Prior Elution Research Articles

Ceria nanoparticles deposited on graphene nanosheets for adsorption of copper(II) and lead(II) ions and of anionic species of arsenic and selenium

A nanocomposite prepared from graphene nanosheets and cerium nanoparticles (G/CeO2) was applied to the extraction of Se(IV), As(V), As(III), Cu(II) and Pb(II). The structure of G/CeO2 was investigated by scanning electron microscopy, X-ray diffraction and Raman spectroscopy. The optimal pH values for extraction are 4.0 for As(V), 3.0 for Se(IV), and 6.0 for both Cu(II) and Pb(II). The maximum adsorption capacity of G/CeO2 (expressed as mg·g−1) were calculated by the Langmuir model and are found to be 8.4 for As(V), 14.1 for Se(IV), 50.0 for Cu(II) and 75.6 for Pb(II). The sorbent was applied to dispersive solid phase microextraction prior to direct quantitation by energy-dispersive X-ray fluorescence spectrometry without the need for prior elution. The limits of detection (in ng·mL−1 units) are 0.10 for As(V), 0.11 for Se(IV), 0.19 for Cu(II) and 0.21 for Pb(II). The precisions (RSDs) are <4.5%. The accuracy of the method (1 - 4%) was verified by analysis of the certified reference material (CRM 1640a - natural water). The method was successfully applied in ultratrace element determination and to the speciation of selenium in environmental waters.Graphical abstractThe method gives possibility of simultaneous preconcentration and determination in environmental waters of both anionic (As(V) and Se(IV)) and cationic (Cu(II) and Pb(II)) forms of selected metals using graphene nanosheets and cerium nanoparticles. Se(IV) can be selective determined in the presence of Se(VI).

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI).

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Isolation of DNA using magnetic nanoparticles coated with dimercaptosuccinic acid

Lately, the isolation of DNA using magnetic nanoparticles has received increased attention owing to their facile manipulation and low costs. Although methods involving their magnetic separation have been extensively studied, there is currently a need for an efficient technique to isolate DNA for highly sensitive diagnostic applications. We describe herein a method to isolate and purify DNA using biofunctionalized superparamagnetic nanoparticles synthesized by a modified polyol method to obtain the desired monodispersity, followed by surface modification with meso-2,3-dimercaptosuccinic acid (DMSA) containing carboxyl groups for DNA absorption. The DMSA-coated magnetic nanoparticles (DMSA-MNPs) were used for the isolation of DNA, with a maximum yield of 86.16%. In particular, we found that the isolation of DNA using small quantities of DMSA-MNPs was much more efficient than that using commercial microbeads (NucliSENS-easyMAG, BioMérieux). Moreover, the DMSA-MNPs were successfully employed in the isolation of genomic DNA from human blood. In addition, the resulting DNA–nanoparticle complex was directly subjected to PCR amplification without prior elution, which could eventually lead to simple, rapid, sensitive and integrated diagnostic systems.

Simultaneous in vivo monitoring of multiple brain metals using an online microdialysis-in-loop solid phase extraction-inductively coupled plasma mass spectrometry system

Using a non-functionalized small-bore polytetrafluoroethylene (PTFE) sample loop as a preconcentrator, a novel hyphenated system—comprising microdialysis sampling, online automatic in-loop solid phase extraction (SPE), and inductively coupled plasma mass spectrometry (ICP-MS)—for monitoring the levels of trace metal ions in living rat brains was developed. Taking advantage of the selective polymer–ion interaction to online separate metal ions from highly saline operating in the in-loop PTFE SPE system, it could online remove the salt matrix selectively from the microdialysate of rat brain extracellular fluid after slightly adjusting the pH; the next analytical cycle could be performed without the need for a pre-conditioning procedure after the prior elution with HNO3. Owing to the simplicity and convenience of the developed operation sequence, this method combines extremely low blank level and detection limits (0.003–0.5 μg L−1) and acceptable spike recoveries (90–98%) with the ability to analyze multiple elements. To demonstrate analytical reliability and compatibility, the analysis of standard reference material NIST 1643e (trace elements in water) and 2670a (trace elements in human urine) as well as continuous long-term monitoring of the variations of multiple trace metal ions in rat brain were performed in this work. According to the analytical results, it showed that the developed hyphenation system had the ability to analyze the SRM samples accurately, and the basal values of Ni (0.36 ± 0.16 μg L−1), Zn (3.09 ± 0.31 μg L−1), and Mn (1.31 ± 0.06 μg L−1) in rat brain extracellular fluid could also be measured.

