Solid-phase Support Research Articles

Magnetic Nanoparticle-Mediated Extraction and Electrochemical Detection of E. coli O157:H7 Genomic DNA

We report a rapid magnetic nanoparticle-mediated protocol to extract and detect DNA of E. coli O157:H7. Bare iron oxide nanoparticles (Fe3O4 NPs) were used as solid phase support to extract high purity DNA from concentrated and spiked water samples. Electrochemical detection of the DNA is achieved by employing polyaniline-functionalized magnetic nanoparticles (PANI-MNPs) which serve as DNA pre-concentrator and transducer. The DNA-based nanobiosensor shows good selectivity and sensitivity (detection limit of 0.10 ng/µL DNA) and offers faster and comparable results relative to conventional methods. This DNA extraction and detection method is expected to be applicable in food, water, and environmental samples.

Nitrogen removal performance of improved subsurface wastewater infiltration system under various influent carbon-nitrogen ratios.

Subsurface wastewater infiltration system (SWIS) has been recognized as a simple operation and environmentally friendly technology for wastewater purification. However, effectively removing nitrogen (N) remains a challenge, hindering the widespread application of SWIS. In this study, zero-valent iron (ZVI) and porous mineral material (PMM) were applied in SWIS to improve the soil matrix. Our results suggested that the addition of ZVI and PMM could simultaneously enhance N removal efficiency and reduce nitrous oxide emissions. This could be attributed to the abundant electrons generated by ZVI alleviating the electronic limitation of denitrification and the porous structure of PMM providing solid phase support for microbial growth. In addition, the abundance of microbial functional genes increased in modified SWIS, which could further explain the higher pollutant removal efficiency. Overall, this study provides new insights into the mitigation of wastewater pollution and greenhouse gas emissions in SWIS. PRACTITIONER POINTS: ZVI and PMM can adapt to different C loads and enhance pollutant removal efficiency in SWIS. Increasing C-N ratios positively affected the nitrate removal performance and negatively affected ammonium removal performance in SWIS. The amending soil matrix promoted the reduction of the N2 O to N2 and greenhouse gas emissions were well controlled. The abundance of microbial functional genes increased with the improvement of the soil matrix.

High-Efficiency Enrichment by Saturating Nanoliters of Protein Affinity Media.

Affinity-purification mass spectrometry (AP-MS) is an established technique for identifying protein-protein interactions (PPIs). The basic technology involves immobilizing a high-specificity ligand to a solid-phase support (e.g., an agarose or magnetic bead) to pull down protein(s) of interest from cell lysates. Although these supports are engineered to minimize interactions with background protein, the conventional method recovers mostly nonspecific binders. The law of mass action for dilute solutions has taught us to use an excess of beads to capture all target proteins, especially weakly interacting ones. However, modern microbead technology presents a binding environment that is much different from a dilute solution. We describe a fluidic platform that captures and processes ultralow nanoliter quantities of magnetic particles, simultaneously increasing the efficiency of PPI detection and strongly suppressing nonspecific binding. We demonstrate the concept with synthetic mixtures of tagged protein and illustrate performance with a variety of AP-MS experiment types. These include a BioID experiment targeting lamin-A interactors from HeLa cells and pulldowns using GFP-tagged proteins associated with a double-strand DNA repair mechanism. We show that efficient extraction requires saturation of the solid-phase support and that <10 nL of beads is sufficient to generate comprehensive protein interaction maps.

TL-532, a novel specific Toll-like receptor 3 agonist rationally designed for targeting cancers: discovery process and biological characterization.

