In the RP, chromatographic runs performed in nano-liquid chromatography, phenols, alkylbenzenes, and PAHs were used as the analytes. Among the synthesized monoliths, retention-independent plate height behavior and the smallest plate heights were obtained with 1-octadecanethiol-attached organosilicon monolith for the analytes in a wide polarity range. With this monolith, the mobile phases prepared with ACN contents ranging between 35 and 85% v/v could be used for satisfactory separation of analytes in a wide polarity range.

Organic acidurias in adults: late complications and management.

Organic acidurias (synonym, organic acid disorders, OADs) are a heterogenous group of inherited metabolic diseases delineated with the implementation of gas chromatography/mass spectrometry in metabolic laboratories starting in the 1960s and 1970s. Biochemically, OADs are characterized by accumulation of mono-, di- and/or tricarboxylic acids ("organic acids") and corresponding coenzyme A, carnitine and/or glycine esters, some of which are considered toxic at high concentrations. Clinically, disease onset is variable, however, affected individuals may already present during the newborn period with life-threatening acute metabolic crises and acute multi-organ failure. Tandem mass spectrometry-based newborn screening programmes, in particular for isovaleric aciduria and glutaric aciduria type 1, have significantly reduced diagnostic delay. Dietary treatment with low protein intake or reduced intake of the precursor amino acid(s), carnitine supplementation, cofactor treatment (in responsive patients) and nonadsorbable antibiotics is commonly used for maintenance treatment. Emergency treatment options with high carbohydrate/glucose intake, pharmacological and extracorporeal detoxification of accumulating toxic metabolites for intensified therapy during threatening episodes exist. Diagnostic and therapeutic measures have improved survival and overall outcome in individuals with OADs. However, it has become increasingly evident that the manifestation of late disease complications cannot be reliably predicted and prevented. Conventional metabolic treatment often fails to prevent irreversible organ dysfunction with increasing age, even if patients are considered to be "metabolically stable". This has challenged our understanding of OADs and has elicited the discussion on optimized therapy, including (early) organ transplantation, and long-term care.

New vinylester-based monoliths as a new stationary phase for capillary electrochromatography.

Vinyl ester-based monoliths are proposed as a new group of stationary phase for CEC. The capillary monolithic columns were prepared by using two vinyl ester monomers, vinyl pivalate (VPV), and vinyl decanoate (VDC) by using ethylene dimethacrylate (EDMA) as the cross-linking agent, and 2-acrylamido-2-methylpropane sulfonic acid as the charge-bearing monomer. The monoliths with different pore structures and permeabilities were obtained by varying the type and composition of the porogen mixture containing isoamyl alcohol and 1,4-butanediol. The electrochromatographic separation of alkylbenzenes was successfully performed by using an acetonitrile/aqueous buffer system as the mobile phase in a CEC system. Vinyl ester monoliths with short alkyl chain length (i.e. poly(VPV-co-EDMA) exhibited better separation performance compared with the monolith with long alkyl chain length (i.e. poly(VDC-co-EDMA). In the case of VPV-based monoliths, the theoretical plate numbers higher than 250 000 plates/m were achieved by using a porogen mixture containing 33% v/v of isoamyl alcohol. For both VDC and VPV-based monoliths, the column efficiency was almost independent of the superficial velocity in the range of 2-12 cm/min.

In Vitro Synergistic Photodynamic, Photothermal, Chemodynamic, and Starvation Therapy Performance of Chlorin e6 Immobilized, Polydopamine-Coated Hollow, Porous Ceria-Based, Hypoxia-Tolerant Nanozymes Carrying a Cascade System.

