Dimethylditetradecylammonium bromide (2C14DAB) as a self-assembled surfactant coating for detection of protein–dye complexes by CE-LIF

A self-assembled column coating for capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF) has been evaluated for the separation and quantitation of protein–dye complexes. This semi-permanent coating, composed of dimethylditetradecyl-ammonium bromide (2C₁₄DAB), is inexpensive and easily assembled onto the column and it allows for better peak resolution and greater control over electroosmotic flow. The versatility of long-chained surfactant coatings was determined particularly with respect to their use with fluorescent probes, different pH buffers, and different proteins. Studies were performed to determine the stability of the coating under various pH and buffer conditions. Red-1c, a red luminescent squarylium dye, was used for on-column protein labeling concurrently with the surfactant coating and LIF detection. Protein–Red-1c complexes were excited with a 650-nm diode laser and their emission detected by a photomultiplier tube with a 664-nm filter. A comparison of pre-column labeling and on-column labeling of a two-model protein system (human serum albumin and β-lactoglobulin A) revealed higher efficiencies and greater sensitivities for both proteins using on-column labeling and coated columns. A linear relationship between peak height and protein concentration was obtained by CE-LIF for this on-column labeling method with 2C₁₄DAB-coated columns and the Red-1c probe.     

Protein determination by microchip capillary electrophoresis using an asymmetric squarylium dye: noncovalent labeling and nonequilibrium measurement of association constants

In response to a growing interest in the use of smaller, faster microchip (mu-chip) methods for the separation of proteins, advancements are proposed that employ the asymmetric squarylium dye Red-1c as a noncovalent label in mu-chip CE separations. This work compares on-column and precolumn labeling methods for the proteins BSA, beta-lactoglobulin B (beta-LB), and alpha-lactalbumin (alpha-LA). Nonequilibrium CE of equilibrium mixtures (NECEEM) represents an efficient method to determine equilibrium parameters associated with the formation of intermolecular complexes, such as those formed between the dye and proteins in this work, and it allows for the use of weak affinity probes in protein quantitation. In particular, nonequilibrium methods employing both mu-chip and conventional CE systems were implemented to determine association constants governing the formation of noncovalent complexes of the red luminescent squarylium dye Red-1c with BSA and beta-LB. By our mu-chip NECEEM method, the association constants K(assoc) for beta-LB and BSA complexes with Red-1c were found to be 3.53 x 10(3) and 1.65 x 10(5) M(-1), respectively, whereas association constants found by our conventional CE-LIF NECEEM method for these same protein-dye systems were some ten times higher. Despite discrepancies between the two methods, both confirmed the preferential interaction of Red-1c with BSA. In addition, the effect of protein concentration on measured association constant was assessed by conventional CE methods. Although a small decrease in K(assoc) was observed with the increase in protein concentration, our studies indicate that absolute protein concentration may affect the equilibrium determination less than the relative concentration of protein-to-dye.     

Indocyanine green as a noncovalent, pseudofluorogenic label for protein determination by capillary electrophoresis

Indocyanine green (ICG)--a negatively charged, polymethine dye--can interact noncovalently with proteins to form fluorescent complexes, with excitation and emission maxima near 780 and 820 nm, respectively. This behavior was realized utilizing either a 100 mM phosphate buffer or a 25 mM citric acid buffer, both at pH 3.1. The behavior of ICG under these conditions, termed pseudofluorogenic, rendered the dye suitable for use as a label for protein determination in capillary electrophoresis with diode laser-induced fluorescence detection (CE-LIF). To this end, pseudofluorogenic ICG was used both as an on-column label for human serum albumin (HSA) and as a precolumn label for a model mixture of proteins, including ribonuclease A, transferrin, and cytochrome c. These ICG-labeled proteins were successfully resolved in less than 11 min, with no interference from excess, unbound dye.     