Toll-like receptor 3 (TLR3) is an innate immune receptor that recognizes double-stranded RNA (dsRNA) and induces inflammation in immune and normal cells to initiate anti-microbial responses. TLR3 acts also as a death receptor only in cancer cells but not in their normal counterparts, making it an attractive target for cancer therapies. To date, all of the TLR3-activating dsRNAs used at preclinical or clinical stages have major drawbacks such as structural heterogeneity, toxicity, and lack of specificity and/or efficacy. We conducted the discovery process of a new family of TLR3 agonists that are chemically manufactured on solid-phase support and perfectly defined in terms of sequence and size. A stepwise discovery process was performed leading to the identification of TL-532, a 70 base pair dsRNA that is potent without transfection reagent and is highly specific for TLR3 without activating other innate nucleic sensors such as RIG-I/MDA5, TLR7, TLR8, and TLR9. TL-532 induces inflammation in murine RAW264.7 myeloid macrophages, in human NCI-H292 lung cancer cells, and it promotes immunogenic apoptosis in tumor cells in vitro and ex vivo without toxicity towards normal primary cells. In conclusion, we identified a novel TLR3 agonist called TL-532 that has promising anticancer properties.

Genetic fusion of mussel foot protein to ZZ protein improves target detection in solid-phase immunoassays

In the process of a solid-phase immunoassay, the stability and binding orientation between the antibody and the solid matrix can substantially influence the results. ZZ protein is a modified peptide of the B domain of Staphylococcus aureus protein A, which can bind to the Fc fragment of an antibody. It is often used for oriented immobilization of antibodies during solid-phase immunoassay. However, the conjugate is often not retained during the process, for example during washing steps. The resulting low stability detracts from reproducibility and sensitivity. Mfp-5 protein comes from mussel, is one of the components of mussel foot silk protein, and has good adhesion and biocompatibility. In this paper, the fusion protein of ZZ and Mfp-5 was constructed and expressed in Escherichia coli. In this method, the ZZ domain was firmly attached to the solid-phase support by Mfp-5, the directional fixation of IgG was realized by binding the ZZ protein to an Fc fragment, and then a Fab fragment was bound to the antigen to realize the solid-phase immunoassay. In addition, a protein adsorption assay confirmed that the adhesion of ZZ-Mfp-5 was significantly higher than that of ZZ protein, and the presence of Mfp-5 improved the ability of ZZ protein to capture antibodies. In conclusion, compared with the passively immobilized ZZ protein, the ZZ-Mfp-5 protein had stronger immobilization and antibody capture, a 10-fold increase in sensitivity and wider linear range, and better stability of detection. This may be an attractive strategy for solid-phase immunoassays or biosensing assays.

Attomolar sensitivity microRNA detection using real-time digital microarrays

MicroRNAs (miRNAs) are a family of noncoding, functional RNAs. With recent developments in molecular biology, miRNA detection has attracted significant interest, as hundreds of miRNAs and their expression levels have shown to be linked to various diseases such as infections, cardiovascular disorders and cancers. A powerful and high throughput tool for nucleic acid detection is the DNA microarray technology. However, conventional methods do not meet the demands in sensitivity and specificity, presenting significant challenges for the adaptation of miRNA detection for diagnostic applications. In this study, we developed a highly sensitive and multiplexed digital microarray using plasmonic gold nanorods as labels. For proof of concept studies, we conducted experiments with two miRNAs, miRNA-451a (miR-451) and miRNA-223-3p (miR-223). We demonstrated improvements in sensitivity in comparison to traditional end-point assays that employ capture on solid phase support, by implementing real-time tracking of the target molecules on the sensor surface. Particle tracking overcomes the sensitivity limitations for detection of low-abundance biomarkers in the presence of low-affinity but high-abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to sim 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min.

DMTMM-mediated methylamidation for MALDI mass spectrometry analysis of N-glycans with structurally conserved sialic acid residues in biological fluids "via direttissima".