A synergistic therapy agent (STA) with photothermal, photodynamic, chemodynamic, and starvation therapy (PTT, PDT, CDT, and ST) functions was developed. Hollow, mesoporous, and nearly uniform CeO2 nanoparticles (H-CeO2 NPs) were synthesized using a staged shape templating sol-gel protocol. Chlorin e6 (Ce6) was adsorbed onto H-CeO2 NPs, and a thin polydopamine (PDA) layer was formed on Ce6-adsorbed H-CeO2 NPs. Glucose oxidase (GOx) was bound onto PDA-coated Ce6-adsorbed H-CeO2 NPs to obtain the targeted STA (H-CeO2@Ce6@PDA@GOx NPs). A reversible photothermal conversion behavior with the temperature elevations up to 34 °C was observed by NIR laser irradiation at 808 nm. A cascade enzyme system based on immobilized GOx and intrinsic catalase-like activity of H-CeO2 NPs was rendered on STA for enhancing the effectiveness of PDT by elevation of ROS generation and alleviation of hypoxia in a tumor microenvironment. Glucose-mediated generation of highly toxic hydroxyl radicals (·OH) was evaluated for CDT. The effectiveness of PDT on glioblastoma T98G cells was markedly enhanced by O2 generation started by the decomposition of glucose. A similar increase in cell death was also observed when ST and CDT functions were enhanced by photothermal action. The viability of T98G cells decreased to 10.6% by in vitro synergistic action including ST, CDT, PDT, and PTT without using any antitumor agent.

"Hide and seek": Misleading transferrin variants in PMM2-CDG complicate diagnostics.

PURPOSE: Congenital disorders of glycosylation (CDG) are one of the fastest growing groups of inborn errors of metabolism. Despite the availability of next-generation sequencing techniques and advanced methods for evaluation of glycosylation, CDG screening mainly relies on the analysis of serum transferrin (Tf) by isoelectric focusing, HPLC or capillary electrophoresis. The main pitfall of this screening method is the presence of Tf protein variants within the general population. Although reports describe the role of Tf variants leading to falsely abnormal results, their significance in confounding diagnosis in patients with CDG has not been documented so far. Here, we describe two PMM2-CDG cases, in which Tf variants complicated the diagnostic. EXPERIMENTAL DESIGN: Glycosylation investigations included classical screening techniques (capillary electrophoresis, isoelectric focusing and HPLC of Tf) and various confirmation techniques (two-dimensional electrophoresis, western blot, N-glycome, UPLC-FLR/QTOF MS with Rapifluor). Tf variants were highlighted following neuraminidase treatment. Sequencing of PMM2 was performed. RESULTS: In both patients, Tf screening pointed to CDG-II, while second-line analyses pointed to CDG-I. Tf variants were found in both patients, explaining these discrepancies. PMM2 causative variants were identified in both patients. CONCLUSION AND CLINICAL RELEVANCE: We suggest that a neuraminidase treatment should be performed when a typical CDG Tf pattern is found upon initial screening analysis.

Butyl methacrylate based monoliths with different cross-linking agents using DMF-aqueous buffer as porogen.

A simple porogen containing only DMF and aqueous buffer was used for synthesis of monolithic stationary media for CEC). Butyl methacrylate (BMA)-based capillary monoliths were obtained using proposed porogen together with acrylic/methacrylic cross-linking agents with different alkyl chain lengths. For this purpose, ethylene glycol dimethacrylate, butanediol dimethacrylate and hexanediol diacrylate (HDDA) were used. The monoliths with better electrochromatographic separation performance were obtained when the acrylic cross-linking agent with the longest alkyl chain length (i.e. HDDA) was used with the proposed porogen. The electrochromatographic separation of alkylbenzenes, phenols and benzoic acids were sucessfully performed in CEC particularly using poly(BMA-co-HDDA) monolithic stationary phase with the column efficiency up to 270 000 plates/m.

The Novel Polymethacrylate Based Hydrophilic Stationary Phase for Ion Chromatography.