Fluorimetric studies and noncovalent labeling of protein with the near-infrared dye HITCI for analysis by CE-LIF

1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITCI) is a commercially available, positively charged, indocarbocyanine dye used typically as a laser dye in the near infrared (NIR). The absorbance and fluorescence properties of HITCI in a variety of solvent systems were determined. Results indicate that the fluorescence of HITCI is not significantly affected by the pH. Titration of HITCI with human serum albumin (HSA) and trypsinogen was carried out to investigate the interactions between this dye and proteins. These studies revealed that the absorbance and fluorescence properties of the dye change upon binding to protein in a wide range of solution pH's. The potential use of HITCI as a noncovalent protein labeling probe, therefore, was explored. Determination and separation of HITCI and HITCI-protein complexes was performed by capillary electrophoresis with diode-laser induced fluorescence detection (CE-LIF). Both pre-column and on-column noncovalent labeling methods are demonstrated.     

Analysis of Bacillus globigii spores by CE

It is imperative in today's world that harmful airborne or solution-based microbes can be detected quickly and efficiently. Bacillus globigii (Bg) spores are used as a simulant for Bacillus anthracis (Ba) due to their similar shape, size, and cellular makeup. The utility of CE to separate and detect low levels of Bg spore concentrations will be evaluated. To differentiate spores from background particulates, several dyes, including fluorescamine, C-10, NN-127, Red-1c, and indocyanine green (ICG), were utilized as noncovalent labels for proteins on the Bg spore surface, as well as for HSA and homoserine standards. On-column labeling, with dye present in the running buffer, was utilized to obtain greater sensitivity and better separation. CE with LIF detection enables interactions between the dye and spore surface proteins to be observed, with enhanced fluorescence occurring upon binding of the dye to surface protein. Resulting electropherograms showed unique fingerprints for each dye with Bg spores. Migration times were under 10 min for all dye-spore complexes, with net mobilities ranging from 3.5x10(-4) to 6.9x10(-4) cm(2) V(-1) s(-1), and calibration curves yielded correlation coefficients of 0.98 or better for four of the dyes studied.     

Determination of protein-dye association by near infrared fluorescence-detected circular dichroism

Near-infrared (NIR) squarylium dye spectral properties were evaluated by absorption, fluorescence, circular dichroism (CD), and fluorescence-detected circular dichroism (FDCD). Substituents of the two NN dyes differed at R₁ and R₂, located symmetrically on the chromophore. The side chains of NN525 are R₁=hexanoic acid, R₂=butyl sulfonate and R₁=R₂=ethyl for NN127. FDCD signals were first confirmed by denaturing BSA with 2–8 M urea showing a diminution of dye FDCD peaks, but no change occurred in spectral properties of the dyes in urea. This indicated that the observed cotton effects occurred by noncovalent interactions with the secondary structure of the protein. The average BSA–dye association constants found by fluorescence, absorbance, and FDCD were 1.27×10⁶ (n=1) and 3.3×10⁶ M⁻¹ (n=1) for NN127 and NN525 respectively. These values were in good agreement when calculated by the three spectroscopic methods validating the use of NIRFDCD for optical parameter calculations. These results are useful to describe NIR squarylium dye labeling of BSA.     

Novel monofunctional and bifunctional boronic acid‐functionalized squarylium dyes as precolumn and on‐column labels for protein analysis by capillary electrophoresis with laser‐induced fluorescence

This article presents a continuous capillary electrophoresis with laser‐induced fluorescence (CE‐LIF) following spectral studies of the noncovalent interactions between novel Squarylium Boronic Acid 4 (SQ‐BA4) & Squarylium Diboronic Acid 2 (SQ‐DBA2) squarylium dyes and human serum albumin (HSA). Two protocols were used wherein the on‐column‐labeling protocol was found to be more sensitive than the precolumn one by showing a better enhancement in the peak area of the HSA–dye complex besides lower limits of detection (LODs) for HSA. Also, stability studies were conducted with or without HSA using precolumn‐labeling mode over one week exhibiting the superiority of SQ‐BA4 to SQ‐DBA2. Then, a mixture containing three model proteins, HSA, β‐lactoglobulin B, and transferrin, was labeled on‐column with both dyes and completely resolved by CE‐LIF after optimization of several parameters. Both dyes provided lower LODs for HSA than those of β‐lactoglobulin B and transferrin with higher sensitivities. In addition, the SQ‐BA4 dye showed again greater sensitivities with all the three proteins than SQ‐DBA2.     

合成的近红外花菁染料测定痕量血清蛋白质

染料结合法测定蛋白质在生化及临床分析中已有广泛应用,常用的染料有溴甲酚绿、溴酚蓝、考马斯亮蓝及铬天青S等,这些染料的最大吸收波长均处于可见光区.近年来,近红外染料越来越引起人们的注意.     

