Ming-Feng Huang

Analysis of amino acids and biogenic amines in breast cancer cells by capillary electrophoresis using polymer solutions containing sodium dodecyl sulfate

We describe simultaneous analysis of naphthalene-2,3-dicarboxaldehyde (NDA)-amino acid and NDA-biogenic amine derivatives by CE in conjunction with light-emitting diode-induced fluorescence detection using poly(ethylene oxide) (PEO) solutions containing sodium dodecyl sulfate (SDS). After sample injection, via EOF 0.1% PEO prepared in 100mM TB solution (pH 9.0) containing 30 mM SDS entered a capillary filled with 0.5M TB solution (pH 10.2) containing 40 mM SDS. Under this condition, 14 NDA-amino acid and NDA-amine derivatives were separated within 16 min, with high efficiency ((1.0-3.2)x10(5) theoretical plates) and sensitivity (LODs at S/N=3 ranging from 2.06 to 19.17 nM). In the presence of SDS and PEO, analytes adsorption on the capillary wall was suppressed, leading to high efficiency and reproducibility. The intraday analysis RSD values (n=3) of the mobilities for the analytes are less than 0.52%. We have validated the practicality of this approach by quantitative determination of 9 amino acids in breast cancer cells (MCF-7) and 10 amino acids in normal epithelial cells (H184B5F5/M10). The concentrations of Tau and Gln in the MCF-7 cells were different than those in the H184B5F5/M10 cells, respectively. Our results show the potential of this approach for cancer study.

Detection of carbohydrates using surface-assisted laser desorption/ionization mass spectrometry with HgTe nanostructures

We have optimized a previously developed method, combining surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) with HgTe nanostructures, for the detection of sucrose, cyclodextrins, pullulan, and dextran without the need for derivatization. For these analytes, [M + Na]+ ions were the dominant species detected in the mass spectra. We adjusted several parameters, including the concentration of the HgTe nanostructures, the pH and salt concentration of the sample matrices, to optimize the sensitivity of the detection of the analytes. This SALDI-MS approach allowed quantitative determination of the concentrations of sucrose, α-cyclodextrin (CD), β-CD, γ-CD, PL-6k, and PL-10k over the ranges 0.5–25, 0.5–50, 0.5–50, 0.5–75, 0.01–5, and 0.05–10 μM, respectively, with limits of detection (at a signal-to-noise ratio of 3) of 100, 28, 30, 22, 8.5, and 25 nM, respectively. The MS profiles of PL-6k and PL-10k were symmetric, revealing the absence of degradation during the SALDI-MS analysis process. From the MS data, we determined the polydispersity indices (PDIs) of PL-6k and PL-10k to be 1.03 and 1.05, respectively. The spot-to-spot and sample-to-sample variations for these analytes were both less than 13%; for example, they were less than 6 and 9%, respectively, for PL-6k. This simple, sensitive, accurate, and reproducible SALDI-MS approach using HgTe nanostructures allows determination of the molecular weights of polysaccharides up to 12 000 Da and quantitative determination of neutral carbohydrates.

Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions

Separations of φX‐174/HaeIII DNA restriction fragments have been performed in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose. During the separation, polymer solutions entered the capillary by EOF. When using LPA solutions, bulk EOF is small due to adsorption on the capillary wall. On the other hand, separation is faster and better for the large DNA fragments (> 872 base pairs, bp) using derivative celluloses and PEO solutions. Several approaches to optimum resolution and speed by controlling EOF and/or altering electrophoretic mobility of DNA have been developed, including (i) stepwise changes of ethidium bromide (0.5–5 µg/mL), (ii) voltage programming (125–375 V/cm), (iii) use of mixed polymer solutions, and (iv) use of high concentrations of Tris‐borate (TB) buffers. The DNA fragments ranging from 434 to 653 bp that were not separated using 2% PEO (8 000 000) under isocratic conditions have been completely resolved by either stepwise changes of ethidium bromide or voltage programming. Compared to PEO solutions, mixed polymer solutions prepared from PEO and HEC provide higher resolving power. Using a capillary filled with 600 mM TB buffers, pH 10.0, high‐speed (< 15 min) separation of DNA (pBR 322/HaeIII digest, pBR 328/BglI digest and pBR 328/HinfI digest) has been achieved in 1.5% PEO.