Rapid determination of strontium-90 in seawater

It is shown that Y2O3, YF3, LaF3, and CeF3 help concentrate 90Y from seawater. To determine 90Sr, YF3 is the best reagent because the effect of interfering lead and thorium radioisotopes is minimum in this case. It is proposed to preconcentrate 210Bi on PbS to eliminate its interference. To determine 90Sr we measure the Cherenkov radiation of 90Y concentrated on YF3 without prior elution.

Cytotoxicity of Two Autopolymerized Acrylic Resins Used in Orthodontics

To test in vitro the null hypothesis that the toxic effect of different acrylic resins used in orthodontics cannot be reduced when a prior elution is performed. Three established cell lines were used (HeLa, NIH3T3, and Hep2) and cultured under standard conditions. Resin segments were immersed in a culture medium and left to elute for 24 and 48 hours. Cells were exposed to medium containing eluates for 24 or 48 hours. The 3-(4,5 dimethyl-2-thiazolyl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) assay was used as the cytotoxicity test. Control cells contained standard medium with no eluate. Analysis of variance and Tukey test were used for statistical analysis. Fibroblastic viability was not affected when the elution time was 24 hours, but treatments showed higher cell viability than controls when the elution time was 48 hours. When left to elute for 24 hours, both resins had a cytotoxic effect on epithelial cells, but this effect was not observed when the elution time was 48 hours. The hypothesis was rejected as both tested materials showed lower cytotoxic effect when treated with 48-hour elutes compared with 24-hour elutes, which indicates that a longer elution time reduces resin toxicity.

Characterization of Phosphopeptides Using a Combination of Immobilized Metal Ion Affinity Media and Direct Analysis by MALDI-TOF-MS: Figure 1.

INTRODUCTIONPhosphoproteins and peptides can be bound with high specificity to immobilized metal ions such as Fe(3+), Ni(2+), and Ga(3+). This technique can be used with either on-line or off-line MS analysis. However, elution of the phosphopeptides from the metal ion column prior to MS analysis can result in sample loss. Affinity-bound analytes, including phosphopeptides, can be directly analyzed by MALDI-MS without prior elution from the affinity media. Also, consecutive enzymatic reactions, such as phosphatase or carboxypeptidase Y digestion, can be carried out on affinity-bound peptides. When the affinity-bound phosphopeptides are treated with phosphatase, the number of phosphorylation sites can be determined based on the observation of 80-Da (or multiples of 80 Da) mass shifts in the MALDI-MS of the reaction mixture. Carboxypeptidase Y treatment of the affinity-bound phosphopeptides can also be used to cleave amino acids from the carboxyl terminus, with subsequent direct analysis of the enzymatic products by MALDI-MS to locate the phosphorylation sites on the bound phosphopeptides. This protocol details the preparation and use of Fe(3+) or Ga(3+) metal IMAC with the on-bead analysis of phosphopeptides by MALDI-MS. Enzymatic digestion of affinity-bound peptides is also described.

Radio-LC-MS for the characterization of 99mTc-labeled bioconjugates.

