Influence of pH on formation and stability of phosphatidylcholine/phosphatidylserine coatings in fused-silica capillaries

The effect of pH on the formation and stability of phospholipid coatings in fused-silica capillaries in electrophoresis was investigated. A liposome solution consisting of 3 mM of 80:20 mol% phosphatidylcholine/phosphatidylserine (PC/PS) in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer was used as coating material. The coating was prepared by a method described earlier and five steroids were used as neutral model analytes. First, the effect of pH of the coating solution on the formation and stability of phospholipid coatings was studied at pH 6.5-8.5. The pH of the background electrolyte (BGE) solution (HEPES) was either kept constant at pH 7.4 or made similar to the pH of the liposome coating solution. Results showed that attachment of the coating on the fused-silica wall mostly depends on the protonation of amines of the phospholipids and HEPES. The ability of the phospholipid coating to withstand changes in pH was then investigated by coating at pH 7.5 and separating steroids with acetic acid, 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), HEPES, or glycine BGE, adjusted to pH between 4.5 and 10.8. The results showed that with use of BGE solution at pH 10.8, the separation of steroids was not successful and the electroosmotic flow was high because of leakage of the phospholipid coating during preconditioning of the capillary with BGE solution. There was no phospholipid leakage with a BGE solution of pH 4.5, indicating that the protonated form of the functional groups of PS and HEPES participating in the attachment of the phospholipid coating to the capillary play an essential role in the success of the coating.     

Anionic phospholipid coatings in capillary electrochromatography. Binding of Ca2+ to phospholipid phosphate group

Anionic phospholipids phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS) were examined for their effect on 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine (POPC)-containing liposomes used as coating material in capillary electrochromatography. Liposome solvent was N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer at pH 7.4 with and without 3 mM of CaCl2. The background electrolyte solution was HEPES buffer at pH 7.4. The net charge, size, and short-term stability of the liposomes were measured with a Zetasizer. Results showed that calcium interacts with all liposomes but most strongly with POPC/PA. The relative migration times, retention factors, and resolution of the model analytes (one cationic, three uncharged ions, and one anionic) were studied. All liposomes successfully coated the silica capillary. Without calcium the strongest interaction and best separation of the analytes were with the POPC/PI and POPC/PS coatings, while interactions with the POPC/PA coating were weak. Calcium enhanced the interactions of the model analytes with all coatings, and the interactions were then strongest with the POPC/PA coating. In the presence of calcium there appears to be a slight reorganization of the coating with increasing number of runs. Our results indicate strong interactions between calcium and the phosphate groups in phospholipids and demonstrate the significant role of the phospholipid polar head group in phospholipid coatings on silica surfaces.     

Preparation of Fritless Packed Silica Columns for Capillary Electrochromatography

Fritless packed silica gel columns were prepared using sol‐gel technology. A part of a 75 μm i.d. fused silica capillary was filled with a mixture of tetramethoxysilane and poly(ethylene glycol). After gelling at 40°C and heating at 300°C, the resultant silica gel was derivatized with dimethyloctadecylchlorosilane. A scanning electron micrograph of a cross‐section of the capillary column showed that the gel took the form of a spherical particle aggregate and adhered to the column inner wall. The column performance was evaluated for electrochromatography using acetonitrile–50 mM HEPES buffer (pH 6.6) (60/40 or 40/60, v/v) as the mobile phase. An electroosmotic flow of 1.0 mm/s was generated with (60/40, v/v) acetonitrile/HEPES buffer at a field strength of 546 V/cm. Using a sol‐gel‐derived packed column at an electroosmotic flow of 0.5 mm/s, efficiencies of up to 1.1×10⁵ plates/m were obtained for retained solutes.     

