Albumin Samples Research Articles

Sensitive Detection of Glycated Albumin in Human Serum Albumin Using Electrochemiluminescence.

Monitoring of blood glucose content is vital for diabetes patients. The conventional widely used method involves an invasive procedure for blood sampling. In addition, blood glucose measured by this way is affected by immediate food consumption and it does not show accurate baseline blood glucose measurement. Thus, monitoring blood glucose by a noninvasive method that accurately reflects baseline blood glucose content is important. Glycated albumin (GA), a biomarker for diabetes indicating the average blood glucose over 2 weeks, can be used for semilong-term blood glucose monitoring. Detection of GA in saliva is a noninvasive method that alleviates the use of needles for diabetic patients; however, its content in saliva is in the nanomolar range. Therefore, the GA enzymatic detection method was combined with the ECL method for a highly sensitive detection of GA in human serum albumin and in the saliva sample. Here, the standard curve was constructed using model substrate, FZK, between 0.1 and 2 μM, and GA in human serum albumin was measured in this range. Also, we successfully demonstrated the detection limit of 0.1 μM GA in human serum albumin sample using ECL, which has seen improvement of about 70 times more than the colorimetric methods. The detection of GA in real saliva sample suggested that sample dilution of 5 times may be necessary to suppress the ECL quenching effect by impurities.

Analysis of Nicotine Metabolisms in Chicken Development

Studies indicate that 12.7% women are smoking during pregnancy, and a significant proportion of the United States population has been exposed to maternal smoking in utero. The early chick embryo is an established model of the first month of embryonic development in humans, including for nicotine effects. At the same time, it is likely that the drug pharmacokinetics in chickens has important distinctions from the one described in humans. Here we attempted to characterize the nicotine metabolism in chickens by measuring changes in nicotine concentrations maintained in ovo after a single nicotine dose. Nicotine (nicotine hydrogen bitartrate solutions) was injected in 24 eggs prior to incubation to match blood plasma levels observed in heavy smokers (30-50 ng/ml). After injections, eggs were sealed and incubated for 5 days. The eggs were harvested on day 5 after injections. Embryos were homogenized and sampled; corresponding yolk and albumin samples were also collected. The nicotine concentrations in all samples were measured using MEPS analysis. The important differences in nicotine metabolic pathways in chicks and humans are discussed.

Development of an enzymatic reactor applying spontaneously adsorbed trypsin on the surface of a PDMS microfluidic device.

Herein, a microfluidic device (MD) containing immobilized trypsin for rapid and efficient proteolysis was described. Trypsin was immobilized via non-specific protein adsorption onto the hydrophobic poly(dimethylsiloxane) (PDMS) channel wall of the MD. Peptide mapping of bovine serum albumin (BSA) samples was carried out to estimate the stability of trypsin adsorbed on PDMS surface. Peptide maps of BSA samples were obtained by capillary zone electrophoresis (CZE), the RSD% for migration times were under 1%. Several proteins (hemoglobin, myoglobin, lysozyme, and BSA) in a wide molecular size range (15-70kDa) were digested efficiently with ∼50s contact time. The number of separated peaks correlated well with the expected number of peptides formed in the complete tryptic digestion of the proteins. Peptide mass fingerprinting of BSA and human serum was carried out. Trypsin retained its activity for 2h; within this period, the MD can be used for multiple digestions. The main properties of this device are simple channel pattern, simple immobilization procedure, regenerability, and disposability; all these features make this MD one of the simplest yet applicable enzymatic microreactors. Graphical abstract Development of microfluidic device including a serpentine channel as an enzyme reactor for protein digestion.