This report describes the first example of using radio-LC-MS for determining the composition of (99m)Tc radiopharmaceuticals at the tracer level. The in-line radiometric detector is a useful addition to a standard LC-MS and provides direct correlation between the MS data and the radioactive species in a radiopharmaceutical kit. Complexes [(99m)Tc(HYNICtide)(tricine)(L)] (RP444, L = TPPTS; RP445, L = TPPDS; and RP446, L = TPPMS) were prepared using a decayed generator eluant. All the ternary ligand (99m)Tc complexes show the expected monoprotonated molecular ions, (M + 1)(+), and diprotonated molecular ions, (M + 2)(2+). The LC-MS spectral data support the proposed structure and are consistent with those obtained for their corresponding (99)Tc analogues. Ternary ligand complexes [(99m)Tc(HYNICtide)(tricine)(L)] (L = ISONIC-HE and ISONIC-Sorb) are neutral, and the molecular weights are also lower than that of RP444. Using a fresh generator eluant (24 h prior elution), only 1-2 mCi of (99m)Tc [(7 x 10(-)(12))-(1.5 x 10(-)(11)) mol of technetium complex] are required to obtain a reasonably clean mass spectrum. Radio-LC-MS is a quick and accurate analytical tool for characterization of (99m)Tc radiopharmaceuticals at the tracer level.

Immunoaffinity Chromatography

AbstractThis technique involves elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. Antibodies are coupled to Sepharose and the cell lysate is passed over the column. After washing, the specific antigen is eluted from the column using one of a number of methods: brief exposure to either high or low pH, or using the detergent octyl beta‐D‐glucoside (easily removable by dialysis). All these methods are described in this unit along with a procedure for covalently linking an antibody to Sepharose using the cyanogen bromide activation method.

Immunoaffinity Chromatography

AbstractImmunoaffinity purification is a powerful technique for isolating proteins. Purifications of 10,000‐fold or more can often be achieved in one step. This unit describes the isolation of soluble or membrane‐bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. The technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. An alternate protocol describes the use of batch purification of antigens which can shorten the column loading time.

Immunoaffinity chromatography.

This unit describes the isolation of soluble or membrane-bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. The technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. To elute the bound antigen from the immunoaffinity matrix, the antibody-antigen interaction is destabilized by brief exposure to high-pH or low-pH buffer. The use of batch purification of antigens is an alternate procedure and results in shorter column loading times. The detergent octyl beta-D-glucoside can be used instead of Triton X-100 for elution. Because octyl beta-D-glucoside has a high critical micelle concentration (CMC), it can be readily removed by dialysis, as described.

Immunoaffinity Chromatography

AbstractThis unit describes the isolation of soluble or membrane‐bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. This technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. Specifically, antibodies are coupled to Sepharose (an insoluble, large‐pore‐size chromatographic matrix). High‐molecular‐weight antigens pass freely into and out of the pores and bind to antibodies covalently bound to the matrix. To elute the bound antigen from the immunoaffinity matrix, the antibody‐antigen interaction is destabilized by brief exposure to high‐ or low‐pH buffer. Batch purification of antigens is provided as an alternate procedure that shortens the column loading time. The detergent octyl β‐D‐glucoside can be used instead of Triton X‐100 for elution. Because octyl β‐D‐glucoside has a high critical micelle concentration (CMC), a protocol is provided for its removal by dialysis. The procedure for covalently linking an antibody to Sepharose using the cyanogen bromide activation method is given in a support protocol.

Sensitive Trace-First Liquid Chromatographic Determination of 4-Aminopyridine in 3,4- Diaminopyridine

For the treatment of human neuromuscular diseases, 3, 4-diaminopyridine (DAP) is six to ten times more effective than 4-aminopyridine (AP), but only half as convulsant and toxic. Therefore, there is a need for the determination of AP in DAP. With only conventional equipment, high-pressure liquid chromatography can be used for the extremely sensitive detection of a trace contaminant under one condition: that is, the trace must be eluted before the major component it contaminates. Prior elution presents a trace peak in a fully exploitable form that is maximally efficient and maximally observable. This has already been demonstrated with a Pirkle-concept chiral stationary phase for determination of a chiral trace. However, its application to determination of a nonchiral trace with a reversed phase has not previously been reported. Such an application is reported here. In this demonstrative study, selectivity and loading capability were iteratively improved. Ion pairing with dodecanesulfonate maximized selectivity. It was again shown that using a less concentrated sample in greater volume maximizes loading capability without obscuring the peak of the trace. Eventually, the ability to detect 0.005% AP in DAP was demonstrated. Whether that sensitivity might be improved still more, perhaps with a larger column, was not established.