Molecular interactions between glycopeptide vancomycin and bacterial cell wall peptide analogues

The molecular interactions of the glycopeptide antibiotic vancomycin (Van) with bacterial cell wall analogues N,N'-diacetyl-L-Lys-D-Ala-D-Ala (Ac(2) KdAdA) and N,N'-diacetyl-L-Lys-D-Ala-D-Lac (Ac(2) KdAdL) were investigated in neat water, phosphate buffer and HEPES buffer by using fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. The FCS determined dissociation constants (k(d)) show that the intrinsic binding affinity between Van and the drug-sensitive peptide ligand Ac(2)KdAdA remains invariant when the solvent is changed from neat water to either PBS or HEPES buffer; this demonstrates that there are no obvious solvent effects on the association between Van and Ac(2)KdAdA due to the strong intermolecular interaction between the two moieties. When compared to Ac(2)KdAdA, a significantly larger k(d) value was observed for the binding between the drug-resistant peptide ligand Ac(2)KdAdL and Van. Furthermore, the k(d) increased by about 8- to 11-times when the solvent was changed from neat water to 10 mM phosphate/HEPES buffer. The stability of the Ac(2)KdAdL-Van complex was dependent on the concentration of the buffer and k(d) increases as the concentration of either phosphate ions or HEPES increased until an equilibrium was attained. Both FCS and MD simulation studies clearly showed that the components constituting the buffer solution (e.g., phosphate ions and HEPES) are involved in molecular interactions with the binding pocket of Van and they profoundly affect the intrinsic stability of the complex formed between the low-affinity Ac(2)KdAdL and Van. These results could help us to better understand the detailed structure and activity of glycopeptide antibiotic derivatives toward bacterial cell wall peptide analogues, and can further facilitate the development of new drug candidates against drug-resistant bacterial strains.     

Detection of duck hepatitis B virus DNA fragments using on-column intercalating dye labeling with capillary electrophoresis-laser-induced fluorescence.

A rapid on-column DNA labeling technique is used to detect viral restriction DNA fragments by capillary electrophoresis-laser induced fluorescence detection. Intercalating dyes such as POPO3 or ethidium homodimer-2 are incorporated into the detection buffer. The cationic dyes migrate into the capillary during electrophoresis and bind to the oppositely migrating DNA fragments. A post-column sheath-flow fluorescence detector is used in the experiment. Excellent labeling efficiency is achieved at minimal background fluorescence by diluting the dyes to between 1 x 10(-7) M and 5 x 10(-7) M in a buffer with low ionic strength relative to the running buffer within the capillary. This dilute sheath-flow buffer allows stacking of dye molecules inside the capillary when an electric field is applied. Calibration curves using a series of DNA size markers (between 72 and 1353 base pairs) were linear over an order of magnitude in DNA concentration. Sensitivity also increased linearly with fragment length, and detection limits ranged from 4 x 10(-14) M to 5 x 10(-13) M for the size-standards. Analysis of cloned viral DNA using duck hepatitis B virus demonstrated a concentration detection limit of 3.9 x 10(-16) M. Last, the technique produced very high separation efficiency, 14 x 10(6) theoretical plates which is greater than 47 x 10(6) plates m-1, for the duck hepatitis B viral genome.     

The interference of HEPES buffer during amperometric detection of ATP in clinical applications

HEPES-based biological buffer is subject to photooxidation upon exposure to fluorescent illumination. Thereby hydrogen peroxide is generated, which interferes with amperometric oxidoreductase-based biosensors for glucose or adenosine triphosphate (ATP). These biosensors operate at an oxidation potential above 500 mV vs. the standard calomel electrode (SCE) and involve hydrogen peroxide as the electroactive molecule detected at the electrode surface. False-positive detection of ATP was observed in HEPES buffer utilizing an amperometric microbiosensor based on the co-immobilization of glucose oxidase and hexokinase for detection of ATP in biological specimens. Electrochemical, mass spectrometric, ³¹P NMR, and ¹H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca²⁺ in HEPES buffer decreases the photooxidation of HEPES. Consequently, the hydrogen peroxide background concentration is reduced, thereby leading to erroneous ATP detection at the dual-enzyme microbiosensor, which determines an increase in ATP via a reduced hydrogen peroxide signal.     