Microalbuminuria screening in diabetes

Microalbuminuria screening in diabetes

Determination of tetracycline using imprinted polymethacrylates along with fluorescent CdTe quantum dots on plastic substrates

Molecular imprints (MIPs) composed of CdTe quantum dots and tetracycline-imprinted polymethacrylates were synthesized on the surface of flexible plastic polyimide sheets from methacrylic acid, allyl mercaptan, and ethylene glycol dimethacrylate. The amount of aqueous CdTe solution, ethanol diluent, the reaction time and temperature of the radical-initiated polymerization were optimized. The MIP-QD composites were then copolymerized on the methacrylated polyimides. The resulting films were then immersed into a supersonic water-ethanol bath to strip off the tetracycline (Tc) templates. The fluorescence quenching intensity measured at 565 nm under 315 nm excitation before and after dropping a Tc sample on the film was used to optimize the processes. The calibration plot is linear in the 70 μM to 2.2 mM Tc concentration range at pH 7.5, and the LOD is 8.8 μM. The relative standard deviation is 8.2% (for n = 10). The method was applied to the determination of Tc in spiked samples of bovine serum albumin and fetal bovine serum and gave recoveries of 98% (at 200 μg⋅mL−1) and 97% (at 1.0 ppt), respectively.

Compact Ultrahigh-Pressure Nanoflow Capillary Liquid Chromatograph.

A compact ultrahigh-pressure nanoflow liquid chromatograph (LC) was developed with the purpose in mind of creating a portable system that could be easily moved to various testing locations or placed in close proximity to other instruments for optimal coupling, such as with mass spectrometry (MS). The system utilized innovative nanoflow pumps integrated with a very low volume stop-flow injector and mixing tee. The system weighed only 5.9 kg (13 lbs) or 4.5 kg (10 lbs) without a controller and could hold up to 1100 bar (16000 psi) of pressure. The total volume pump capacity was 60 μL. In this study, the sample injection volume was determined by either a 60 nL internal sample groove machined in a high-pressure valve rotor or by a 1 μL external sample loop, although other sample grooves or loops could be selected. The gradient dwell volume was approximately 640 nL, which allowed significant reduction in sample analysis time. Gradient performance was evaluated by determining the gradient step accuracy. A low RSD (0.6%, n = 4) was obtained for day-to-day experiments. Linear gradient reproducibility was evaluated by separating a three-component polycyclic aromatic hydrocarbon mixture on a commercial 150 μm inner diameter capillary column packed with 1.7 μm particles. Good retention-time reproducibility (RSD < 0.17%) demonstrated that the pumping system could successfully generate ultrahigh pressures for use in capillary LC. The system was successfully coupled to an LTQ Orbitrap MS in a simple and efficient way; LC-MS of a trypsin-digested bovine serum albumin (BSA) sample provided narrow peaks, short dwell time, and good peptide coverage.

Stable isotopes predict reproductive performance of European starlings breeding in anthropogenic environments

AbstractUnderstanding reproductive performance in ecologically impoverished vs. more sustained anthropogenic habitats is critical to assess population health status and to develop land use and conservation management strategies. We compared resource‐based maternal effects, reproductive performance, and offspring quality in a model migratory passerine bird, the European starling (Sturnus vulgaris). We assessed female condition, quantity of egg constituents, quality of diet consumed during egg formation, and nestling growth and survival in two habitats: cultivated farmland and meadows. Egg, albumin, and shell mass were greater at the meadow site, while yolk mass did not differ significantly between sites; albumin mass most strongly predicted egg mass. Stable isotope enrichment (δ15N and δ13C) in yolk but not albumin suggests a broader range of diet for yolk formation at the meadow site but could also reflect different hydric conditions between sites. δ13C and δ15N enrichment did not predict yolk, albumin, or egg mass. Concentration of yolk testosterone was higher at the meadow site and correlated with δ13C enrichment in yolk. Nestling survival was higher in the meadow than in the farmland site and corresponded to egg mass and δ13C enrichment in lipid‐free yolk. Surviving nestlings were larger in the meadow than in the farmland site. Results indicate that agricultural practice influences reproductive output through resource‐based maternal effects. The analyses of isotopic and biochemical composition of small samples of yolk and albumin may provide a minimally invasive tool to assess individual reproductive performance and predict impacts of habitat quality on population health.