Indirect fluorescence of aliphatic carboxylic acids in nonaqueous capillary electrophoresis using merocyanine 540

A method for the analysis of aliphatic carboxylic acids (ACAs) in nonaqueous capillary electrophoresis (NACE) in conjunction with indirect laser‐induced fluorescence (ILIF) using merocyanine 540 (MC 540) is described. Performing the analysis in organic solvent is advantageous when using MC 540, because of its greater quantum yield in aprotic solvent. To achieve a high dynamic reserve (DR) and optimize resolution, we have tested a number of aqueous mixtures containing alcohols and acetonitrile (ACN). The optimum buffer for the analysis of C2‐C18 ACAs, in terms of sensitivity, resolution, and speed, is an aqueous mixture of 40% ACN, 30% ethanol, and 1 mM Tris at apparent pH 7.4 (adjusted with ascorbic acid). Under this condition, the DR is greater than 1000, thereby the limits of detection for acids are in the range of sub‐νM to νM. Linear plots show that the dynamic ranges for the analysis of ACAs are at least two decades in concentration, with regression coefficients all greater than 0.98. The relative standard deviations of the migration times and peak heights for all ACAs are less than 2.0%. Furthermore, this simple and cost‐effective method has been applied to the analysis of marine lipid concentrate, with the concentrations of 1.67 ± 0.03 and 4.50 plusmn; 0.05 mM (n = 5) for C14 and C16 acids, respectively, in a tablet of marine lipid concentrate sample.

Indirect Fluorescence of Amines in Capillary Electrophoresis, Using Cresyl Violet

Abstract This paper describes the analysis of amines under acidic conditions by capillary electrophoresis (CE) in conjunction with indirect laser‐induced fluorescence (ILIF), using cresyl violet as a probe. In the system, a small pinhole (0.2 mm) and an interference filter (589 nm) were used to confine the light and to minimize the plasma interference from a He–Ne laser, respectively, improving the baseline stability. Adding lithium ions to the background electrolytes (BGEs) is effective to achieve narrower peak profiles, leading to better resolution. The analysis of six amines by CE–ILIF using an aqueous solution, pH 3.5, containing 5.0% methanol, 0.1 mM sulfuric acid, 0.1 mM cresyl violet, and 0.3 mM lithium was complete in 5 min, with the limits of detection (LOD) on the level of µM. Negative peak profiles are for amines with greater electrophoretic mobility than that of lithium ions, but positive peaks for the slower ones. To further improve the sensitivity, on‐line concentration based on pH junction has been demonstrated. When injecting the sample prepared in a solution of 0.2 mM sulfuric acid, pH 3.3, at 15 kV for 60 s, and conducting the separation using the above‐mentioned condition, the sensitivity improvements are greater than 10‐fold compared to that injecting at 15 kV for 5 s. With the advantages of rapidity, sensitivity, and low cost, this method has proven potential for the analysis of trace amines in biological samples.

Capillary electropherograms for restriction fragment length polymorphism of Helicobacter pylori.

Rapid identification of Helicobacter pylori strains is of importance for diagnosis and then treatment of duodenal and gastric ulcers. We developed a CE approach for the analysis of RFLP of the PCR products of urease (UreAB) gene and flagellin A (FlaA) gene fragments. Prior to CE analysis, the 2.4‐kbp UreAB and 1.5‐kbp FlaA PCR products were digested with the restriction enzymes HaeIII and HhaI, respectively. The DNA fragments were then separated by CE in conjunction with laser‐induced fluorescence detection using poly(ethylene oxide) in the presence of electroosmotic flow. The DNA fragments range in sizes 259–1831 bp and 12–827 bp for UreAB and FlaA restriction fragments, respectively. Of 27 samples, the CE approach provided five and ten different RFLP patterns of the HaeIII and HhaI digests. The RFLP of PCR products of the two genes allow great sensitivity of identification of H. pylori strains. When compared with slab gel electrophoresis, the present CE approach provides advantages of rapidity (within 6 min per run), simplicity, and automation. The preliminary results have shown great practicality of the CE approach for screening H. pylori strains.