Determination of HEPES in <Superscript>68</Superscript>Ga-labeled peptide solutions

A practical and reliable HPLC method was used for the determination of 2-[4-N-(2-hydroxyethyl)-1-piperazinyl]-N′-ethanesulfonic acid (HEPES) content in the ⁶⁸Ga-labeled [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-1-Nal3-octreotide (DOTANOC). Linearity of this method was observed in a concentration range of 0.01–10 mg mL⁻¹ and the quantitative limit (signal to noise = 11) was determined as 10 μg mL⁻¹. The HEPES concentration in the final products of ⁶⁸Ga-DOTANOC was typically lower than the detection limit. Pure water and HEPES buffer as reaction medium were investigated using various activities of gallium-68. It was demonstrated that the presence of HEPES buffer consistently furnished very high radiochemical purity of ⁶⁸Ga-DOTANOC, which remained stable for several hours post-labeling. Evidence is provided that in addition to its role as a buffer, HEPES also functions as a radioprotectant agent.     

Effects of electrolyte modification and capillary coating on separation of glycoprotein isoforms by capillary electrophoresis

The capillary electrophoresis (CE) running electrolyte composition was optimized for the separation of selected glycoproteins. A good separation of the ovalbumin (OV) and alpha-acid glycoprotein (AAG) isoforms was achieved in 20 mmol x L(-1) N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) at pH 7.0, in 20 mmol x L(-1) phosphate, pH 7.0, or in 25 mmol x L(-1) borate, pH 7.9. Various ways of suppression of the glycoprotein adsorption onto the capillary wall were compared. Alpha, omega-diamine alkanes and bis(aminoalkyl) amines were added to the CE buffers, the optimized concentration being 1 mmol x L(-1) in 20 mmol x L(-1) phosphate buffer. The OV and AAG isoforms could be separated using all the alpha,omega-diamine alkanes or bis(2-aminoethyl)amine. The length of the alkyl chain in the diaminoalkane did not influence the separation. The separation of the isoforms of pollen allergens was also tested. The effects of modification of the capillary wall by succinyl-poly-L-lysine and hydrophilic CElect-P1 capillary were compared. A decrease in the glycoprotein and protein adsorption resulted in an improved separation of the isoforms.     

Hydrothermal Synthesis of Platinum‐Group‐Metal Nanoparticles by Using HEPES as a Reductant and Stabilizer

Platinum‐group‐metal (Ru, Os, Rh, Ir, Pd and Pt) nanoparticles are synthesized in an aqueous buffer solution of 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) (200 mM , pH 7.4) under hydrothermal conditions (180 °C). Monodispersed (monodispersity: 11–15 %) metal nanoparticles were obtained with an average particle size of less than 5 nm (Ru: 1.8±0.2, Os: 1.6±0.2, Rh: 4.5±0.5, Ir: 2.0±0.3, Pd: 3.8±0.4, Pt: 1.9±0.2 nm). The size, monodispersity, and stability of the as‐obtained metal nanoparticles were affected by the HEPES concentration, pH of the HEPES buffer solution, and reaction temperature. HEPES with two tertiary amines (piperazine groups) and terminal hydroxyl groups can act as a reductant and stabilizer. The HEPES molecules can bind to the surface of metal nanoparticles to prevent metal nanoparticles from aggregation. These platinum‐group‐metal nanoparticles could be deposited onto the surface of graphite, which catalyzed the aerobic oxidation of alcohols to aldehydes.     

