Elution Buffer Research Articles

Acidic Chitinase-Chitin Complex Is Dissociated in a Competitive Manner by Acetic Acid: Purification of Natural Enzyme for Supplementation Purposes.

Acidic chitinase (Chia) has been implicated in asthma, allergic inflammations, and food processing. We have purified Chia enzymes with striking acid stability and protease resistance from chicken and pig stomach tissues using a chitin column and 8 M urea (urea-Chia). Here, we report that acetic acid is a suitable agent for native Chia purification from the stomach tissues using a chitin column (acetic acid-Chia). Chia protein can be eluted from a chitin column using 0.1 M acetic acid (pH 2.8), but not by using Gly-HCl (pH 2.5) or sodium acetate (pH 4.0 or 5.5). The melting temperatures of Chia are not affected substantially in the elution buffers, as assessed by differential scanning fluorimetry. Interestingly, acetic acid appears to be more effective for Chia-chitin dissociation than do other organic acids with similar structures. We propose a novel concept of this dissociation based on competitive interaction between chitin and acetic acid rather than on acid denaturation. Acetic acid-Chia also showed similar chitinolytic activity to urea-Chia, indicating that Chia is extremely stable against acid, proteases, and denaturing agents. Both acetic acid- and urea-Chia seem to have good potential for supplementation or compensatory purposes in agriculture or even biomedicine.

Testing for Human Norovirus and Recovery of Process Control in Outbreak-Associated Produce Items

The development of rapid and sensitive detection methods for human noroviruses (HuNoV) in produce items is critical, especially with the recent rise in outbreaks associated with this food commodity. In this study, 50-g portions of various produce items linked to a norovirus outbreak (celery, cucumber, lettuce, grapes, and radish) were artificially inoculated with murine norovirus (MNV-1) and concentrated either by ultracentrifugation or polyethylene glycol (PEG) precipitation after elution with an alkaline Tris-glycine-beef extract buffer supplemented with pectinase. As a viral concentration step following virus elution and clarification, ultracentrifugation yielded a faster method (<8 h, including reverse transcription quantitative PCR), with MNV-1 recoveries similar to or better, than those obtained with PEG precipitation. The addition of polyvinylpyrrolidone to the elution buffer, to remove polyphenolic inhibitors, improved MNV-1 recoveries by over two- and fivefold for cucumber and grapes, respectively. However, despite MNV-1 recoveries ranging from 10 to 38% as calculated with 10-fold diluted RNA, contaminating HuNoV was not detected in any of the outbreak-associated samples tested. For store-bought produce samples, the limit of detection for artificially seeded HuNoV GII.4 was determined to be 103 copies per 50 g, with reproducible detection achieved in grapes, radish, and celery. The results support the use of ultracentrifugation as an alternative approach to PEG precipitation to concentrate norovirus from a variety of produce items.

Purification of influenza virus-like particles using sulfated cellulose membrane adsorbers.

ABSTRACTBACKGROUNDVaccines based on virus‐like particles (VLPs) are an alternative to inactivated viral vaccines that combine good safety profiles with strong immunogenicity. In order to be economically competitive, efficient manufacturing is required, in particular downstream processing, which often accounts for major production costs. This study describes the optimization and establishment of a chromatography capturing technique using sulfated cellulose membrane adsorbers (SCMA) for purification of influenza VLPs.RESULTSUsing a design of experiments approach, the critical factors for SCMA performance were described and optimized. For optimal conditions (membrane ligand density: 15.4 µmol cm−2, salt concentration of the loading buffer: 24 mmol L‐1 NaCl, and elution buffer: 920 mmol L‐1 NaCl, as well as the corresponding flow rates: 0.24 and 1.4 mL min−1), a yield of 80% in the product fraction was obtained. No loss of VLPs was detected in the flowthrough fraction. Removal of total protein and DNA impurities were higher than 89% and 80%, respectively.CONCLUSIONUse of SCMA represents a significant improvement compared with conventional ion exchanger membrane adsorbers. As the method proposed is easily scalable and reduces the number of steps required compared with conventional purification methods, SCMA could qualify as a generic platform for purification of VLP‐based influenza vaccines. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

BackgroundChromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored.ResultsWe compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents.ConclusionsThis study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

Phosphopeptide enrichment with cross-linked Os(II)(dmebpy)2 Cl-derivatized acrylamide and vinylimidazole copolymer.