Characterization of serum glycoprotein N-glycans with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in positive-ion mode needs a derivatization step to stabilize and neutralize the negative charge on sialic acids. The acidic sugars are attached to the end of glycoproteins, glycolipids or gangliosides. Here, we present a method for sialic acid stabilization via modification based on derivatization of carboxylic acid group activated with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) with methylamine. DMTMM substitutes in many processes N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS) as activation reagent due to its better performance and higher stability in water. Glycosylated proteins are used as solid phase support for glycan derivatization and purification from excess of derivatization reagents. We evaluated our glycan analysis method in murine sera and intestinal lavages. The stabilization of sialic acid enables a complete conservation of the glycan structures, in contrast to other methods where sialic acids are partially lost. In BALB/c mouse sera, we detected predominantly mono- and di-sialylated N-glycans with mostly N-Glycolylneuraminic acid (Neu5Gc) and only trace amounts of N-Acetyl neuraminic acid (Neu5Ac). BALB/c mouse intestinal lavages glycoproteins contained asialo N-glycans. DMTMM-mediated methylamidation of N-glycans for MALDI mass spectrometry analysis is a fast and cheap method for structurally conserved glycan derivatization.

On-line Micro-Packed Column Solid-Phase Extraction of Cadmium Using Metal-Organic Framework (MOF) UiO-66 with Posterior Determination by TS-FF-AAS

Metal-organic framework (MOF) UiO-66 was synthesized and evaluated as solid-phase extractor support for cadmium preconcentration in a micro-flow injection system coupled to thermospray flame furnace atomic absorption spectrometry (TS-FF-AAS) detection. The adsorbent was characterized by X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), Raman spectroscopy, and textural data from N2 adsorption/desorption isotherm data. The optimized conditions were achieved by loading 10.0 mL of a sample, containing 0.05 mol L-1 of phosphate buffer solution at pH 8.0, at a high flow rate of 10.0 mL-1 through 20.0 mg of UiO-66 packed into a mini-column, followed by elution with 1.0 mol L-1 HCl. At these conditions, the method presented a preconcentration factor of 35.7, limit of detection of 0.03 μg L-1 and a dynamic linear range from 0.1 to 8.0 μg L-1. The adsorption performance of UiO-66 towards cadmium was not influenced by Pb2+, Hg2+, Fe2+, Co2+, Cu2+, Ni2+, Zn2+, Mg2+ and Ca2+ ions. Analysis of different waters samples (tap water, physiological solution, mineral water, and lake water) was carried out without matrix interference, yielding recovery values ranging from 92.0 to 111.9%.

Biomass-Derived Adsorbent for Dispersive Solid-Phase Extraction of Cr(III), Fe(III), Co(II) and Ni(II) from Food Samples Prior to ICP-MS Detection

A biomass-derived adsorbent was simply prepared and applied as efficient and low-cost solid-phase supports. The adsorbent material was characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), surface area analysis and Fourier transform infrared (FTIR) spectroscopy. The amorphous structure of the prepared adsorbent was indicated from the XRD. The prepared adsorbent exhibited surface functional groups such as carbonyl and hydroxyl groups, which enhance the application of DSPE. An accurate separation and preconcentration of Cr(III), Fe(III), Co(II) and Ni(II) prior to ICP-MS detection was achieved using the biomass-derived adsorbent. The extraction process was performed at pH 4 using 1 mL of 0.5 N nitric acid for elution and recovery of ions. The prepared biomass-derived adsorbent showed efficient performance for extraction application, exhibiting a preconcentration factor of 50 and LODs of 1.4, 2.4, 1.9 and 3.0 µg.L−1 for Cr(III), Fe(III), Co(II) and Ni(II), respectively, while the LOQs were reported as 4.1, 7.3, 5.7 and 8.9 µg.L−1 for Cr(III), Fe(III), Co(II) and Ni(II), respectively. The DSPE procedure presented was successfully applied to the determination of the Cr(III), Fe(III), Co(II) and Ni(II) contamination in some food samples.