Sequencing of Protein from a Single Spot of a 2-D Gel Pattern: N-Terminal Sequence of a Major Wheat LMW-Glutenin Subunit

Kasarda, D. D., Tao, H. P., Evans, P. K., Adalsteins, A. E. and Yuen, S. W. 1988. Sequencing of protein from a single spot of a 2-D gel pattern: N-terminal sequence of a major wheat LMW-glutenin subunit—J. exp. Bot. 39: 899–906. Proteins extracted from wheat (Triticum aestivum L.) flour were fractionated by two-dimensional electrophoresis (isoelectric focusing in the first dimension and SDS-polyacrylamide gel electro-phoresis in the second). The proteins in the gel were electroblotted onto a polyvinylidene difluoride membrane and the pattern was developed by staining with amido black. There was considerable variation in the degree of transfer for various proteins; ω-gliadins did not transfer at all, most high-molecular-weight glutenin subunits transferred poorly or not at all, but most other gliadins and some glutenin subunits transferred fairly well. A single spot corresponding to the major low-molecular-weight (LMW) glutenin subunit of the wheat (T. aestivum L.) cultivar ‘Anza’ was cut out and subjected to automatic amino acid sequencing without prior elution from the membrane. The sequence was identical at 12 positions of the 14 cycles to the major sequence reported earlier for a mixture of LMW-glutenin subunits. Yields of phenylthiohydantoin-amino acids ranged from about one to five picomoles for the correctly identified amino acids.

Direct cell culture assay of thin-layer-chromatographed material without prior substance elution

A method enabling the direct assay in a cell culture system of material separated by thinlayer chromatography (TLC) without prior elution of active material from the TLC sheet is described. After chromatographic development on a plastic cellulose sheet, small disks were punched out from the sheet and transferred to microwells used for cell culture. Lymphoid cells in culture medium were incubated with the disks and the DNA synthesis was measured by [ 3H]thymidine incorporation. The reliability of the method was tested with l-alanine and thymidine, which are known to enhance the DNA synthesis of lymphoid cells and to interfere with the uptake of labeled thymidine, respectively. The incorporation of [ 3H]thymidine was stimulated with disks containing l-alanine and inhibited with disks containing unlabeled thymidine, completely in agreement with the expected results. Elution experiments showed that more than 75% of l-alanine and thymidine on the TLC disks was eluted into the culture medium within 1 h. Elution of material of higher molecular weight was also demonstrated. The method was used for the separation and assay of a thymocyte-specific growth factor isolated from calf thymus and greatly facilitated the detection of the active material after TLC.

Paper chromatographic separation of phosphate esters, tricarboxylic cycle acids and amino acids in extracts from malaria parasites

A paper chromatographic method for the separation and identification of complex mixtures of phosphate esters, tricarboxylic cycle acids and amino acids in biological extracts is described. The method has been applied to the investigation of carbohydrate metabolism pathways in the intraerythrocytic, simian malaria parasite Plasmodium knowlesi; on treatment of parasitized erythrocytes with [U-C 14]glucose, a perchloric acid extract was prepared and separated by chromatography on Whatman 31 ET filter-paper. A simple procedure for the measurement of the specific activities of radioactive compounds without prior elution from the chromatographic support is also described.