Composition of phacoemulsificated human lenses analyzed by infrared spectroscopy

PurposePhacoemulsification is one of the most popular techniques of treating cataract. During the surgery the vibrating phaco‐probe emulsifies the lens into pieces that are vacuumed into a cassette. Afterwards the shredded lens material is usually disposed. Currently spectroscopic methods are ordinarily applied for studying biological materials due to their high sensitivity, reliability and non‐destructive character. The study attempts to determine whether the infrared spectroscopy technique is appropriate to reveal the differences in the structure of the lenses, especially focusing on the content of substances indicating changes in the secondary protein structure or mineralization process.MethodsIn the study 99 randomly selected dispersed human lenses were analyzed. The procedures of phacoemulsification were performed at the Military Hospital in Cracow and Military Institute in Warsaw. Obtained shredded lenses were forwarded to the Institute of Nuclear Physics at Polish Academy of Sciences in Cracow for further investigation. After appropriate preparation, the material was analyzed by FTIR spectroscopy using Nicolet spectrometer equipped with DTGS detector and ATR attachment. For the accurate interpretation of the spectra of lenses, exemplary samples of albumin, DNA, glucose and hydroxyapatite were also analyzed.ResultsThe FTIR spectra of lenses are characteristic for natural tissues and are dominated by intense bandwidth derived from amides and lipids. The FTIR spectra of the lenses are most similar to the spectrum of albumin.ConclusionsThe material obtained during phacoemulsification is suitable for infrared spectroscopic investigation. The FTIR method allows for the determination of human eye lenses structure and enables to indicate the differences in their composition.

A portable sample concentrator on paper-based microfluidic devices

Microfluidic paper-based analytical devices (µPADs) provide a promising solution for low-cost point-of-care diagnostic applications. However, much work remains to be done in optimizing their design and performance. Accordingly, this study investigates the preconcentration performance of µPADs comprising one, two and three convergent channels, respectively. The performance of the three devices is evaluated experimentally using fluorescein and a fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) sample with an initial concentration of 10−5 M. It is shown that the single-channel µPAD achieves a 20-fold improvement in the sample concentration in approximately 2 min. By contrast, the double- and triple-channel µPADs achieve preconcentration factors of 60- and 140-fold, respectively. Finally, a portable concentrator device is proposed. The experimental results show that a 100-fold improvement in the FITC-BSA sample concentration can be obtained in approximately 110 s given the use of four 16-V batteries, yielding a practical point-of-care diagnostic device.

Simultaneous determination of estrogens (ethinylestradiol and norgestimate) concentrations in human and bovine serum albumin by use of fluorescence spectroscopy and multivariate regression analysis

The endocrine disruption property of estrogens necessitates the immediate need for effective monitoring and development of analytical protocols for their analyses in biological and human specimens. This study explores the first combined utility of a steady-state fluorescence spectroscopy and multivariate partial-least-square (PLS) regression analysis for the simultaneous determination of two estrogens (17α-ethinylestradiol (EE) and norgestimate (NOR)) concentrations in bovine serum albumin (BSA) and human serum albumin (HSA) samples. The influence of EE and NOR concentrations and temperature on the emission spectra of EE-HSA EE-BSA, NOR-HSA, and NOR-BSA complexes was also investigated. The binding of EE with HSA and BSA resulted in increase in emission characteristics of HSA and BSA and a significant blue spectra shift. In contrast, the interaction of NOR with HSA and BSA quenched the emission characteristics of HSA and BSA. The observed emission spectral shifts preclude the effective use of traditional univariate regression analysis of fluorescent data for the determination of EE and NOR concentrations in HSA and BSA samples. Multivariate partial-least-squares (PLS) regression analysis was utilized to correlate the changes in emission spectra with EE and NOR concentrations in HSA and BSA samples. The figures-of-merit of the developed PLS regression models were excellent, with limits of detection as low as 1.6×10−8M for EE and 2.4×10−7M for NOR and good linearity (R2>0.994985). The PLS models correctly predicted EE and NOR concentrations in independent validation HSA and BSA samples with a root-mean-square-percent-relative-error (RMS%RE) of less than 6.0% at physiological condition. On the contrary, the use of univariate regression resulted in poor predictions of EE and NOR in HSA and BSA samples, with RMS%RE larger than 40% at physiological conditions. High accuracy, low sensitivity, simplicity, low-cost with no prior analyte extraction or separation required makes this method promising, compelling, and attractive alternative for the rapid determination of estrogen concentrations in biomedical and biological specimens, pharmaceuticals, or environmental samples.

