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.

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.

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.

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.

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).

Ni(II)-decorated porous titania microspheres as a stationary phase for column chromatography applications: Highly selective purification of hemoglobin from human blood.

Titania (TiO2)-based monodisperse-porous stationary phase/sorbent was synthesized by decoration of Ni(II) ions onto TiO2 microspheres 4.2 µm in size, obtained by a staged-shape template hydrolysis and condensation protocol. Ni(II) ions were attached onto iminodiacetic acid-3-glycidoxypropyltrimethoxysilane (IDA-GPTMS) bound-titania microspheres by metal-chelate complex formation. The appropriate mean size, sufficiently high surface area and high porosity providing an appropriate column permeability make Ni(II)-decorated TiO2 microspheres a good sorbent/stationary phase for batch/continuous-column chromatography applications. Ni(II)-decorated TiO2 microspheres were investigated as a sorbent for purification of a typical histidine-rich protein, hemoglobin (Hb) via immobilized metal affinity chromatography (IMAC) in batch fashion, by including bovine serum albumin (BSA) as reference. The saturation capacities of batch adsorption runs performed with bovine Hb and BSA were determined as 137 ±â€¯9 and 45 ±â€¯3 mg/g, respectively. Human Hb with the purity of > 95% was recovered from whole blood by IMAC conducted in batch-fashion. Ni(II)-decorated microspheres were also evaluated as a stationary phase in a microfluidic-IMAC system, in which, human Hb was recovered from whole blood with a purity of 85%. The microfluidic-IMAC system constructed here, based on monodisperse-porous TiO2 microspheres, is a promising tool for genomics/proteomics applications involving isolation of valuable biomolecules from low-volume samples.

Aggregation-resistant nanozyme containing accessible magnetite nanoparticles immobilized in monodisperse-porous silica microspheres for colorimetric assay of human genomic DNA.

Bridging-induced aggregation of individual nanozyme particles by long-chain biomacromolecules causes loss of peroxidase mimetic activity for various nanozymes. When a common nanozyme (Fe3O4 nanoparticles) was used for colorimetric assay of a long-chain biomacromolecule, human genomic DNA (hgDNA, 14.000 kDa, containing 22 kilobases), peroxidase-like activity was absent owing to the irreversible aggregation of Fe3O4 nanoparticles in the presence of hgDNA. We synthesized an aggregation-resistant nanozyme containing Fe3O4 nanoparticles immobilized in 5.1 µm monodisperse-porous silica microspheres. Fe3O4 nanoparticles were immobilized in a form accessible to the substrate and large biomolecules in the solution within the monodispersed silica microspheres including both mesopores and macropores. An appreciable and stable spectrophotometric response was obtained, originating from the satisfactorily high peroxidase-like activity of the synthesized nanozyme. No aggregation was observed in the aqueous dispersion of the nanozyme in the presence of hgDNA. Large particle size, low particle number density, high surface area, and the presence of macropores were evaluated as factors contributing to the adsorption of hgDNA chains onto the synthesized nanozyme without interparticle bridge formation between the individual microspheres causing aggregation. Here, for the first time a colorimetric assay was developed based on the enhancement of peroxidase-mimetic activity of non-aggregated porous silica microspheres to determine hgDNA concentration up to 300 ng/µL. hgDNA could be also isolated with 47% yield and an equilibrium hgDNA adsorption of 19.000 ng/mg, using the same nanozyme. Hence, a material acting as a nanozyme for colorimetric determination of hgDNA was also evaluated as a magnetic, solid phase extraction sorbent for the first time.

In-situ photopolymerized C4-functionalized organosilicon monoliths for reversed-phase protein separation in nano-liquid chromatography.