Reversible phosphorylation of proteins catalyzed by kinases and phosphatases plays a key regulatory role in intracellular biological processes. Protein phosphorylation profiling is still a challenge due to its low stoichiometry, diversity of phosphorylated protein forms, and dynamic nature of phosphorylation states. Mass spectrometry (MS) has been widely used for the characterization of protein phosphorylation, due to its high sensitivity and MS/MS sequencing capability. However, the low abundance and ionization efficiency of phosphorylated peptides and interference from their non-phosphorylated counterparts and other peptides in the enzymatic digests of proteins complicate the localization of phosphorylation sites in liquid chromatography (LC)/MS analysis. So the enrichment of phosphopeptides from the digests is often required before LC/MS. Immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) are the two most commonly used enrichment techniques for phosphopeptides prior to MS analysis. Cross-linked Os(II)(4,4'-dimethyl-2,2'-bipyridine)2 Cl-derivatized acrylamide and vinylimidazole copolymer were applied for the enrichment of phosphopeptides. Under neutral loading buffer conditions phosphopeptides bind on the Os-polymer without nonspecific binding of acidic peptides. Differential binding of monophosphorylated and multiply phosphorylated peptides can be achieved under different concentrations of imidazole. Sequential elution of bound phosphopeptides can be obtained with elution buffers of different pH values below 3. The loading buffers with imidazole can be aqueous or 7/3 H2 O/ACN. Once phosphopeptides bind onto the Os-polymer, washing with water, 0.1% acetic acid (pH~3) or 1/1 H2 O/ACN 0.05% acetic acid (pH ~3) does not elute phosphopeptides. The Os-polymer does not show bias of binding and elution toward phosphopeptide standards with singly, doubly and triply phosphorylated sites. Cross-linked Os(II)(dimethylbipyridine)2 Cl-derivatized poly(acrylamide)-poly(vinylimidazole) copolymer is proven to be a new efficient IMAC resin for phosphopeptide enrichment and shows some unique properties for differential binding and sequential elution of phosphopeptides. It could become a better alternative to traditional IMAC and TiO2 for phosphopeptide enrichment.

Recovery Optimization and Survival of the Human Norovirus Surrogates Feline Calicivirus and Murine Norovirus on Carpet.

Carpets have been implicated in prolonged and reoccurring outbreaks of human noroviruses (HuNoV), the leading cause of acute gastroenteritis worldwide. Viral recovery from environmental surfaces, such as carpet, remains undeveloped. Our aim was to determine survival of HuNoV surrogates on an understudied environmental surface, carpet. First, we measured the zeta potential and absorption capacity of wool and nylon carpet fibers, we then developed a minispin column elution (MSC) method, and lastly we characterized the survival of HuNoV surrogates, feline calicivirus (FCV) and murine norovirus (MNV), over 60 days under 30 and 70% relative humidity (RH) on two types of carpet and one glass surface. Carpet surface charge was negative between relevant pH values (i.e., pH 7 to 9). In addition, wool could absorb approximately two times more liquid than nylon. The percent recovery efficiency obtained by the MSC method ranged from 4.34 to 20.89% and from 30.71 to 54.14% for FCV and MNV on carpet fibers, respectively, after desiccation. Overall, elution buffer type did not significantly affect recovery. Infectious FCV or MNV survived between <1 and 15 or between 3 and 15 days, respectively. However, MNV survived longer under some conditions and at significantly (P < 0.05) higher titers compared to FCV. Albeit, surrogates followed similar survival trends, i.e., both survived longest on wool then nylon and glass, while 30% RH provided a more hospitable environment compared to 70% RH. Reverse transcription-quantitative PCR signals for both surrogates were detectable for the entire study, but FCV genomic copies experienced significantly higher reductions (<3.80 log10 copies) on all surfaces compared to MNV (<1.10 log10 copies).IMPORTANCE Human noroviruses (HuNoV) are the leading cause of acute gastroenteritis worldwide. Classical symptoms of illness include vomiting and diarrhea which could lead to severe dehydration and death. HuNoV are transmitted by the fecal-oral or vomitus-oral route via person-to-person contact, food, water, and/or environmental surfaces. Published laboratory-controlled studies have documented the environmental stability of HuNoV on hard surfaces, but there is limited laboratory-based evidence available about survival on soft surfaces, e.g., carpet and upholstered furniture. Several epidemiological reports have suggested soft surfaces may be HuNoV fomites illustrating the importance of conducting a survival study. The three objectives of our research were to demonstrate techniques to characterize soft surfaces, develop a viral elution method for carpet, and characterize the survival of HuNoV surrogates on carpet. These results can be used to improve microbial risk assessments, the development of much-needed soft surface disinfectant, and standardizing protocols for future soft surface studies.