Analysis of the Non-Specific Binding Proteins in the RNA Pull-Down Experiment

Background: To investigate the interactions between RNA and proteins is essential to understand how these macromolecule complexes exert their functions. RNA pull-down is a classic technique to enrich RNA binding proteins, however, a large number of non-specific binding proteins may be enriched during sample preparation, interfering with the downstream mass spectrometric analyses and also causing false positives. Objective: In this study we examined the background contaminates in RNA pull-down experiment using mass spectrometric analysis. Method Antisense MALAT1 was first synthesized using in vitro transcription and incubated with cellular proteins extracted from HepG2 cells. The non-specific binding proteins were isolated using streptavidin conjugated magnetic beads and separated on SDS-PAGE. Each gel lane was divided into nine bands and digested with trypsin for the downstream LC-MS/MS analyses. Methods: Antisense MALAT1 was first synthesized using in vitro transcription and incubated with cellular proteins extracted from HepG2 cells. The non-specific binding proteins were isolated using streptavidin conjugated magnetic beads and separated on SDS-PAGE. Each gel lane was divided into nine bands and digested with trypsin for the downstream LC-MS/MS analyses. Results: 191 protein groups were identified as non-specific binding proteins in RNA pull-down samples. In addition, a comparison between different sample preparation conditions showed that the level of background contaminates was mostly induced by the solid phase support rather than the studied RNA. In addition, using more stringent detergent and streptavidin magnetic beads with smaller size could reduce the amount of background interfering proteins. Conclusion: This study provides a reference to distinguish bona fide RNA interacting proteins from the background contaminants. The results also demonstrate that different sample preparation conditions have great impacts on the level of enriched background contaminates, shedding new light on the optimization of RNA pull-down experiments.

Detection of millennial Rosin in Empress Xiao's Crown by ELISA

Rosin, which is composed primarily of abietic acid, is widely used in adhesives, glazing agents, and varnishes, etc. Historically, traces of rosin have been hard to detect in relic samples using traditional techniques. In this work, the carbodiimide method is employed to couple abietic acid with keyhole limpet hemocyanin, which serves as the immunizing antigen to prepare anti-abietic acid polyclonal antibodies. Experiments show that anti-abietic acid polyclonal antibodies have good specificity and sensitivity for rosin. In optimized conditions, where ovalbumin coupled with abietic acid is bound with a solid-phase support as a competitor, abietic acid as low as 10−2 μg/ml can be detected by competitive Enzyme-linked Immunosorbent Assay. Afterward, this method was successfully implemented to semi-quantitatively detect abietic acid in micro-relic-samples from Pei's Crown and Empress Xiao's Crown, which not suitable for routine identification of rosin for too many impurities. It indicates that rosin was commonly used as an adhesive in the manufacture of these noble crowns, during the Sui and Tang Dynasties. In this study, specific anti-abietic polyclonal antibodies were first prepared and applied to identify rosin in historical relic successfully. It expands the range of immunoassay-detectable adhesives in the field of archaeology, from proteins to small organic molecules.

Application of a new type of Si–Al porous clay material as a solid phase support for immobilizing Acidovorax sp. PM3 to treat domestic sewage

A novel Si–Al porous clay material W (reprocessed from ceramic waste) was used for Acidovorax sp. strain PM3 immobilization to promote the growth of strains and improve nitrogen and phosphorus removal performance in water treatment systems. The porous clay material W was characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, and scanning electron microscopy indicating that porous clay material W was a type of mullite with 63.52 m2/g specific surface area. After immobilization, the maximum biomass increased 2.7 times the specific growth rate and the removal rates of chemical oxygen demand (COD), ammonia (NH4+–N), and total phosphorus (TP) by the immobilized PM3 were 42.99, 29.19, and 11.76% higher than the free strain after 24 h. The Monod equation showed that the growth rate and processing speed of immobilized PM3 increased. The maximum adsorption capacities of COD and NH4+–N onto porous clay material W were 2.33 and 0.32 mg/g on the basis of Langmuir isotherm. The removal capacities of COD, NH4+–N, and TP by the immobilized PM3 were 588.24, 20.37, and 5.06 mg/l, respectively, as shown by kinetic studies. These results demonstrated that porous clay material W could improve the efficiency of microbial nitrogen and phosphorus removal, and the immobilized microorganism system could effectively treat domestic sewage. The adsorption isotherms can well describe the adsorption process. The maximum adsorption capacity of COD and NH4+–N on porous clay material W is 2.33 and 0.32 mg/g, respectively. Kinetic studies showed that the removal capacity of immobilized PM3 to COD, NH4+–N, and TP was 58.824, 20.37, and 5.06 mg/l, respectively.