Determination of phycobiliproteins by capillary electrophoresis with laser-induced fluorescence detection

Phycobiliproteins are derived from the photosynthetic apparatus of cyanobacteria and eukaryotic algae. They are composed of a protein backbone to which linear tetrapyrrole chromophores are covalently bound. Furthermore, they are water-soluble highly fluorescent, and relatively stable at room temperature and neutral pH. For this reason, capillary electrophoresis-laser induced fluorescence (CE-LIF) seems the idea method for determination of these important proteins. The effects of buffer additives such as sodium dodecyl sulfate (SDS)and putrescine on the separation of the three major phycobiliprotein types, namely allophycocyanin, phycocyanin, and phycoerythrin, with excitation and emission maxima at 652/660, 615/647, and 565(494)/575 nm, respectively, are considered. Detection limits for these proteins by CE-LIF are some 60-500 times better than by absorbance detection. The development of a fast and sensitive CE-LIF assay such as this is of potential significance to our understand ing of chemical and biological oceanographic processes.     

Polymer-Enhanced Capillary Transient Isotachophoresis with Boronic Acid-Functionalized Squarylium Dyes for the Fluorescent Determination of Digoxin and Digoxigenin

Here is reported a new application of polymer-enhanced capillary transient isotachophoresis for the separation and quantification of the drug digoxin and its primary metabolite digoxigenin coupled with laser-induced fluorescence (LIF) detection facilitated by labeling with two boronic acid-functionalized squarylium dyes of different alkyl side chain lengths, SQ-BA1 and SQ-BA2. The conditions for drug–dye complex formation were optimized, as determined by absorbance and fluorescence spectra, according to solution pH and buffer composition. As digoxin has a digitoxose sugar moiety in its structure, it was shown to exhibit better enhancement in the fluorescence intensity of both dyes than digoxigenin, which lacks this moiety, presumably through the formation of a cyclic boronate ester complex. A comparison of analyte labeling in pre-column and on-column modes was conducted in subsequent capillary electrophoresis-LIF studies, with the latter labeling mode yielding superior sensitivity. However, to achieve the complete resolution of labeled digoxin and digoxigenin analytes, it was necessary to use the modified isotachophoresis method, with added borate ions that may differentially interact with the drug and its metabolite, hence affecting their mobilities. Limits of quantification of the method for the determination of digoxin with SQ-BA1 and SQ-BA2 were 2.61?×?10^?3 and 2.82?×?10^?3?M and limits of detection were 7.83?×?10^?4 and 8.47?×?10^?4?M while sensitivities were as great as 5.06?×?10⁹ and 2.89?×?10⁹?M^?1, respectively, indicating that the method is suitable for practical analysis.     

新型水溶性不对称五甲川菁染料的合成、光稳定性及蛋白质的荧光标记

合成并表征了系列水溶性五甲川菁染料, 研究了其在不同溶剂中的光谱性能. 结果表明, 染料在水中的最大吸收和荧光光谱在647~665 nm波长范围内, 荧光量子产率达到0.1左右. 考察了N位取代基对染料水溶液光稳定性的影响, 结果表明, 在N原子上引入带有苯环结构和大体积的磺酸基, 可以提高染料的光稳定性. 高效液相色谱(HPLC)分析结果表明, 染料4a的N-羟基琥珀酰亚胺(NHS)活性酯标记牛血清白蛋白(BSA)的检测限为1.2×10^-8 mol/L, 与紫外检测相比, 检测灵敏度提高了近2个数量级.     

Noncovalent labeling of biomolecules with red and near- infrared dyes

Biopolymers such as proteins and nucleic acids can be labeled with a fluorescent marker to allow for their detection. Covalent labeling is achieved by the reaction of an appropriately functionalized dye marker with a reactive group on a biomolecule. The recent trend, however, is the use of noncovalent labeling that results from strong hydrophobic and/or ionic interactions between the marker and biomolecule of interest. The main advantage of noncovalent labeling is that it affects the functional activity of the biomolecule to a lesser extent. The applications of luminescent cyanine and squarylium dyes are reviewed.     