Antiglycation and cell protective actions of metformin and glipizide in erythrocytes and monocytes

Chronic hyperglycaemia causes glycation which subsequently results in the long-term complications of diabetes. Albumin, the major plasma protein is more sensitive to glycation resulting in structural, biological and physiological modifications. The long-term benefits of commonly used anti-diabetic drugs such as metformin and glipizide in diabetic patients are well understood. However, no extensive study has been performed to assess their role in the glycation induced albumin modifications and cellular protection. We carried out the glycation of bovine serum albumin using methylglyoxal as a glycating agent in absence or presence of metformin and glipizide to establish their anti-glycation action. Different glycation markers (fructosamine, carbonyl groups, free thiol groups and β-amyloid aggregation) and protein structural markers (absorption spectroscopy and native-polyacrylamide gel electrophoresis) were examined. Further THP-1 cells (monocytes) and erythrocytes were treated with drugs that were exposed to glycated albumin samples for 24 h, respectively at 37 °C to investigate the cytoprotective actions of drugs against glycation. After the treatment different anti-oxidant indices (catalase, glutathione, superoxide dismutase and nitric oxide), cell viability, lipid peroxidation and erythrocyte hemolysis were determined. Treatment with metformin and glipizide during in vitro albumin glycation significantly reduced the formation of glycation adducts and inhibited structural modifications. They restored the level of antioxidants in THP-1 and erythrocytes cells treated with glycated albumin thus protecting cells. Our results suggested protection mode of albumin glycation through inhibition by metformin and glipizide. Additionally, they exerted inhibitory actions on glycation-induced cellular damage by restoring cellular antioxidant defense.

Preconcentration of diluted mixed-species samples following separation and collection in a micro-nanofluidic device.

A microfluidic device consisting of a nanoscale Nafion membrane and a polydimethylsiloxane microchannel is proposed for the preconcentration of diluted multi-mixed species samples then following separation and collection. When an electric field is applied across the microchip, an accumulation of the mixed-species sample occurs at the junction between the microchannel and the membrane by means of ion concentration polarization effect. A separation of the sample then takes place due to the difference in the electrophoretic mobilities of the sample components. Finally, the component of interest is guided to a collection reservoir by manipulating the external potential configuration and is trapped in place by means of a magnetically actuated valve. The preconcentration performance of the proposed device is evaluated in both straight and convergent microchannels using a fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) sample. It is shown that a preconcentration factor of 40 times can be achieved using a straight microchannel. By contrast, the preconcentration factor increases to 50 times when using a convergent channel. The practical feasibility of the proposed device is demonstrated by performing the preconcentration, separation, and collection of a mixed FITC-BSA and Tetramethylrhodamine sample.

Sample pre-concentration with high enrichment factors at a fixed location in paper-based microfluidic devices.

The lack of sensitivity is a major problem among microfluidic paper-based analytical devices (μPADs) for early disease detection and diagnosis. Accordingly, the present study presents a method for improving the enrichment factor of low-concentration biomarkers by using shallow paper-based channels realized through a double-sided wax-printing process. In addition, the enrichment factor is further enhanced by exploiting the ion concentration polarization (ICP) effect on the cathodic side of the nanoporous membrane, in which a stationary sample plug is obtained. The occurrence of ICP on the shallow-channel μPAD is confirmed by measuring the current-voltage response as the external voltage is increased from 0 to 210 V (or the field strength from 0 to 1.05 × 10(4) V m(-1)) over 600 s. In addition, to the best of our knowledge, the electroosmotic flow (EOF) speed on the μPAD fabricated with a wax-channel is measured for the first time using a current monitoring method. The experimental results show that for a fluorescein sample, the concentration factor is increased from 130-fold in a conventional full-thickness paper channel to 944-fold in the proposed shallow channel. Furthermore, for a fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) sample, the proposed shallow-channel μPAD achieves an 835-fold improvement in the concentration factor. The concentration technique presented here provides a novel strategy for enhancing the detection sensitivity of μPAD applications.