A Novel Nanoparticle-Based Method to Isolate DNA From Dried Saliva and Semen Samples

Objectives: Saliva and semen stains may be found at crime scene (suicide, homicide, kidnapping, rape, sexual assault, poisoning, etc.). Through, which DNA can be isolated and individual can be identified. DNA can be of great importance in forensic science investigation to identify individuals. Currently, there are enormous methods to isolate DNA from biological fluids. The current study employed nanoparticle–biomolecule conjugate method to isolate DNA. Materials & Methods: A nanoparticle-biomolecule conjugate is a magnetic nanoparticle with biomolecules attached to its surface. Saliva and semen were collected using cotton swabs from different surfaces (tile, steel, aluminum, glass, iron, wood, etc.) on which saliva and semen were poured and allowed to dry earlier as to create simulated crime scene. Magnetic nanoparticles (Fe3O4) act as solid phase support to isolate DNA from saliva and semen stains. In the current study, cells and spermatozoa were extracted from saliva and semen swabs, respectively. Cell lysis was performed to extract epithelial cells from saliva and spermatozoa from semen and magnetic nanoparticles and biomolecule conjugate were added to the lysed cells to bind to DNA. Results: Application of external magnetic field helped to settle down nanoparticle-biomolecule conjugate and thus separate DNA from other cell components. Spectrophotometer Nanodrop technology was employed to perform the readings, which confirmed the presence of DNA at 260/280 ratio and quality of DNA was compared with the DNA isolated using the two different methods.Conclusion: The current study aimed at developing all new methods to isolate DNA from dried saliva and semen samples using magnetic nanoparticles by reviewing the methods developed earlier to isolate DNA from blood and tissue using magnetic nanoparticles. With modifications in the method and chemical preparation, a new method was successfully developed. DNA was isolated successfully; and confirmation was made by analyzing DNA through Nanodrop spectrophotometer.

Abstract NT-119: TARGET DISCOVERY OF NATURAL PRODUCT INSPIRED PHYLLANTHUSMINS FOR TREATMENT OF HIGH GRADE SEROUS OVARIAN CANCER

Abstract BACKGROUND: High grade serous ovarian cancer (HGSOC) is a lethal gynecological malignancy with a need for new therapeutic agents. Many of the most widely used chemotherapeutic drugs are either derived from or are semi-synthetic derivatives of natural products. We developed potent synthetic analogs (PHYs) of the phyllanthusmin class inspired by prior natural product isolated from Phyllanthus poilanei Beille. MATERIALS &amp; METHODS: HGSOC cell lines, OVCAR3 and OVCAR8, and non-tumorigenic controls, IOSE80 and FT33, were used in this study. Cytotoxicity assays included sulforhodamine B assay, and annexin X/PI staining and Western blotting for confirmation of apoptosis induction. A photo affinity labeling method was used to attach PHY analogs to solid phase support. Targets were isolated using a pulldown technique and mass spectrometry. CRISPR-Cas9 genome editing was used to knockout and confirm putative targets. RESULTS: The most potent analog, PHY34, has nanomolar potency in HGSOC cell lines in vitro and displayed cytotoxic activity through late-stage autophagy inhibition and activation of apoptosis. PHY34 was readily bioavailable through intraperitoneal administration in vivo where it significantly reduced HGSOC tumor burden. Targets were identified using photo affinity labeling-aided protein pulldown and mass spectrometry, and confirmed by generating knockout cell lines of targets. CONCLUSIONS: This class of compounds holds promise as a potential, novel chemotherapeutic approach and demonstrates the effectiveness of pleiotropically targeting autophagy and apoptosis as a viable strategy for combating high grade serous ovarian cancer. Citation Format: Alexandria N. Young, Steven Kurina, Andrew Huntsman, Rathnayake A. Rathnayake, Melissa Korkmaz, A. Douglas Kinghorn, Leslie Aldrich, Stephanie Cologna, James R. Fuchs, Joanna E. Burdette. TARGET DISCOVERY OF NATURAL PRODUCT INSPIRED PHYLLANTHUSMINS FOR TREATMENT OF HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-119.