Ion chromatography is widely used as a useful and powerful tool for the analysis of anionic and cationic components found in waters and aqueous media. The performance and selectivity of ion chromatography are based on the stationary phase column packed material. In this study, it is aimed to develop new column material with quaternary ammonium functional group based on monodisperse polymeric particles for ion chromatography and to investigate their chromatographic performance. For the analysis of inorganic anions by ion chromatography, new stationary phases macroporous monodisperse particles based on 3-chloro-2-hydroxy propylmethacrylate-co-ethylene dimethacrylate are obtained as column packing material. 3-chloro-2-hydroxy propylmethacrylate and ethylene glycol dimethacrylate are transformed to porous monodisperse particle form by using glycidyl methacrylate as the seed latex and ethyl benzene as the porogen solvent via micro-suspension polymerization technique. Then macroporous monodisperse particles surface is functionalized by triethylamine so strong anion exchange is obtained for ion chromatography packing material. A series of stationary phases prepared from polymer particles containing different amounts of porogen solvent were tested. The results show that column packing material is successful to separate inorganic anions mixture such as F-, Cl-, NO2-, Br-, NO3- by using the carbonate and bicarbonate solutions as mobile phases.

A new multimodal magnetic nanozyme and a reusable peroxymonosulfate oxidation catalyst: Manganese oxide coated-monodisperse-porous and magnetic core-shell microspheres.

Monodisperse-porous, polydopamine and manganese oxide coated, core-shell type, magnetic SiO2 (MagSiO2@PDA@MnO2) microspheres 6.4 µm in size were synthesized for the first time, using magnetic, monodisperse-porous SiO2 (MagSiO2) microspheres 6.2 µm in size as the starting material. MagSiO2 microspheres were obtained by a recently developed method namely "staged shape templated hydrolysis and condensation protocol". In the synthesis, MagSiO2 microspheres were consecutively coated by polydopamine (PDA) and then by a MnO2 layer in the aqueous medium. The pore volume and the specific surface area of monodisperse-porous MagSiO2@PDA@MnO2 microspheres were measured as 0.59 cm3 g-1 and 154 m2 g-1, respectively. Their Mn and Fe contents were determined as 66 ± 1 mg g-1 and 165 ± 5 mg g-1 respectively. MagSiO2@PDA@MnO2 microspheres exhibited multimodal enzyme mimetic behavior with highly superior catalase-like, oxidase-like and peroxidase-like activities. The effective production of singlet oxygen (1O2) and superoxide anion (O2-*) radicals in MagSiO2@PDA@MnO2-peroxymonosulfate (PMS) system was demonstrated by ESR spectroscopy. By evaluating this property, MagSiO2@PDA@MnO2 microspheres were tried as a reusable catalyst for dye removal via peroxymonosulfate (PMS) activation in batch experiments for the first time. The degradation runs were made with, rhodamine B (Rh B), methyl orange (MO) and methylene blue (MB) as the pollutant. The core-shell type design allowing the deposition of porous MnO2 layer onto a large surface area provided very fast, instant removals with all dyes, via both physical adsorption and degradation via PMS activation. In the reusability experiments, the removal yields of MO and Rh B decreased 1.8% and 8.9% over five consecutive runs in batch fashion. MagSiO2@PDA@MnO2 microspheres exhibited very good functional and structural stability in consecutive dye degradations. No significant change was observed in Fe content of microspheres while Mn content exhibited a decrease of 7.4% w/w over 5 consecutive degradation runs.

Ni(II) functionalized polyhedral oligomeric silsesquioxane based capillary monolith for purification of histidine-tagged proteins by immobilized metal affinity micro-chromatography.

A new capillary monolithic stationary phase was synthesized for the purification of histidine tagged proteins by immobilized metal affinity micro-chromatography (µ-IMAC). For this purpose, mercaptosuccinic acid (MSA) linked-polyhedral oligomeric silsesquioxane [MSA@poly(POSS-MA)] monolith 300 µm in diameter was obtained by thiol-methacrylate polymerization using methacryl substituted-polyhedral oligomeric silsesquioxane (POSS-MA) and MSA as the thiol functionalized agent in a fused silica capillary tubing. Ni(II) cations were immobilized onto the porous monolith via metal-chelate complex formation with double carboxyl functionality of bound MSA segments. µ-IMAC separations aiming the purification of histidine tagged-green fluorescent protein (His-GFP) from Escherichia coli extract were carried out on Ni(II)@MSA functionalized-poly(POSS-MA) [Ni(II)@MSA@poly(POSS-MA)] capillary monolith. His-GFP was succesfully isolated by µ-IMAC on Ni(II)@MSA@poly(POSS-MA) capillary monolith with the isolation yield of 85 % and the purity of 92 % from E. coli extract. Higher His-GFP isolation yields were obtained with lower His-GFP feed concentrations and lower feed flow rates. The monolith was used for consecutive His-GFP purifications with a tolerable decrease in equilibrium His-GFP adsorption over five runs.