Comparative studies on biophysical interactions between 4-dicyanomethylene-2,6-dimethyl-4H-pyran (DDP) with bovine serum albumin (BSA) and human serum albumin (HSA) via photophysical approaches and molecular docking techniques

Photophysical studies of 4-Dicyanomethylene-2,6-Dimethyl-4H-Pyran (DDP) dye with globular proteins, Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) were carried out in aqueous solution. An isosbestic point resulted on the addition of serum albumins, which signifies a complex or an equilibrium state of DDP dye with albumin. Addition of BSA to DDP dye results in a fluorescence enhancement accompanied with a significant hypsochromic shift, whereas with that of HSA, a fluorescence quenching with a considerable blue shift resulted. Excited state studies of DDP dye with serum albumins portray that the role of binding sites of dye with albumins vary considerably and the nature of interaction is presumably attributed to combined hydrogen-bonding and hydrophobic interactions. Molecular docking studies of DDP dye with albumins and two other derivatives 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM) dye and 4-(Dicyanomethylene)-2-methyl-6-(4-t-buyl)-4H-pyran (DCT) dyes with BSA and HSA elucidates that the hydrogen-bonding interaction accompanied with several hydrophobic, pi–pi an pi–alkyl interactions coexist between dye and albumins. The binding energy, intermolecular energy and stability of the DDP, DCM and DCT dyes through docking techniques with albumins authenticate that the dye predominantly acts as hydrogen-bonding acceptor site and the protein molecule as the donor. DDP dye prefers to exist in four different binding sites of HSA, whereas, in the case of BSA, the most preferred site is found to be hydrophobic domain (site I). Interestingly, the most preferred site of DCT dye is III A subdomain of HSA, whereas DCM dye is oriented towards I B subdomain. DDP and DCT are smaller in size and reside in the domain preferred for smaller ligands (II A and IIIA) as resulted in several drugs-HSA interaction whereas DCM dye which is categorized as medium to larger ligand based on the extended structure resides in the most favoured site IB. Fluorescence techniques in combination with molecular docking methods elucidate binding characteristics and the domain in which the dye resides in a micro heterogeneous environment is established in this study.     

Thiol-reactive dyes for fluorescence labeling of proteomic samples

Covalent derivatization of proteins with fluorescent dyes prior to separation is increasingly used in proteomic research. This paper examines the properties of several commercially available iodoacetamide and maleimide dyes and discusses the conditions and caveats for their use in labeling of proteomic samples. The iodoacetamide dyes BODIPY TMR cadaverine IA and BODIPY Fl C(1)-IA were highly specific for cysteine residues and showed little or no nonspecific labeling even at very high dye:thiol ratios. These dyes also showed minimal effects on pI's of standard proteins. Some iodoacetamide dyes, (5-TMRIA and eosin-5-iodoacetamide) and some maleimide dyes (ThioGlo I and Rhodamine Red C(2) maleimide) exhibited nonspecific labeling at high dye:thiol ratios. Labeling by both iodoacetamide and maleimide dyes was inhibited by tris(2-carboxyethyl)phosphine (TCEP); interactions between TCEP and dye were also observed. Thiourea, an important component of sample solubilization cocktails, inhibited labeling of proteins with iodoacetamide dyes but not with maleimide dyes. Maleimide dyes may serve as an alternative for labeling proteins where it is essential to have thiourea in the solubilization buffer. Covalent derivatization by BODIPY TMR cadaverine IA, BODIPY Fl C(1)-IA or Rhodamine Red C(2) maleimide was also demonstrated to be compatible with in-gel digestion and peptide mass fingerprinting by matrix assisted laser desorption/ionization-mass spectrometry and allowed successful protein identification.     

四羧基镍酞菁共振散射法测定蛋白质

建立了一种测定蛋白质的新方法．在pH3．6的Britton-Robinson（B-R）缓冲溶液中,蛋白质与四羧基镍酞菁NiPc（COOH）4发生相互作用,使体系在λ=388nm处的共振散射（RLS）增强,并且增强的散射强度（IRLS）与蛋白质的含量成比例,据此利用四羧基镍酞菁NiPc（COOH）4为光谱探针共振散射法测定人血清中的总蛋白质,同时优化了体系光散射检测的实验参数．在最佳的实验条件下,对牛血清白蛋白（BSA）、人血清白蛋白（HSA）、人血清总蛋白（TP）的线性范围分别为0．00～1．20mg／L、0．00～1．00mg／L、0．00～1．00mg／L,相应检测限分别为5．97×10^-4mg／L、2．90×10^-4mg／L、4．76×10^-4mg／L．将该方法应用于实际人血清样品中总蛋白的测定,结果与考马斯亮蓝法比较,令人满意．     

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.     