A Quantitative Point-of-Need Assay for the Assessment of Vitamin D3 Deficiency

Vitamin D is necessary for the healthy growth and development of bone and muscle. Vitamin D deficiency, which is present in 42% of the US population, is often undiagnosed as symptoms may not manifest for several years and long-term deficiency has been linked to osteoporosis, diabetes and cancer. Currently the majority of vitamin D testing is performed in large-scale commercial laboratories which have high operational costs and long times-to-result. Development of a low-cost point-of-need assay could be transformative to deficiency analysis in limited-resource settings. The best biomarker of vitamin D status, 25hydroxyvitamin D3 (25(OH)D3), however, is particularly challenging to measure in such a format due to complexities involved in sample preparation, including the need to separate the marker from its binding protein. Here we present a rapid diagnostic test for the accurate, quantitative assessment of 25(OH)D3 in finger-stick blood. The assay is accompanied by a smartphone-assisted portable imaging device that can autonomously perform the necessary image processing. To achieve accurate quantification of 25(OH)D3, we also demonstrate a novel elution buffer that separates 25(OH)D3 from its binding protein in situ, eliminating the need for sample preparation. In human trials, the accuracy of our platform is 90.5%.

Efficacy of Combination Treatment with Sodium Metasilicate and Sodium Hypochlorite for Inactivation of Norovirus on Fresh Vegetables.

In recent years, fresh vegetables have frequently been associated with the foodborne transmission of enteric viruses, such as human norovirus (NoV). Therefore, several studies have focused on developing methods to inactivate foodborne viruses for preventing outbreaks of foodborne illnesses. Sodium hypochlorite (NaOCl) is commonly used as a disinfectant, but results in undesirable effects on the appearance and taste of foods and can generate toxic byproducts when it exceeds the allowable concentration. Here, we evaluated the efficacy of a range of NaOCl concentrations (50-1000 ppm) for reducing the amounts of human NoV (NoV GII.4) on lettuce (Lactuca sativa), celery (Apium graveolens L.), and white cabbage (Brassica oleracea ssp. capitata). In addition, the combination treatment of NaOCl and sodium metasilicate (SMS, 0.1-0.5%) pentahydrate was evaluated for its ability to decrease the populations of NoV GII.4 in the three food samples. An immunomagnetic separation procedure combined with reverse transcription quantitative polymerase chain reaction was used for virus detection. For lettuce, celery, and cabbage, the NoV GII.4 recovery rates were 57.3% ± 6.5%, 52.5% ± 1.7%, and 60.3% ± 3.9%, respectively, using a glycine/NaCl elution buffer (0.25 M glycine/0.14 M NaCl, pH 9.5). The reductions of NoV GII.4 were 3.17, 3.06, and 3.27 log10 genomic copies/μL for lettuce, celery, and cabbage, respectively, at 1000 ppm NaOCl, while a reduction of ∼3 log10 genomic copies/μL was obtained when the samples were treated with a combination of 100 ppm NaOCl and 0.4% SMS pentahydrate. Taken together, these results demonstrated that combined treatment with NaOCl and SMS pentahydrate was an efficient strategy to reduce the concentration of NaOCl for control of NoV GII.4 contamination in fresh vegetables.