Determination of the kinetic parameters of rhodanese by electrophoretically mediated microanalysis in a partially filled capillary

Electrophoretically mediated microanalysis (EMMA) was applied for the study of kinetic parameters of the bisubstrate enzymatic reaction of rhodanese. The Michaelis constants (K(m)) for both substrates and the effect of temperature on rhodanese reaction were evaluated by means of the combination of the EMMA methodology with a partial filling technique. In this setup, the part of the capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. The enzymatic reaction was performed in 25 mM N-(2-hydroxymethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer (pH 8.5) while the low pH background electrolyte 100 mM beta-alanine-HCl (pH 3.5) was used for separation of substrates and products that are the inorganic anions. The estimated value of K(m) for thiosulfate of 1.30 x 10(-2) M was consistent with previously published values; the K(m) for cyanide of 7.6 x 10(-3) M was determined for the first time. In addition, the type of kinetic mechanism of enzymatic reaction was also elucidated. The finding of the double displacement (ping-pong) mechanism is in accordance with previous literature data. Also, the experimentally determined temperature optimum of the rhodanese-catalyzed reaction around 20-25 degrees C agreed with literature values.     

Small diamines as modifiers for phosphatidylcholine/phosphatidylserine coatings in capillary electrochromatography

Greater stability of liposome coatings and improved resolution of model steroids in capillary electrochromatography (CEC) were sought by adding small diamines (ethylenediamine, diaminopropane, bis-tris-propane, or N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid, HEPES)) to the liposome solution before coating of fused silica capillaries. The phospholipid coatings consisted of 1 mM of 8:2 mol% phosphatidylcholine (PC)/phosphatidylserine (PS) and 5 mM of modifier in buffer solutions (acetate, phosphate, or Tris) at pH 4.0-7.4. The coating was based on a published procedure, and five steroids were used as neutral model analytes in evaluation of the coating. The results showed that under optimal conditions, the small linear diamines increased the packing density of anionic phospholipids, leading to improved separations. In addition, the choice of buffer for the liposome coating and separation appeared to influence the performance of the coatings. While buffers with amino groups take part in the phospholipid bilayer formation, buffers like phosphate may even have negative effect on coating formation. The factors affecting phospholipid coatings with diamines as modifiers are clarified.     

Open-tubular capillary electrochromatography of bovine beta-lactoglobulin variants A and B using an aptamer stationary phase.

DNA aptamers that form a G-quartet conformation were covalently attached to a capillary surface for open-tubular capillary electrochromatographic separation of bovine beta-lactoglobulin variants A and B, which vary by 2 of their 162 amino acid residues. Separation was achieved using a 4-plane, G-quartet aptamer stationary phase with tris(hydroxymethyl)aminomethane (Tris) or phosphate buffer as the mobile phase. In control experiments, separation did not occur using either an oligonucleotide of similar base composition but which does not form a G-quartet structure, or using capillary zone electrophoresis on a bare capillary under similar experimental conditions. Separation was achieved using a capillary coated only with the covalent linker molecule. In phosphate buffer, the separations were similar for aptamer-coated and linker-only stationary phases, while in Tris buffer, retention times were almost doubled for the linker-only capillary. When Tris buffer is the mobile phase, there appears to be weaker interactions between the proteins and the stationary phase that may result in a gentler, less denaturing separation than is commonly achieved using hydrocarbon-based stationary phases.     

Capillary electrophoretic discrimination of single nucleotide polymorphisms using an oligodeoxyribonucleotidepolyacrylamide conjugate as a pseudo-immobilized affinity ligand: optimum ligand length predicted by the melting temperature values.

We developed a weak-affinity separation system for single-nucleotide polymorphisms (SNPs) based on capillary electrophoresis. In this approach, single-stranded DNA (ssDNA)-polyacrylamide (polyAAm) conjugate was used as a pseudo-immobilized affinity ligand to separate the target DNA, cytochrome P450 2C9 (CYP2C9), and its point mutant. The ligand DNA was designed to be complementary to the normal DNA, and the target DNA was electrophoretically separated by the difference in the affinity with the pseudo-immobilized ligand in the capillary. We showed that the separation efficiency was closely associated with the Tm value of double-stranded DNA (dsDNA) consisting of the target and ligand DNA, which depends on the measurement conditions, such as the base number of the ligand DNA and the concentration of Mg2+ in the buffer solution.     