Resonance light scattering technique for the determination of protein with rutin and cetylpyridine bromide system

A new resonance light scattering (RLS) assay of protein is presented. In Tris-NaOH (pH = 10.93) buffer, the RLS of rutin-cetylpyridine bromide (CPB) system can be greatly enhanced by protein, including bovine serum albumin (BSA) and human serum albumin (HSA). The enhanced RLS intensities are in proportion to the concentration of proteins in the range of 5 x 10(-9) to 2.5 x 10(-6) g ml(-1) for BSA and 2.5 x 10(-8) to 3.5 x 10(-6) g ml(-1) for HSA. The detection limits (S/N = 3) are 3.0 ng ml(-1) for BSA and 10.0 ng ml(-1) for HSA. Samples are determined satisfactorily.     

Handling and detection of 0.8 amol of a near-infrared cyanine dye by capillary electrophoresis with laser-induced fluorescence detection

We are interested in the detection of DNA adducts and other trace analytes by labeling them with a fluorescent tag followed by use of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for high resolution and sensitivity. Towards this goal, here we report the following: (1) synthesis and handling properties of a near-IR, carboxyl-substituted heptamethine cyanine dye; (2) modification of an existing ball lens LIF detector to provide near-LIF detection with excitation at 785 nm for CE; and (3) corresponding handling and detection of as little as 0.8 amol of the dye by enrich-injection of 4.7 microl of 1 x 10(-13) mol/l dye in methanol from an 8-microl volume into a corresponding CE-LIF system. The electrolyte for the separation was methanol-40 mmol/l aqueous sodium borate (98:2, v/v). This finding encourages further exploration of the dye by functionalization of its carboxyl group for chemical labeling purposes.     

Evaluation of Antifungal Activity and Potential Application as Fluorescent Probes of Indolenine and Benzo[e]Indole-Based Squarylium Dyes

The antifungal performance and the possible use as fluorescent probes of a series of squarylium dyes derived from indolenine and benzo[e]indole previously synthesized was evaluated. Some photophysical properties were performed in ethanol and phosphate buffer, and the type of aggregates form in phosphate buffer was analyzed. Using the 1,3-diphenylisobenzofuran assay, a qualitative assessment of the capacity of dyes to produce singlet oxygen after irradiation was performed. Regarding the antifungal activity, this was studied through a broth microdilution assay using Saccharomyces cerevisiae PYCC 4072 as a biological model. The effect of irradiation of the dyes, with an appropriate light emitting diode system, on the antifungal activity was also evaluated, and it was verified that some of the dyes improve their activity after irradiation. Using fluorescence microscopy techniques, the colocalization of dyes in S. cerevisae cells was investigated and it was possible to verify that some of the squarylium dyes with a barbituric moiety in the four-membered central ring stained and accumulated preferentially in the mitochondrial web and perinuclear membrane of the cells. The possible use as a fluorescent probe for the detection of HSA was also evaluated for one of the dyes of the series, demonstrating a linear variation in the fluorescence intensity accompanied by the increase in the protein concentration.     

Study of the noncovalent interaction of squarylium dyes with serum albumins

The noncovalent interaction of zwitterionic indolium squarylium dyes (hydrophilic and hydrophobic) and a structurally analogous ionic indodicarbocyanine (hydrophilic) dye with serum albumins was studied by spectral and fluorescent methods. It has been found that the hydrophilic squarylium dye with sulfonate groups most efficiently interacts with albumins, which is probably due to the double negative charge of the dye molecule at the expense of the sulfonate groups and the possibility to form hydrogen bonds with albumin. The hydrophobic squarylium dye, as well as the hydrophilic indodicarbocyanine dye without the squarylium fragment in its structure, bind with albumins much weaker than the structurally relevant hydro- philic squarylium dye. The properties of the latter dye permit us to recommend it for using as a spectral and fluorescent probe for serum albumins in extracellular media of living organisms.