Extraction of microRNAs from biological matrices with titanium dioxide nanofibers.

MicroRNAs (miRNAs) are small RNAs that bind to mRNA targets and regulate their translation. A functional study of miRNAs and exploration of their utility as disease markers require miRNA extraction from biological samples, which contain large amounts of interfering compounds for downstream RNA identification and quantification. The most common extraction methods employ silica columns or the TRIzol reagent but give out low recovery for small RNAs probably due to their short strand lengths. Herein, we fabricated the titanium dioxide nanofibers using electrospinning to facilitate miRNA extraction and developed the optimal buffer conditions to improve miRNA recovery from biological matrices of cell lysate and serum. We found that our TiO2 fibers could obtain a recovery of 18.0±3.6% for miRNA fibers while carrying out the extraction in the more complex medium of cell lysate, much higher than the 0.02±0.0001% recovery from the commercial kit. The much improved extraction of miRNAs from our fibers could be originated from the strong coordination between TiO2 and RNA's phosphate backbone. In addition, the binding, washing, and elution buffers judiciously developed in the present study can achieve selective extraction of small RNA shorter than 500 nucleotides in length. Our results demonstrate that TiO2 nanofibers can work as a valuable tool for extraction of miRNAs from biological samples with high recovery. Graphical abstract Schematic for extraction of small RNAs using TiO2 nanofibers.

Plasmid DNA purification by zirconia magnetic nanocomposite

A zirconia magnetic nanocomposite was prepared by a co-precipitation method in one step. The synthesized nanocomposite was characterized by scanning electron microscopy, energy-dispersive X-ray and Fourier transform infrared spectroscopy. The nanocomposite was applied for the adsorption/desorption of tobacco plant DNA (as a model). Experimental parameters controlling adsorption efficiency, including pH of binding solution, extraction time, the amount of nanocomposite and ionic strength, were optimized. To obtain high desorption efficiency, the effects of pH, the ionic strength of elution buffer and desorption time were investigated. The nanocomposite provided the adsorption capacity of 53.5 mg g−1 for DNA. The adsorption and desorption efficiency of the sorbent was found to be greater than 98 and 81%, respectively. The zirconia magnetic nanocomposite was used for the purification of plasmid DNA (pDNA) from Escherichia coli cell culture. The yield and purity of pDNA obtained by the method were compared to those obtained by the phenol-chloroform solvent extraction and a commercial kit.

Development of a fast and efficient method for hepatitis A virus concentration from green onion

Hepatitis A virus (HAV) can cause serious liver disease and even death. HAV outbreaks are associated with the consumption of raw or minimally processed produce, making it a major public health concern. Infections have occurred despite the fact that effective HAV vaccine has been available. Development of a rapid and sensitive HAV detection method is necessary for an investigation of an HAV outbreak. Detection of HAV is complicated by the lack of a reliable culture method. In addition, due to the low infectious dose of HAV, these methods must be very sensitive. Current methods rely on efficient sample preparation and concentration steps followed by sensitive molecular detection techniques. Using green onions which was involved in most recent HAV outbreaks as a representative produce, a method of capturing virus particles was developed using carboxyl-derivatized magnetic beads in this study. Carboxyl beads, like antibody-coated beads or cationic beads, detect HAV at a level as low as 100 pfu/25g of green onions. RNA from virus concentrated in this manner can be released by heat-shock (98°C 5min) for molecular detection without sacrificing sensitivity. Bypassing the RNA extraction procedure saves time and removes multiple manipulation steps, which makes large scale HAV screening possible. In addition, the inclusion of beef extract and pectinase rather than NP40 in the elution buffer improved the HAV liberation from the food matrix over current methods by nearly 10 fold. The method proposed in this study provides a promising tool to improve food risk assessment and protect public health.