A naphthalene-based Al3+ selective fluorescent sensor for living cell imaging

An efficient fluorescent Al(3+) receptor, N-(2-hydroxy-1-naphthalene)-N'-(2-(2-hydroxy-1-naphthalene)amino-ethyl)-ethane-1,2-diamine (L) has been synthesized by the condensation reaction between 2-hydroxy naphthaldehyde and diethylenetriamine. High selectivity and affinity of L towards Al(3+) in ethanol (EtOH) as well as in HEPES buffer at pH 7.4, makes it suitable to detect intracellular Al(3+) with fluorescence microscopy. Metal ions, viz. Li(+), Na(+), K(+), Mg(2+), Ca(2+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Ag(+), Cd(2+), Hg(2+) and Pb(2+) do not interfere. The lowest detection limit for Al(3+) is 3.0 × 10(-7) M and 1.0 × 10(-7) M in EtOH and HEPES buffer respectively.     

Capillary electrophoretic assay of human acetyl‐coenzyme A carboxylase 2

Human acetyl‐coenzyme A carboxylase 2 catalyzes the carboxylation of acetyl coenzyme A to form malonyl coenzyme A, along with the conversion of magnesium‐adenosine triphosphate complex to magnesium‐adenosine diphosphate complex. A simple off‐column capillary electrophoresis assay for human acetyl‐coenzyme A carboxylase 2 was developed based on the separation of magnesium‐adenosine triphosphate complex, magnesium‐adenosine diphosphate complex, acetyl coenzyme A and malonyl coenzyme A with detection by ultraviolet absorption at 256 nm. When Mg²⁺ was absent from the separation buffer, the zones due to magnesium‐adenosine triphosphate complex and magnesium‐adenosine diphosphate complex both split and migrated as two separate peaks. With Mg²⁺ added to the separation buffer, magnesium‐adenosine triphosphate complex and magnesium‐adenosine diphosphate complex produced single peaks, and the reproducibility of peak shape and area improved for human acetyl‐coenzyme A carboxylase 2 assay components. The final separation buffer used was 30.0 mM HEPES, 3.0 mM MgCl₂, 2.5 mM KHCO₃, and 2.5 mM potassium citrate at pH 7.50. The same buffer was used for the enzyme‐catalyzed reaction (off‐column). Inhibition of human acetyl‐coenzyme A carboxylase 2 by CP‐640186, a known inhibitor, was detected using the capillary electrophoresis assay.     

Open-tubular electrochromatography of organic phosphates on a sapphyrin-modified capillary.

Sapphyrin coating of the inner wall of the capillary results in a distinct interaction of the phosphate residue-possessing compounds as proven by a seven-membered model mixture of nucleoside mono- and diphosphates and ATP. Modification of the inner surface of the capillary not only alters the endoosmotic flow (as would have been expected) but brings about an electrochromatographic effect based on the interaction of tested phosphate moiety-bearing solutes with the immobilized sapphyrin layer. Elution of the sample can be achieved by using either 25 mM borate-acetate buffer in which monophosphates are not only separated from each other, but also selectively separated from di- and triphosphates (ATP). With the other two buffer systems tested, i.e. borate-phosphate and Tris-HCl, better selectivity (though smaller interaction with the capillary coating) was observed. The coating is relatively stable (can be used for 20 subsequent runs at least), simple to materialize, and in spite of a strong UV absorbancy of sapphyrin at the wavelength used (254 nm), decreases the limit of detection by no more than one order of magnitude as compared to the untreated capillary. Resolution factors (calculated to the preceding peak) are in most cases better in the electrochromatographic separation mode as compared to the separation in the untreated capillary, which reflects both the decrease in the electroosmotic flow and the interaction with the capillary wall coating.     