Synthesis of a monolithic, micro-immobilised enzyme reactor via click-chemistry.

An immobilised enzyme reactor (IMER) in the form of capillary monolith was developed for a micro-liquid chromatography system. The plain monolith was obtained by in situ thermal copolymerisation of glycidyl methacrylate and ethylene dimethacrylate in a fused silica capillary (200 × 0.53 mm ID) by using n-propanol/1,4-butanediol as porogen. The enzyme, α-chymotrypsin (CT), was covalently attached onto the monolith via triazole ring formation by click-chemistry. For this purpose, the monolithic support was treated with sodium azide and reacted with the alkyne carrying enzyme derivative. CT was covalently linked to the monolith by triazole-ring formation. The activity behaviour of monolithic IMER was investigated in a micro-liquid chromatography system by using benzoyl-L-tyrosine ethyl ester (BTEE) as synthetic substrate. The effects of mobile-phase flow rate and substrate feed concentration on the final BTEE conversion were investigated under steady-state conditions. In the case of monolithic IMER, the final substrate conversion increased with increasing feed flow rate and increasing substrate feed concentration. Unusual behaviour was explained by the presence of convective diffusion in the macropores of monolith. The results indicated that the monolithic-capillary IMER proposed for micro-liquid chromatography had significant advantages with respect to particle-based conventional high-performance liquid chromatography-IMERs.

Polymethacrylate-based monolithic capillary column with weak cation exchange functionalities for capillary electrochromatography.

Polymethacrylate-based monolith with weak cation exchange functionalities was prepared in capillary column (i.d. 100 µm, o.d. 375 µm) by in situ polymerization of butyl methacrylate, ethylene dimethacrylate and N-methacryloyl-L-glutamic acid in presence of porogens. The porogen mixture included N,N-dimethyl formamide and phosphate buffer. The preparation procedure of stationary phase contained the synthesis of monomer, silanization of capillary inner wall and in situ polymerization. The use of amino acid based monomer for the monolith synthesis is one of the originalities of this novel approach. N-methacryloyl-L-glutamic acid has two carboxyl functionalities. The separation of the solutes were performed at different acetonitrile/phosphate buffer and acetonitrile/sodium hydroxide contents. The applied voltage for the alkyl benzenes was changed between +5 and +30 kV. CEC separations of alkyl benzenes, acidic, basic, phenolic and some polycylic aromatic compounds were succesfully performed under capillary-electrochromatography mode with cathodic electroosmotic flow.

Glutathione detection in human serum using gold nanoparticle decorated, monodisperse porous silica microspheres in the magnetic form.

A nanozyme for glutathione (GSH) detection in a broad concentration range was synthesized. GSH is usually detected up to an upper limit of 100 µM using current noble metal nanozymes due to the sharp decrease in the colorimetric response with the increasing GSH concentration. Strong inhibition of colorimetric reactions by GSH adsorbed onto noble metal based nanozymes in the form of non-porous, nanoscale particulate materials dispersed in an aqueous medium is the reason for the sharp decrease in the colorimetric response. In the present study, a new magnetic nanozyme synthesized by immobilization of Au nanoparticles (Au NPs) on magnetic, monodisperse porous silica microspheres (>5 µm) obtained by a "staged-shape templating sol-gel protocol" exhibited peroxidase-like activity up to a GSH concentration of 5000 µM. A more controlled linear decrease in the peroxidase-like activity with a lower slope with respect to that of similar nanozymes was observed with the increasing GSH concentration. The proposed design allowed the GSH detection in a broader concentration range depending on the adsorption of GSH onto the Au NPs immobilized on magnetic, monodisperse porous silica microspheres. A calibration plot allowing the detection of GSH in a broad concentration range up to 3300 µM was obtained using the magnetic nanozyme. The GSH concentration was also determined in human serum by elevating the upper detection range and adjusting the sensitivity of detection via controlling the nanozyme concentration.