Ca2+-induced stabilization of the nucleoside 2′- deoxyribosyltransferase from Lactobacillus hilgardii ZJS01: Characteristics and application in nucleosides synthesis

A novel nucleoside 2′-deoxyribosyltransferase (LhNDT) from Lactobacillus hilgardii ZJS01 was cloned and expressed in Escherichia coli BL21 (DE3). The amino acid sequence of LhNDT displayed the highest identity of 26% to other reported NDTs. LhNDT was a monomer as revealed by gel filtration chromatography with or without addition of Ca2+ in elution buffer. This enzyme showed a high activity and stability over a broad pH range and was sensitive to temperature. With the addition of 15mM Ca2+, the thermostability of LhNDT was greatly promoted by 63 times and 46 times at 30°C and 35°C, respectively. With the use of 2′-deoxyuridine as sugar donor at an economic molar ratio of 1:1, 5-aza-2′-deoxycytidine (46%) and 5-fluoro-2′- deoxycytidine (58%) were efficiently synthesized from 5-azacytosine and 5-fluorocytosine, respectively, by LhNDT for 2h.

Acid/Salt/pH Gradient Improved Resolution and Sensitivity in Proteomics Study Using 2D SCX-RP LC-MS.

The usage of strong cation exchange (SCX) chromatography in proteomics is limited by its poor resolution and nonspecific hydrophobic interactions with peptides, which lead to peptide overlap across fractions and change of peptide retention, respectively. The application of high concentration of salt (up to 1000 mM) in SCX also restricted its use in online 2D SCX-RP LC. In the present research, we first exploited the chromatographic ability of online 2D SCX-RP LC by combination of acid, salt, and pH gradient, three relatively independent modes of eluting peptides from SCX column. 50% ACN was added to elution buffer for eliminating hydrophobic interactions between SCX matrix and peptides, and the concentration of volatile salt was reduced to 50 mM. Acid/salt/pH gradient showed superior resolution and sensitivity as well as uniform distribution across fractions, consequently leading to significant improvements in peptide and protein identification. 112 191 unique peptides and 7373 proteins were identified by acid/salt/pH fractionation, while 69 870 unique peptides and 4536 proteins were identified by salt elution, that is, 62.5 and 60.6% more proteins and unique peptides, respectively, identified by the former. Fraction overlap was also significantly minimized by acid/salt/pH approach. Furthermore, acid/salt/pH elution showed more identification for acidic peptides and hydrophilic peptides.

Immunoaffinity purification of bluetongue virus group specific antibody using recombinant protein adsorbed to polystyrene wells

Most of the common viral diseases can be diagnosed by serological assays and these assays mostly depend on the purity and quality of antibody used. Such specific antibodies can be generated by hybridoma technology. Alternatively, the virus-specific antibodies can be purified from polyclonal serum by immunoaffinity purification technique. Based on this immune affinity purification technique we have purified group-specific antibody against Bluetongue virus (BTV) using recombinant protein VP7 of BTV bound to polystyrene wells. Elution buffer consisting of 100 mM Glycine-HCl, pH 3.0 was found optimum for dissociation of the antibody from recombinant antigen and also to maintain the integrity of antigen. The reactivity of eluted antibody was tested in enzyme-linked immunosorbent assay (ELISA). The purified antibody will be useful in other serological assays like ELISA, fluorescent assay, and agar gel immunodiffusion (AGID) for Bluetongue disease diagnosis.

Immunoaffinity purification of bluetongue virus group specific antibody using recombinant protein adsorbed to polystyrene wells

Most of the common viral diseases can be diagnosed by serological assays and these assays mostly depend on the purity and quality of antibody used. Such specific antibodies can be generated by hybridoma technology. Alternatively, the virus-specific antibodies can be purified from polyclonal serum by immunoaffinity purification technique. Based on this immune affinity purification technique we have purified group-specific antibody against Bluetongue virus (BTV) using recombinant protein VP7 of BTV bound to polystyrene wells. Elution buffer consisting of 100 mM Glycine-HCl, pH 3.0 was found optimum for dissociation of the antibody from recombinant antigen and also to maintain the integrity of antigen. The reactivity of eluted antibody was tested in enzyme-linked immunosorbent assay (ELISA). The purified antibody will be useful in other serological assays like ELISA, fluorescent assay, and agar gel immunodiffusion (AGID) for Bluetongue disease diagnosis

Optimization of expression JTAT protein with emphasis on transformation efficiency and IPTG concentration.