Impact of glycation on structural and antioxidant function of human serum albumin: Relevance in diabetic complications

AimNon-enzymatic glycation impairs the structural and functional characterstics of human serum albumin (HSA) native conformation. Prolonged hyperglycemia causes cross links formation in proteins that may contribute to progression of diabetic complications. MethodsHSA (20μM) was incubated with different concentration of d-glucose100, 200, 300 and 400mg/dl for a period of 40 days in phosphate buffer saline (20mM pH=7.4) under sterile conditions. Incubated samples were extensively dialyzed and structural changes were analyzed by far and near UV circular dichroism spectra measurement. Fructosamine assay with nitroblue tetrazolonium was performed to confer isomerisation between glucose and protein. Aggregations of the glycated product (AGEs) formed during reduction of nitrobluetetrazolium dye were evaluated by transmission electron microscopy. Crosslinks aggregates were investigated by in-situ Congo red binding assay. Red blood cells hemolysis test was performed to decipher the antioxidant activity of albumin samples. ResultsFructosamine content in glycated albumin demonstrates the non-enzymatic addition of glucose to HSA and confers the formation of monoformazone (marker of glycation). Significant changes were found in the glycated samples of HSA compared to native (unmodified) in far and near UV circular dichroism. Transmission electron microscopy, Congo red staining, showed the formation of crosslink's aggregated mass in glycated HSA. Glycation of albumin reduces the antioxidant capacity of native albumin confirmed by red blood cells hemolysis test. ConclusionThe finding of present study brings new evidences on the detrimental alterations of on albumin vital functions after non-enzymatic glycation with glucose. These results may emphasize the albumin associated diabetic complications under glycemic range of diabetes mellitus.

A New Application for Albumin Dialysis in Extracorporeal Organ Support: Characterization of a Putative Interaction Between Human Albumin and Proinflammatory Cytokines IL-6 and TNFα.

Albumin dialysis in extracorporeal organ support is often performed in the treatment of liver failure as it facilitates the removal of toxic components from the blood. Here, we describe a possible effect of albumin dialysis on proinflammatory cytokine levels in vitro. Initially, albumin samples were incubated with different amounts of cytokines and analyzed by enzyme-linked immunosorbent assay (ELISA). Analysis of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) levels indicated that increased concentrations of albumin reduce the measureable amount of the respective cytokines. This led to the hypothesis that the used proinflammatory cytokines may interact with albumin. Size exclusion chromatography of albumin spiked with cytokines was carried out using high-performance liquid chromatography analysis. The corresponding fractions were evaluated by immunoblotting. We detected albumin and cytokines in the same fractions indicating an interaction of the small-sized cytokines IL-6 and TNFα with the larger-sized albumin. Finally, a two-compartment albumin dialysis in vitro model was used to analyze the effect of albumin on proinflammatory cytokines in the recirculation circuit during 6-h treatment. These in vitro albumin dialysis experiments indicated a significant decrease of IL-6, but not of TNFα, when albumin was added to the dialysate solution. Taken together, we were able to show a putative in vitro interaction of human albumin with the proinflammatory cytokine IL-6, but with less evidence for TNFα, and demonstrated an additional application for albumin dialysis in liver support therapy where IL-6 removal might be indicated.

Abstract 4747: Genomic alterations in ductal carcinoma in situ compared with Invasive breast cancer: a quantitative real-time PCR study