A convenient and efficient total solid-phase synthesis of DOTA-functionalized tumor-targeting peptides for PET imaging of cancer

IntroductionAn efficient and cost-effective synthesis of the metal chelating agents that couple to tumor-targeting peptides is required to enhance the process of preclinical research toward the clinical translation of molecular imaging agents. DOTA is one of the most widely used macrocyclic ligands for the development of new metal-based imaging and therapeutic agents owing to its ability to form stable and inert complexes under physiological conditions. Although solid-phase synthesis compatible DOTA-tris-(t-Bu ester) is a commercial product, it is expensive and contain chemical impurities. There is a need to explore new and cost-effective methods for the preparation of metal chelating agents, i.e., DOTA, directly on solid support to facilitate rapid, cost-effective, and high purity preparation of DOTA-linked peptides for imaging and therapy. In the present study, we describe a facile synthetic strategy of DOTA preparation and its linkage to peptides directly on solid-phase support.MethodsBombesin (BN) peptides were functionalized with DOTA chelator prepared from cyclen precursor on solid-phase and from commercial DOTA-tris and radiolabeled with 68Ga. In vitro BN/GRP receptor binding affinities of the corresponding radiolabeled peptides were determined by saturation binding assays on human breast MDA-MB-231, MCF7, T47D, and PC3 prostate cancer cells. Pharmacokinetics were studied in Balb/c mice and in vivo tumor targeting in MDA-MB-231 tumor-bearing nude mice.ResultsDOTA was prepared successfully from cyclen on solid-phase support, linked specifically to BN peptides and resultant DOTA-coupled peptides were radiolabeled efficiently with 68Ga. The binding affinities of all the six BN peptides were comparable and in the low nanomolar range. All 68Ga-labeled peptides showed high metabolic stability in plasma. These radiopeptides exhibited rapid pharmacokinetics in Balb/c mice with excretion mainly through the urinary system. In nude mice, MDA-MB-231 tumor uptake profiles were slightly different; the BN peptide with Ahx spacer and linked to DOTA through cyclen exhibited higher tumor uptake (2.32% ID/g at 1 h post-injection) than other radiolabeled BN peptides investigated in this study. The same leading BN peptide also displayed favorable pharmacokinetic profile in Balb/c mice. The PET images clearly visualized the MDA-MB-231 tumor.ConclusionsDOTA prepared from cyclen on solid-phase support showed comparable potency and efficiency to DOTA-tris in both in vitro and in vivo evaluation. The synthetic methodology described here allows versatile, site-specific introduction of DOTA into peptides to facilitate the development of DOTA-linked molecular imaging and therapy agents for clinical translation.

Quantitative Assessment of Affinity Selection Performance by Using DNA-Encoded Chemical Libraries.

DNA-encoded chemical libraries are often used for the discovery of ligands against protein targets of interest. These large collections of DNA-barcoded chemical compounds are typically screened by using affinity capture methodologies followed by PCR amplification and DNA sequencing procedures. However, the performance of individual steps in the selection procedures has been scarcely investigated, so far. Herein, the quantitative analysis of selection experiments, by using three ligands with different affinity to carbonic anhydrase IX as model compounds, is described. In the first set of experiments, quantitative PCR (qPCR) procedures are used to evaluate the recovery and selectivity for affinity capture procedures performed on different solid-phase supports, which are commonly used for library screening. In the second step, both qPCR and analysis of DNA sequencing results are used to assess the recovery and selectivity of individual carbonic anhydrase IX ligands in a library, containing 360 000 compounds. Collectively, this study reveals that selection procedures can be efficient for ligands with sub-micromolar dissociation constants to the target protein of interest, but also that selection performance dramatically drops if 104 copies per library member are used as the input.