In this study, polyhedral oligomeric silsesquioxane (POSS)-based capillary monoliths with short alkyl chain ligand in the form of butyl (C4) were synthesized via two different polymerization routes, namely UV-initiated free radical copolymerization of methacrylate-derivatized POSS (POSS-MA) with butylmethacrylate (BMA) and UV-initiated thiol-methacrylate copolymerization of POSS-MA with butanethiol (BT). An organosilicon monolith with a pore size distribution lying on both mesoporous and macroporous scales, a lower mean pore size and a higher specific surface area was obtained with UV-initiated thiol-methacrylate polymerization. Both monoliths were then comparatively evaluated for gradient separation of proteins under reversed phase conditions in nano-liquid chromatography. The chromatographic performance was defined in terms of peak-resolution and peak capacity. Four carbon (C4) functionalized-poly(POSS-MA) monolith produced by UV-initiated thiol-methacrylate polymerization exhibited better separation performance with higher peak resolutions and peak capacities. Both, the morphological characterization of monoliths and the results of gradient separation of proteins showed that thiol-methacrylate polymerization was more suitable for the synthesis of C4 functionalized organosilicon based stationary phases for reversed-phase protein separation. The monolith prepared by thiol-methacrylate polymerization was also successfully applied for impurity analysis of two important hormones, namely insulin and genotropin. A comparison with a commercial poly(styrene-co-divinylbenzene) monolith documented the good chromatographic performance of the new BT-attached poly(POSS-MA) monolith.

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.

Isolation of RNA and beta-NAD by phenylboronic acid functionalized, monodisperse-porous silica microspheres as sorbent in batch and microfluidic boronate affinity systems.

Monodisperse-porous silica microspheres 5.5 µm in size were obtained by a staged shape templated hydrolysis-condensation method, with a bimodal pore-size distribution. 3-aminophenylboronic acid (APBA) was covalently attached onto the silica microspheres with a capacity of 0.476 mmol APBA/g microspheres. The boronate affinity isolation behaviour of ribonucleic acid (RNA) containing cis-diol at 3'-end was investigated by using APBA attached-silica microspheres as the sorbent in batch fashion. A short-chain diol carrying agent, ß-nicotinamide adenine dinucleotide (ß-NAD) was used as a target molecule with stronger affinity for phenylboronic acid ligand. The maximum equilibrium adsorptions for RNA and ß-NAD were determined as 60 and 159 mg/g sorbent, respectively. By using the synthesized sorbent, phosphate buffer at pH 7.0 containing sorbitol was successfuly used as a mild elution medium for obtaining quantitative desorptions with both RNA and ß-NAD. RNA isolations from mammalian and bacterial cells were successfully performed while protecting the structural integrity of RNA via boronate affinity interaction in batch fashion. A microfluidic boronate affinity system including a microcolumn 300 µm in diameter was also constructed using APBA attached-silica microspheres as the stationary phase. The breakthrough curves of microfluidic system were obtained by studying with different feed concentrations of RNA and ß-NAD. Quantitative desorptions and satisfactory isolation yields were obtained with RNA and ß-NAD in the microfluidic system. The proposed system is useful for boronate affinity applications in genomics or proteomics in which valuable cis-diols at low concentrations are recovered from low-volume samples.

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.

Comparative DNA isolation behaviours of silica and polymer based sorbents in batch fashion: monodisperse silica microspheres with bimodal pore size distribution as a new sorbent for DNA isolation.

Monodisperse silica microspheres with bimodal pore-size distribution were proposed as a high performance sorbent for DNA isolation in batch fashion under equilibrium conditions. The proposed sorbent including both macroporous and mesoporous compartments was synthesized 5.1 µm in-size, by a "staged shape templated hydrolysis and condensation method". Hydrophilic polymer based sorbents were also obtained in the form of monodisperse-macroporous microspheres ca 5.5 µm in size, with different functionalities, by a developed "multi-stage microsuspension copolymerization" technique. The batch DNA isolation performance of proposed material was comparatively investigated using polymer based sorbents with similar morphologies. Among all sorbents tried, the best DNA isolation performance was achieved with the monodisperse silica microspheres with bimodal pore size distribution. The collocation of interconnected mesoporous and macroporous compartments within the monodisperse silica microspheres provided a high surface area and reduced the intraparticular mass transfer resistance and made easier both the adsorption and desorption of DNA. Among the polymer based sorbents, higher DNA isolation yields were achieved with the monodisperse-macroporous polymer microspheres carrying trimethoxysilyl and quaternary ammonium functionalities. However, batch DNA isolation performances of polymer based sorbents were significantly lower with respect to the silica microspheres.

One pot synthesis of carboxyl functionalized-polyhedral oligomeric siloxane based monolith via photoinitiated thiol-methacrylate polymerization for nano-hydrophilic interaction chromatography.