One of small accessory genes between pol and env is tat gene encoding TAT protein. This research was aimed to optimize the expression of Jembrana TAT (JTAT) protein with preparing Escherichia coli (E. coli) in advance using adopted methods of M1 (MgCl2 + CaCl2) and M2 (CaCl2 + Glycerol). The best transformation efficiency resulting from a better transformation method was used to subsequent expression of JTAT protein. A synthetic tat gene encoding protein JTAT was previously cloned into pBT-hisC. Concentration of 200; 400; 600 µM IPTG was induced to a small volume culture (200 ml; OD600 = 4), incubated for 3 h. Pellets were harvested by centrifugation (4000 rpm; 4 °C; 15 min). Buffer B (10 mM Immidazole) was added into pellets, lysed by freeze-thaw followed by sonication. Supernatant was collected by centrifugation (10,000 rpm; 4 °C; 20 min) and purified using Ni-NTA Agarose resin, released by elution buffer (E) containing 400 mM Immidazole to collect purified protein twice (E1, E2). The protein was characterized by SDS-PAGE and Western Blot (WB), quantified (at λ595 nm) with BSA standard method in prior. The result showed that transformation efficiency was better in M2 (2.53 × 106) than M1 (3.10 × 105). The JTAT protein was expressed at a right size of 11.8 kDa. Concentration of 200 µM IPTG produced a significantly better protein yield (1.500 ± 0.089 mg/ml; P < 0.05) than 600 µM IPTG (0.896 ± 0.052 mg/ml) and not different to 400 µM IPTG (1.298 ± 0.080 mg/ml). This research indicated that transformation efficiency needs to be taken account in prior of optimization of the protein expression.

Preparation of an Acridinium Ester-Labeled Antibody and Its Application in GoldMag Nanoparticle-Based, Ultrasensitive Chemiluminescence Immunoassay for the Detection of Human Epididymis Protein 4

An ultrasensitive and rapid sandwich-type chemiluminescence immunoassay (CLIA) was developed for the clinical determination of human epididymis protein 4 (HE4) in human serum, using GoldMag nanoparticles as solid phase and acridinium ester (AE) as chemiluminescence system (GMP-CLIA). The process of AE labeling antibodies was systematically studied and evaluated. The effect of varies factors such as molar ratio of AE to antibodies, labeling time, and the components of elution buffer and trigger solution were optimized. Under the selected conditions, AE labeling experiments were successfully performed with the average labeling efficiency of 1.92 ± 0.08, and antibody utilization rate of 69.77 ± 1.19%. Antibody activity remained unchanged after labeling. The established GMP-CLIA method can detect HE4 in the range of 0.25–50 ng·mL−1 (10–2000 pM) with a detection limit of 0.084 ng·mL−1 (3.36 pM). The sensitivity has reached a high level, comparable with the current commercial detection kits. This proposed method has been successfully applied to the clinical determination of HE4 in 65 human sera. The results showed a good correlation with a clinical method, microplate-based chemiluminescence enzyme immunoassay (CLEIA), with the correlation coefficient of 0.9594.

Low concentration DNA extraction and recovery using a silica solid phase.