pH effect on dynamic coating for capillary electrophoresis of DNA

A buffer consisting of tris(hydroxymethyl)aminomethane, 2-(N-moropholino)ethanesulfonic acid (Mes) and EDTA with constant ion strength was used to investigate the effect of buffer pH on the dynamic coating behavior of poly(N-isopropylacrylamide) (PNIPAM) for DNA separation. The atomic force microscopy (AFM) image illustrated that PNIPAM in lower-pH buffer was much more efficient in covering a silica wafer than that in higher-pH buffer. The coating performance of PNIPAM was also quantitatively analyzed by Fourier transform IR attenuated total reflectance spectroscopy and by measuring the electroosmotic flow (EOF). These results indicated that the stability of the dynamic coating was dependent on the pH of the sieving matrix and was improved by reducing the pH to the weak-acid range. The lower pH of the sieving buffer may induce the polymer more efficiently to adsorb on the capillary wall to suppress EOF and DNA–capillary wall interaction for DNA separation. The enhanced dynamic coating capacity of PNIPAM in lower-pH buffer may be attributed to the hydrogen bonds between the hydroxyl groups of the silica surface and the oxygen atom of the carbonyl groups of PNIPAM.     

Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix

The detection of point and other simple mutations in DNA is important for cancer research and diagnosis and other biological studies. Capillary electrophoresis has been successfully used for separating DNA fragments. However, a low-viscosity polymer sieving buffer for DNA separation with on-line coating has never been reported. In this paper, a new method using capillary electrophoresis with on-line coating and laser-induced fluorescence detection (CE-LIF) for screening for point or simple DNA mutations has been demonstrated. The method uses an on-line dynamic coating technique that increases capillary lifetime and analysis reproducibility, and employs a low-viscosity polymer solution, which allows the user to rinse the capillary rapidly and refill with polymer solution easily. Experiments proved that the additives in the separation buffer for on-line capillary coating do not affect the separation efficiency of the running buffer, and do not interfere with the formation of hydrogen-bonded network between boric acid, mannitol and hydroxypropylmethylcellulose polymers. The stability of the dynamically coated capillary was quantitatively studied; the capillary lifetime was increased 6- to 7-fold compared with that of permanently coated CE columns. Standard DNA fragments containing mutations, with sizes of 209, 219, and 338 bps, were successfully separated and detected with this system, after the mutated DNA fragments were cleaved by CEL-I endonuclease. The technique is very sensitive for the size-separation of low-range, middle-range, and high-range DNA fragments. Results were compared with the HPLC methods developed by Transgenomic, Inc. and were in good agreement. The method should be applicable to mutation detection for all relevant biological and clinical studies. The factors influencing separations and the stability of dynamic capillary coatings are also discussed in the paper.     

Colorimetric detection of carbenicillin using cationic polythiophene derivatives

A water-soluble, polythiophene-based colorimetric sensor was designed for selective and sensitive detection of carbenicillin in HEPES buffer solution. Quaternized quinine was linked to thiophene through bis-functionality benzyl group, which can interact with carbenicillin via electrostatic interaction and geometric match effect. The sensor exhibited a colorimetric signal change upon the addition of carbenicillin because of the formation of more nonplanar structures. However, the addition of other beta-lactam antibiotics or dicarboxylic acids into the sensor solution caused no obvious changes in absorbance intensity ratio. This result may be attributed to the cavity formed by the semi-rigid framework of PTQ2, which is suitable for the special binding with carbenicillin. This novel sensor can effectively distinguish carbenicillin from other beta-lactam antibiotics and has a wide linear range response in HEPES buffer solution. Linear calibration curves are obtained with 0 to 18 μmol/L of HEPES buffer solution, with a limit of detection of 0.54 μmol/L.     

Applying the Yb3+ ion as a simple and sensitive probe to detect phosphate-containing derivatives in aqueous solution

A simple and sensitive detection method for HPO(4)(2-) and phosphate-containing derivatives in aqueous solution with a new ensemble which is prepared by mixing ytterbium chloride and pyrocatechol violet in a 2:1 molar ratio in 10 mM HEPES buffer at pH 7.0.