Selective Removal of Uranium from Rare Earth Leachates via Magnetic Solid-Phase Extraction Using Schiff Base Ligands

This study highlights a new type of extraction procedure combining a magnetic solid-phase support and a Schiff base ligand for the removal of uranium from rare earth leachates. After synthesizing a...

Design of experiments as tools to tailor impregnated polymers specific for radionuclides separation in microsystems

An experimental design is described for optimization of the microscopic morphology of a methacrylate monolith that was elaborated for chromatographic separation of radionuclides in nitric acid media. This paper presents a systematic study of the synthesis of the polymeric porous monolith poly(ethylene glycol dimethacrylate-co-allyl methacrylate) used as solid-phase support and a post-functionalization of the monolith in microsystem with tributyl phosphate extractant. Polymerization time and chemical composition of the polymerizable mixture that comprises water, 1,4-butanediol, 1-propanol, monomers were chosen as the most relevant experimental factors of the photochemical process. Using the globules area as a significant response of an experimental design, the monolith morphology can be predicted. A new versatile and robust impregnation process was developed in microsystem. The designed micro chromatographic system showed a good resistance in concentrated nitric acid and a great loading capacity compared to commercially available solution (150 mg U versus 75 mgU/g resin).

On-nylon membrane detection of nucleic acid molecules by rolling circle amplification

Positively-charged nylon membrane (NM) is a general solid-phase support for nucleic acid detection due to its convenient immobilization of nucleic acid materials by direct electrostatic adherence and simple UV crosslinking. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification technique for nucleic acid detection. Near-infrared fluorescence (NIRF) is a new fluorescence technique with high sensitivity due to low background. This study developed a simple method for detecting nucleic acid molecules by combining the advantages of NM, RCA and NIRF, named NIRF-based solid phase RCA on nylon membrane (NM-NIRF-sRCA). The detection system of this method only need two kinds of nucleic acid molecules: target-specific probes with a RCA primer (P) at their 3′ end and a rolling circle (RC). The detection procedure consists of four steps: (1) immobilizing detected nucleic acids on NM by UV crosslinking; (2) hybridizing NM with specific probes and RC; (3) amplifying by a RCA reaction containing biotin-dUTP; (4) incubating NM with NIRF-labeled streptavidin and imaging with a NIRF imager. The method was fully testified by detecting oligonucleotides, L1 fragments of various HPV subtypes cloned in plasmid, and E.coli genomic DNA. This study thus provides a new facile method for detecting nucleic acid molecules.

Simple Design of an Enzyme-Inspired Supported Catalyst Based on a Catalytic Triad

Summary Enzyme active sites afford an intricate interplay of functional groups to mediate complex organic and inorganic reactions. Many hydrolytic enzymes use a catalytic triad comprising three different functional residues—(Ser(-OH), Hist(-imidazole), Asp(-CO 2 H))—that catalyze the hydrolysis of numerous unique substrates. Inspired by this design, we have developed a simple one-step synthesis for preparing a new supported catalytic system in which the three reactive groups of the catalytic triad (alcohol, imidazole, and carboxylate) are incorporated into a single functional unit. These artificial active sites can be coupled to a solid-phase support (Merrifield resin) by copper(I)-catalyzed azide-alkyne cycloaddition "click chemistry," and their effectiveness as esterolysis catalysts was demonstrated. Furthermore, tuning the local hydrophobicity of the resin particles with an approach analogous to the native enzyme hydrophobic pocket increased the catalytic efficiency. Quantum mechanics and molecular dynamics computational modeling were used to probe the catalytic effect and suggested a concerted two-step mechanism and hydrophobic nanoenvironment similar to that of hydrolytic enzymes.