DNA extraction from clinical samples is commonly achieved with a silica solid phase extraction column in the presence of a chaotrope. Versions of these protocols have been adapted for point of care (POC) diagnostic devices in miniaturized platforms, but commercial kits require a high amount of input DNA. Thus, when the input clinical sample contains less than 1 μg of total DNA, the target-specific DNA recovery from most of these protocols is low without supplementing the sample with exogenous carrier DNA. In fact, many clinical samples used in the development of POC diagnostics often exhibit target DNA concentrations as low as 3 ng/mL. With the broader goal of improving the yield and efficiency of nucleic acid-based POC devices for dilute samples, we investigated both DNA adsorption and recovery from silica particles by using 1 pg– 1 μg of DNA with a set of adsorption and elution buffers ranging in pH and chaotropic presence. In terms of adsorption, we found that low pH and the presence of chaotropic guanidinium thiocyanate (GuSCN) enhanced DNA-silica adsorption. When eluting with a standard low-salt, high-pH buffer, > 70% of DNA was unrecoverable, except when DNA was initially adsorbed with 5 M GuSCN at pH 5.2. Unrecovered DNA was either not initially adsorbed or irreversibly bound on the silica surface. Recovery was improved when eluting with 95°C formamide and 1 M NaOH, which suggested that DNA-silica-chaotrope interactions are dominated by hydrophobic interactions and hydrogen bonding. While heated formamide and NaOH are non-ideal elution buffers for practical POC devices, the salient results are important for engineering a set of optimized reagents that could maximize nucleic acid recovery from a microfluidic DNA-silica-chaotrope system.

Determination of copper total and speciation in food samples by flame atomic absorption spectrometry in association with solid-phase extraction with bamboo (Bambusa vulgaris) fiber loaded with bathocuproine

A bamboo fiber mini-column impregnated with bathocuproine is proposed as an off-line pre-concentration system for determination of copper speciation in food samples using flame atomic absorption spectrometry (FAAS). Sampling flow rate, elution flow rate, pH and buffer concentration were evaluated for their importance in the solid-phase extraction using a 24 full factorial design and were all found to be significant. Afterward, a Doehlert matrix was used to optimize the pre-concentration process. The experimental conditions established with this design were: 8.0mLmin−1 sampling flow rate, 10.0mLmin−1 elution flow rate and 0.02molL−1 buffer at pH5.5. This system allowed the determination of copper with a limit of detection (LOD) of 0.5μgL−1, a limit of quantification (LOQ) of 1.66μgL−1 and a precision expressed as relative standard deviation (RSD) of 2.70 and 2.79% for copper concentrations of 5.0 and 20μgL−1, respectively. A pre-concentration factor of 33 was achieved using a 50.0-mL sample volume. The accuracy was confirmed by analysis of a certified reference material (NIST SRM 1573a Tomato Leaves). Spike/recovery tests were performed with results ranging from 93 to 100%. The developed method was applied to determine copper in corn flour, cassava root flour, oregano leaves and cumin. In addition, a speciation strategy was used for wine samples, in which free copper was evaluated in 40.00-mL aliquots of undigested samples and total copper assessed in samples digested before the pre-concentration step.

Preparation of linear polyacrylamide coating and strong cationic exchange hybrid monolith in a single capillary, and its application as an automated platform for bottom-up proteomics by capillary electrophoresis-mass spectrometry

A linear polyacrylamide (LPA) coating and a sulfonate-silica hybrid strong cation exchange (SCX) monolith were prepared sequentially in a single fused silica capillary. The SCX-LPA capillary was used for sample preconcentration using solid-phase extraction (SPE) followed by capillary zone electrophoresis (CZE) separation and mass spectrometry detection. Samples were prepared in 1 M acetic acid, loaded by pressure onto the SCX SPE monolith, and eluted using 200 mM ammonium bicarbonate (pH 8). The background electrolyte for CZE was 1 M acetic acid. This combination of elution buffer and background electrolyte results in formation of a dynamic pH junction, which further improves the preconcentration performance. All experiments were performed using a CZE autosampler and an electrokinetically pumped nanospray interface. By loading 50 ng of Xenopus laevis eggs protein digest, 330 protein groups and 872 peptides were identified. The system was also applied to the analysis of 11 μL of a 10−4 mg⋅mL−1 (1.1 ng) bovine serum albumin tryptic digest; 12 peptides were identified and the protein coverage was 25%. Finally, by loading 5.5 μL of a 10−3 mg⋅mL−1 (5.5 ng) E. coli digest, 145 protein groups and 365 peptides were identified.