A simple method for the isolation of multiple sclerosis specific brain antigens

A method for isolation of two multiple sclerosis(MS)-specific brain antigens [3] is described. The brain cytosol was passed through a Con A-Sepharose column and the glycoproteins containing MS-specific antigens (MSG) were eluted with 50 mmol/l methyl-α- d-glucopyranoside, pH 6. By fractionation of MSG on DEAE-cellulose, a fraction containing MS-specific antigens (MSG2) was eluted in 0.25 mol/l NaCl, after prior elution of other proteins with distilled water. The MS specific antigens in the MSG2 fraction were traced and characterized by crossed immunoelectrophoresis and isoelectric focusing. A quantitative determination of proteins eluted in MSG2 revealed that the 2 MS-specific antigens had been purified 10 000 times. Using this method to isolate the two MS-specific antigens of autopsy brains, they were found to be present in 7 7 MS brains and absent in 9 9 non-MS brains studied. The method described in the present communication may be used both for the partial purification of the MS-specific antigens as well as for the tracing of the specific antigens in different topographical areas of the MS brains.

Determination of the proportion of cholesteryl ester in plasma.

In normal human plasma the proportion of choles­ terol present as cholesteryl ester is very constant, but in certain clinical conditions, such as liver disease, a reduction may occur. Measurement of plasma cholesteryl ester is usually achieved indirectly as the difference between total cholesterol and free choles­ terol, which inevitably leads to a larger error in its determination. Plasma lipids in human and experi­ mental schistosomiasis (Owen and Gillett, 1978)show small but significant decreases in the percentage of cholesterol esterified. The measurement of such changes is the subject of this note. Thin-layer chromatography was used to separate free cholesterol and cholesteryl ester fractions followed by hydrolysis of both under identical conditions without prior elution from the silica gel. The non-saponifiable material was then extracted into hexane, and ali­ quots were taken for colorimetric estimation. Plasma lipids were extracted and washed accord­ ing to the procedure of Folch, and the neutral lipids were separated on 0·25 mm layers of silica gel G (Merck) using hexane-ether-acetic acid 90:20:1 (by vol) as the developing solvent. Areas correspond­ ing to free cholesterol and cholesteryl ester were located by exposure to iodine vapour and transferred to tubes (125 x 50 mm) fitted with Teflon-lined screw caps (Kimble Products, Ill., USA). Three millilitres of 1M ethanolic KOH was added and, after mixing, the contents were heated at 80°C for one hour. Hexane (5 ml) was added followed by 2% (w/v) Na2S04 (5 ml), the contents were mixed, and aliquots of the hexane phase were taken for cholesterol estimation. In preliminary experiments, recovery and extent of hydrolysis were examined under varying conditions by adding 7- 3H-cholesteryl oleate to the tubes. Determination of the percentage hydrolysis of the labelled cholesteryl ester at different temperatures (40°C, 6Q c C, and 80 c C) and for varying time inter

Quantitative analysis of phenolic compounds after thin-layer chromatographic separation

A method has been developed for the quantitative analysis of phenolic substances having non-substituted phenolic groups after their separation by thin-layer chromatography on silica gel or cellulose. After the clear detection of the phenolic substance on the chromatoplate by means of Folin-Ciocalteu reagent, the quantitative spectrophotometric determination of the substance is carried out by transferring the sorbent area containing the spot into a test-tube and using the Folin-Ciocalteu reagent, without the necessity for carrying out a prior elution for the recovery of the substance. The method appears to have good accuracy and to be suitable for general application.

A new method for determining the distribution of allergenic fractions in biological materials: Its application to grass pollen extracts

Dialyzed whole extracts of seven grass pollens representing six botanical tribes were fractionated with the use of a modified starch gel electrophoretic technique. Without prior elution from the gels, individual protein-containing fractions were analyzed for their allergenic properties by direct skin testing in grass pollen-sensitive individuals and for their antigenic components with the use of immunodiffusion against rabbit antisera to highly purified rye grass fractions and to crude grass pollen extracts. Immunochemically distinct groups of antigens, classified as Groups I, II, and III, were found in most of the grass pollens studied, the principal exception being Bermuda which contained none of these groups. Antigens of a particular group but from different pollens possessed similar allergenic properties in the individuals tested. Each individual could be broadly classified on the basis of his skin reactivity to the different antigenic groups.