Microfluidic immobilized metal affinity chromatography based on Ti(IV)-decorated silica microspheres for purification of phosphoproteins.

A silica-based immobilized metal affinity chromatography (IMAC) sorbent with the morphological properties suitable for purification of large phosphorylated biomolecules was synthesized. The sorbent was designed in the form of monodisperse-porous silica microspheres, 5.3 µm in size, having bimodal pore size distribution with a large median pore size (40 nm) and high surface area (163 m2/g) decorated with Ti(IV) cations (i.e. Ti(IV)@THSPMP@SiO2 microspheres). The decoration of silica microspheres with Ti(IV) cations was made by using 3-(trihydroxysilyl)propyl methylphosphonate (THSPMP) as a bifunctiontional linker, by preserving their bimodal pore size distribution. The mesopores provided a large surface area for parking of adsorbed phosphoproteins as large phosphorylated biomolecules while the intraparticular transport of phosphoproteins was facilitated by the macropores providing a large median pore size. High equilibrium adsorption capacity and high desorption yield in the purification of phosphoproteins were obtained using Ti(IV)@THSPMP@SiO2 microspheres as the sorbent in batch- and microfluidic-IMAC systems. The phosphoproteins, α-casein and ß-casein were isolated from milk and human serum with almost quantitative yields and high purity in the batch IMAC system. The appropriate microcolumn permeability (3.66 × 10-14 m2) originating from its appropriate average diameter (5.3 µm), high porosity (0.948 cm3/g) and high surface area (163 m2/g) of Ti(IV)@THSPMP@SiO2 microspheres makes the synthesized sorbent a promising stationary phase for dynamic chromatography. Hence, a new phosphoprotein enrichment format, a microfluidic IMAC system was constructed and successfully operated for highly selective purification of phosphoproteins from non-fat milk as a complex sample. The microfluidic-IMAC system is a promising tool particularly for phosphoproteomic applications performed using samples in microliter or nanoliter scale, also involving an on-line connection of purification unit to LC-MS for the identification of large phosphorylated biomolecules enriched.

Surface-imprinted silica particles for Concanavalin A purification from Canavalia ensiformis.

Concanavalin A is a representative of the plant protein group known as lectins. Many lectin proteins have useful characteristics for studies on cell division and cell surfaces. In this study, a new adsorbent for the specific separation of Concanavalin A was prepared by applying a silica particle surface imprinting method. First, silica particles were activated via acidic treatment, and then, 3-methacryloyloxypropyl trimethoxysilane (MPTMS) was used for modification. For the preparation of Concanavalin A surface-imprinted silica particles (Con A-MISPs), N-methacryloyl-l-histidine methyl ester (MAH) was used as a functional monomer. The silica particles were characterized using a Zetasizer, scanning electron microscopy equipment (SEM), and Fourier transform infrared spectroscopy (FTIR). The effects of parameters such as the pH, initial concentration of Concanavalin A, and temperature on the adsorption of Concanavalin A were determined. The maximum Concanavalin A adsorption onto Con A-MISPs was observed to be 305.2 mg/g at a pH of 6. The reusability of the Con A-MISPs was approximately 93.5%. The non-imprinted silica particles (NISPs) were prepared in the same manner without Concanavalin A to compare the surface imprinting factor. Selective binding studies were carried out with lysozyme and hemoglobin molecules. The selectivity of the Con A-MISPs was also investigated by isolating Concanavalin A from Canavalia ensiformis. The purity of the Concanavalin A was shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).