Abstract Introduction: Several molecular markers have been investigated as possible prognostic factors for ductal carcinoma in situ (DCIS) progression to invasive ductal cancer (IDC). DCIS is regarded as a non-obligate precursor of IDC as it is frequently found adjacent to or surrounded by invasive breast cancer. Previously, we carried out genomic analyses that demonstrated that DCIS has specific genetic alterations that were conserved in invasive disease. Nevertheless, it is still unknown whether DCIS is merely a marker of increased risk, or actually a driver of the progression to IDC.Thus, it is crucial to better understand the genetic aberrations that underlie DCIS progression to IDC. Methodology: We conducted array chromosomal genomic hybridization on pure DCIS, IDC and DCIS paired with IDC, with 30 cases each (all were microdissected). Three chromosomal regions were selected based on the rate of their detection, which was significantly different (p&amp;lt;0.05) between tumors of different receptor status, whether in combination, or single parameter; ER, PgR, HER2. In total, 9 genes were chosen for further validation, spanning Chromosome 4p15.33, Chromosome 5q11.2 and Chromosome 5q31.3. Quantitative real-time polymerase chain reaction (Q-RT-PCR) was used to study the copy number state of the 9 genes in different pure cases, including 22 DCIS, 29 IDC and 10 DCIS paired with IDC (all were microdissected). All areas contained more than 90% carcinoma or control cells. By normalizing values to the housekeeping gene, human albumin, and normal control DNA samples, we obtained ΔΔCt values for each sample. Results: 61 samples were analyzed by comparing DCIS and IDC copy numbers of genes within the selected chromosomal regions. The median relative gene quantities were 1.66(4p15.33), 1.34(5q11.2) and 1.25(5q31.3). In all regions, IDC showed higher levels of amplification than DCIS with varying degrees of significance. Not all differences between DCIS and IDC in individual genes were significant. This observation may be attributed to focal variations in gene copy numbers and noise from Q-RT-PCR methods. 4p15.33 and 5q31.3 regions demonstrated significant gain in IDC compared to DCIS (p = 0.021). Genes within these regions include miRNA 572 (MIR572) and integrin alpha 2 (ITGA2). MIR572 is known to be involved in post-transcriptional regulation of gene expression, whereas ITGA2 encodes the alpha subunit of a transmembrane receptor for collagens. In the Chromosome 5q31.3 region, paired samples of DCIS and IDC also showed a trend in gene copy numbers, with IDC having a higher value than DCIS. Conclusions: Our findings demonstrate that there are specific genetic alterations associated with DCIS progression to IDC. In particular, MIR572 within Chromosome 4p15.33 and ITGA2 within Chromosome 5q31.3 may serve as candidate regions to predict the progression of DCIS to IDC. Note: This abstract was not presented at the meeting. Citation Format: Trillium E. Chang, Keisha Warren, Ranju Nair, Tian Y. Lu, Adewunmi Adeoye, Vladimir Iakovlev, Susan J. Done. Genomic alterations in ductal carcinoma in situ compared with Invasive breast cancer: a quantitative real-time PCR study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4747. doi:10.1158/1538-7445.AM2015-4747

Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.

Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing.

Size measuring techniques as tool to monitor pea proteins intramolecular crosslinking by transglutaminase treatment

In this work, techniques for monitoring the intramolecular transglutaminase cross-links of pea proteins, based on protein size determination, were developed. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis profiles of transglutaminase-treated low concentration (0.01% w/w) pea albumin samples, compared to the untreated one (control), showed a higher electrophoretic migration of the major albumin fraction band (26kDa), reflecting a decrease in protein size. This protein size decrease was confirmed, after DEAE column purification, by dynamic light scattering (DLS) where the hydrodynamic radius of treated samples appears to be reduced compared to the control one.

Covalent modification of human serum albumin by the natural sesquiterpene lactone parthenolide.

The reactivity of parthenolide (PRT), a natural sesquiterpene lactone from Tanacetum parthenium (Asteraceae), with human serum albumin (HSA) was studied by UHPLC/+ESI-QqTOF MS analysis after tryptic digestion of albumin samples after incubation with this compound. It was found that the single free cysteine residue, C34, of HSA (0.6 mM) reacted readily with PRT when incubated at approximately 13-fold excess of PRT (8 mM). Time-course studies with PRT and its 11β,13-dihydro derivative at equimolar ratios of the reactants revealed that PRT under the chosen conditions reacts preferably with C34 and does so exclusively via its α-methylene-γ-lactone moiety, while the epoxide structure is not involved in the reaction.

Preconcentration of diluted biochemical samples using microchannel with integrated nanoscale Nafion membrane.

A microfluidic preconcentration device comprising a microchannel and a surface-patterned nanoscale Nafion membrane is proposed. Given the application of an electric field across the chip, the nanopore within Nafion membrane becomes ion selective due to an overlapping of the electric double layer. The resulting difference in flux of the co- and counter-ions within the membrane nanopore prompts the formation of a concentration gradient and leads to a gradual accumulation of the co-ions at the micro-nano junction. It is shown experimentally that the rate of concentration and the preconcentration factor both increase with an increasing electrical field intensity. The preconcentration performance in a straight microchannel is compared with that in a convergent microchannel using fluorescein disodium salt dehydrate and Fluorescein isothiocyanate (FITC)-labeled bovine serum albumin samples. The results show that the reduced cross-sectional area of the convergent microchannel increases the preconcentration factor compared to that obtained in a straight microchannel and yields a significant reduction in the preconcentration time.