Determination of thiols by capillary micellar electrokinetic chromatography with laser induced fluorescence detection using 1,3,5,7‐tetramethyl‐8‐phenyl‐(4‐iodoacetamido) difluoroboradiaza‐s‐indacene as labeling reagent

A sensitive and effective micellar electrokinetic capillary chromatography with laser‐induced fluorescence detection approach was described for the determination of low molecular‐mass thiols using 1,3,5,7‐tetramethyl‐8‐phenyl‐(4‐iodoacetamido) difluoroboradiaza‐s‐indacene as the labeling reagent. After precolumn derivatization, baseline separation of six thiol compounds including cysteine, glutathione, N‐acetylcysteine, homocysteine, 6‐mercaptopurine, and penicillamine were achieved within 18 min. The optimal running buffer was composed of mixtures involving 25 mM sodium dodecyl sulfate, 25% (v/v) acetonitrile and 15 mM sodium phosphate buffer, pH 7.5. The detection limits (S/N = 3) were found as low as 40 pM under argon ion laser‐induced fluorescence detector (λ_ex/λ_em = 488/520 nm), which were much better than the reported approaches. The accuracy and specificity of this assay for real samples were assured by a standard addition method. The proposed method has been applied to the analysis of thiols both in human plasma and plum flower samples with recoveries of 92.0–109.4%.     

Quantification of polyamines in human erythrocytes using a new near-infrared cyanine 1-(epsilon-succinimidyl-hexanoate)-1'-methyl-3,3,3',3'-tetramethyl-indocarbocyanine-5,5'-disulfonate potassium with CE-LIF detection

A novel near-infrared (NIR) cyanine 1-(epsilon-succinimidyl-hexanoate)-1'-methyl-3,3,3',3'-tetramethyl-indocarbocyanine-5,5'-disulfonate potassium (MeCy5-OSu) has been developed in our laboratory. Simultaneous determination of MeCy5-OSu-derivatized polyamines spermine (Spm), spermidine (Spd), cadaverine (Cad), and putrescine (Put) based on the separation by CE combined with diode LIF detection has been accomplished. The highest derivatization efficiency was achieved in 0.2 mol/L borate buffer (pH 8.8) for 20 min at 25 degrees C. Polyamine derivatives were separated within 14 min in the phosphate running buffer (pH 3) containing 50 mmol/L phosphoric acid, 40 mmol/L SDS, and 35% methanol v/v. Linearity of response was obtained in the range of 10-200 nmol/L. The detection limits (S/N = 3) for Spm, Spd, Cad, and Put were 0.8, 1, 3, and 2 nmol/L, respectively. The proposed method has been successfully applied to the analysis of polyamines in erythrocytes of two healthy persons and one cancer patient. Average recoveries for erythrocyte samples were 93.6-106% and coefficients of variation ranged from 1.8 to 5.4%. The analysis of polyamines in erythrocytes can be used for studying the relationship between their changes and the carcinogenesis process involved in erythrocytes.     

Effect of permodified β‐cyclodextrin on the photophysical properties of poly[2,7‐(9,9‐dioctylfluorene)‐alt‐(5,5′‐bithiophene)] main chain polyrotaxanes

The photophysical properties of two polyrotaxanes ( PFBTh?PSβCD and PFBTh?PMeβCD ) composed of fluorene and bithiophene encapsulated into permodified β‐cyclodextrin cavities have been investigated and compared with those of the reference PFBTh . Rotaxane formation results in improvements of the thermal stability, solubility in common organic solvents, as well as better film forming ability combined with a high transparency. As expected PFBTh and its encapsulated forms absorb at wavelengths beyond 510 nm, and time‐resolved photoluminescence (PL) in solution shows a well‐define vibronic structures with a predominance of the 0‐0 transitions and an energy difference of 0.16 eV. The fluorescence lifetimes follow a monoexponential decay with a value τ = 630 ± 30 ps. Atomic force microscopy, AFM, indicated a tendency of polyrotaxanes to organize into fibers. The advancing contact angles indicated higher surface hydrophobicity and lower surface free‐energy values for polyrotaxanes compared with their unthreaded analogues. The device based on PFBTh?PSβCD: PCBM in a 1/1 w/w ratio under simulated AM 1.5G illumination at 100 mW cm^?2 exhibited improved photovoltaic parameters of cells, resulted in high V_oc (0.68 V), J_sc (1.65 mA cm^?2), FF (31.6%), and PCE (0.35) values, compared with PFBTh or PFBTh?PMeβCD , respectively. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 460–471     