A hybrid monolith exhibiting almost retention independent separation performance in hydrophilic interaction chromatography (HILIC) was obtained by one-pot photoinitiated thiol-methacrylate polymerization. Polyhedral oligomeric silsesquioxane containing methacrylate units (POSS-MA) was used as the main monomer and crosslinking agent, together with a hydrophilic ligand with two carboxyl groups, mercaptosuccinic acid (MSA) as the thiol agent and chromatographic ligand. The isocratic separation of nucleosides, nucleotides and organic acids on MSA attached-poly(POSS-MA) monolith was investigated in HILIC mode. The van-Deemter plots for obtained for nucleosides, nucleotides and benzoic acids clearly showed that there were two regions in each graph with two different slopes in the studied range of linear flow rate (i.e. 0.2-4.3mm/s). The slope of plate height-linear velocity curve was so small in the low linear velocity region between 0.2-2.1mm/s while the slope in high linear velocity region between 2.1-4.3mm/s was so higher with respect to the first region. The van-Deemter plots sketched for all analyte grous used in HILIC mode obeyed this tendency Almost "retention independent plate height behavior" was demonstrated in HILIC, using nucleotides, nucleotides or benzoic acids as the analytes in the linear velocity range of 0.2-2.1mm/s. This behavior was explained by the porous structure of the synthesized monolith facilitating the convective transport of analytes. The variation of plate height was not retention-independent within high linear velocity range (>3.2mm/s) when nucleosides were separated in HILIC mode.

Human genomic DNA isolation from whole blood using a simple microfluidic system with silica- and polymer-based stationary phases.

Monodisperse-porous silica microspheres 5.1µm in size with a bimodal pore-size distribution (including both mesoporous and macroporous compartments) were obtained using a newly developed staged-shape templated hydrolysis and condensation protocol. Synthesized silica microspheres and monodisperse-porous polymer-based microspheres with different functionalities, synthesized by staged-shape template polymerization, were comparatively tested as sorbents for human genomic DNA (hgDNA) isolation in a microfluidic system. Microcolumns with a permeability range of 1.8-8.5×10-13m2 were fabricated by the slurry-packing of silica- or polymer-based microspheres. The monodisperse-porous silica microspheres showed the best performance in hgDNA isolation in an aqueous buffer medium; >2500ng of hgDNA was recovered with an isolation yield of about 50%, using an hgDNA feed concentration of 100ng/µL. Monodisperse-porous silica microspheres were also evaluated as a sorbent for genomic DNA isolation from human whole blood in the microfluidic system; 14ng of hgDNA was obtained from 10µL of whole blood lysate with an isolation yield of 64%. Based on these results, we conclude that monodisperse-porous silica microspheres with a bimodal pore size distribution are a promising sorbent for the isolation of hgDNA in larger amounts and with higher yields compared to the sorbents previously tried in similar microfluidic systems.

Synthesis of a reactive polymethacrylate capillary monolith and its use as a starting material for the preparation of a stationary phase for hydrophilic interaction chromatography.

Poly(3-chloro-2-hydroxypropyl methacrylate-co-ethylene dimethacrylate), poly(HPMA-Cl-co-EDMA) capillary monolith was proposed as a reactive starting material with tailoring flexibility for the preparation of monolithic stationary phases. The reactive capillary monolith was synthesized by free radical copolymerization of 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl) and ethylene dimethacrylate (EDMA). The mean pore size, the specific surface area and the permeability of poly(HPMA-Cl-co-EDMA) monoliths were controlled by adjusting porogen/monomer volume ratio, porogen composition and polymerization temperature. The porogen/monomer volume ratio was found as the most effective factor controlling the porous properties of poly(HPMA-Cl-co-EDMA) monolith. Triethanolamine (TEA-OH) functionalized polymethacrylate monoliths were prepared by using the reactive chloropropyl group of poly(HPMA-Cl-co-EDMA) monolith via one-pot and simple post-functionalization process. Poly(HPMA-Cl-co-EDMA) monolith reacted with TEA-OH was evaluated as a stationary phase in nano-hydrophilic interaction chromatography (nano-HILIC). Nucleotides, nucleosides and benzoic acid derivatives were satisfactorily separated with the plate heights up to 20µm. TEA-OH attached-poly(HPMA-Cl-co-EDMA) monolith showed a reproducible and stable retention behaviour in nano-HILIC runs. However, a decrease in the column performance (i.e. an increase in the plate height) was observed with the increasing retention factor. Hence "retention-dependent column efficiency" behaviour was shown for HILIC mode using the chromatographic data collected with the polymer based monolith synthesized.

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.