Determination of genomic N3‐methylthymidine in human cancer cells treated with nitrosamines using capillary electrophoresis with laser‐induced fluorescence

Methylating substances alter DNA by forming N3‐methylthymidine (N3mT), a mutagenic base modification. To develop a sensitive analytical method for the detection of N3mT in DNA based on capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF), we synthesized the N3mT‐3’‐phosphate as a chemical standard. The limit of detection was 1.9 amol of N3mT, which corresponds to one molecule of N3mT per 1000 normal nucleotides or 0.1%. With this method, we demonstrated that the carcinogenic nitrosamine N’‐nitrosonornicotine (NNN) induced N3mT in the human lung cancer cell line A549. Treatment with NNN also caused an elevated degree of 5‐hydroxymethylcytidine (5hmdC) in DNA, while the methylation degree (i.e. 5‐methylcytidine; 5mdC) stayed constant. According to our data, NNN could, via yet unknown mechanisms, play a role in the formation of N3mT as well as 5hmdC. In this study we have developed a new sensitive analytical method using CE‐LIF for the simultaneous detection of the three DNA modifications, 5mdC, 5hmdC and N3mT.     

Rapid and sensitive determination of free thiols by capillary zone electrophoresis with near‐infrared laser‐induced fluorescence detection using a new BODIPY‐based probe as labeling reagent

A CZE with near‐infrared (NIR) LIF detection method has been developed for the analysis of six low molecular weight thiols including glutathione, homocysteine, cysteine, γ‐glutamylcysteine, cysteinylglycine, and N‐acetylcysteine. For this purpose, a new NIR fluorescent probe, 1,7‐dimethyl‐3,5‐distyryl‐8‐phenyl‐(4'‐iodoacetamido)difluoroboradiaza‐s‐indacene was utilized as the labeling reagent, whose excitation wavelength matches the commercially available NIR laser line of 635 nm. The optimum procedure included a derivatization step of the free thiols at 45°C for 25 min and CZE analysis conducted within 14 min in the running buffer containing 16 mmol/L pH 7.0 sodium citrate and 60% v/v ACN. The LODs (S/N = 3) ranged from 0.11 nmol/L for N‐acetylcysteine to 0.31 nmol/L for γ‐glutamylcysteine, which are better than or comparable to those reported with other derivatization‐based CE‐LIF methods. As the first trial of NIR CE‐LIF method for thiol determination, the practical application of the proposed method has been validated by detecting thiols in cucumber and tomato samples with recoveries of 96.5–104.3%.     

Estimation of protein concentration at high sensitivity using SDS‐capillary gel electrophoresis‐laser induced fluorescence detection with 3‐(2‐furoyl)quinoline‐2‐carboxaldehyde protein labeling

3‐(2‐furoyl)quinoline‐2‐carboxaldehyde (FQ) is a sensitive fluorogenic dye, used for derivatization of proteins for SDS‐CGE with LIF detection (SDS‐CGE‐LIF) at silver staining sensitivity (ng/mL). FQ labels proteins at primary amines, found at lysines and N‐termini, which vary in number and accessibility for different proteins. This work investigates the accuracy of estimation of protein concentration with SDS‐CGE‐LIF in real biological samples, where a different protein must be used as a standard. Sixteen purified proteins varying in molecular weight, structure, and sequence were labeled with FQ at constant mass concentration applying a commonly used procedure for SDS‐CGE‐LIF. The fluorescence of these proteins was measured using a spectrofluorometer and found to vary with a RSD of 36%. This compares favorably with other less sensitive methods for estimation of protein concentration such as SDS‐CGE‐UV and SDS‐PAGE‐Coomassie and is vastly superior to the equivalently sensitive silver stain. Investigation into the number of labels bound with UHPLC‐ESI‐QTOF‐MS revealed large variations in the labeling efficiency (percentage of labels to the number of labeling sites given by the sequence) for different proteins (from 3 to 30%). This explains the observation that fluorescence per mole of protein was not proportional to the number of lysines in the sequence.