CAPILLARY ELECTROPHORETIC SEPARATION OF DNA FRAGMENTS UNDER STEPWISE CHANGES OF POLYMER SOLUTIONS

ABSTRACT We demonstrated techniques of stepwise changes of poly-ethyleneoxide (PEO) solutions for DNA separations in the presence of the electroosmotic flow (EOF). The stepwise changes were performed by injecting different PEO solutions contained in a number of syringes to a polyethylene tube, from which the polymer solutions entered a capillary filled with 1X TBE buffer by the EOF. The separation of DNA markers V and VI was not complete under isocratic conditions, either using 1.0–2.5% PEO solutions containing 0.5 μg/mL ethidium bromide (EtB) or using 2.0% PEO solutions containing 0.1–2.0 μg/mL EtB. Resolution, sensitivity and time of the DNA separation were varied under conditions of either stepwise changes of PEO concentrations from 1.5 to 2.5% in 0.5 μg/mL EtB solutions or stepwise changes of EtB concentrations from 0.1 to 2.0 μg/mL in 2.0% PEO solutions. The optimal separation was obtained under simultaneous stepwise changes of PEO and EtB: 1.5% PEO containing 0.1μg/mL EtB for 45 s, 1.5% PEO containing 0.5μg/mL EtB for 120 s, 2.0% PEO containing 0.5μg/mL EtB for 45 s, 2.0% PEO containing 1.0μg/mL EtB for 30 s, and 2.5% PEO containing 2.0μg/mL EtB for the rest. The separation was complete in 17 min and the RSD values of migration times were less than 2.0%.

Analyses of functional polymer-modified nanoparticles for protein sensing by surface-assisted laser desorption/ionization mass spectrometry coupled with HgTe nanomatrices.

In this study, we employed HgTe nanostructure-based matrices (nanomartrices; NMs) for surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) for the analyses of polyethylene glycol (PEG) derivatives as well as thiol-PEG-modified gold nanoparticles (PEG-Au NPs). Relative to common organic matrices, the use of HgTe NMs as the matrix for SALDI-MS resulted in more highly efficient analyses of PEG derivatives, in terms of sensitivity and reproducibility. The symmetric MS profiles of PEG (Mw: ca. 8000 Da) obtained through HgTe NMs/SALDI-MS analysis revealed the absence of polymer degradation during this process. Under optimal conditions, the HgTe NMs/SALDI-MS system enabled the detection of PEG sample as low as 100 pg and with molecular weights of up to approximately 42,000 Da. We also used this approach for the analyses of PEG-Au NPs in which various functional groups (carboxymethyl, amine, biotin) were present at the PEG termini, revealing that the combination of SALDI-MS and HgTe NMs have great potential for use in the characterization of modified polymer-ligands on nanomaterials. We also demonstrated the PEG-Au NPs can be coupled with HgTe NMs/SALDI-MS for characterization of biorecognition events. After avidin, the target protein, had been selectively captured by the biotin-PEG-Au NPs, we found that the desorption/ionization efficiency of biotin-PEG from the Au NP surface was suppressed; accordingly, this novel SALDI-MS approach allows rapid detection of avidin with high specificity and sensitivity. Au NP surfaces functionalized with other functional-PEG ligands might also allow amplification of signals from other biological interactions.

Analysis of DNA complexes with small solutes by CE with LIF detection

In this article, we describe the analysis of aptamers for Hg2+ ions through CE with LIF (CE‐LIF) detection using 2% poly(ethylene oxide) solutions containing OliGreen (fluorophore). In the presence of an EOF, DNA strands migrating against the EOF were detected at the cathode end. Four DNA strands – T33, T5C28, T5C5T23, and T15C5T13 – could not be separated through CE‐LIF in the absence of Hg2+. At 0.3 mM Hg2+, however, all four were partially separated within 20 min, with SDs of the migration times all being less than 2.5%. From the CE, fluorescence, and ellipticity data, we concluded that the conformations of these four DNA strands all changed from random‐coil to folded structures as a result of T–Hg2+–T bonding. In addition, we found that this CE approach provided different electropherograms patterns for T7, T15, and T33 in the absence and presence of Hg2+, indicating various interactions of the DNA strands with Hg2+. Using this simple, high‐resolution CE approach, we also demonstrated that adenosine triphosphate has a stronger interaction with the adenosine triphosphate aptamer than with either the platelet‐derived growth factor aptamer or T33. This CE approach holds great potential for screening aptamers for small solutes, studying the catalytic activity of DNAzymes, and evaluating the biological functions of microRNA.

Conformational dynamics of DNA bulge loops investigated by CE-LIF

Capillary electrophoresis with laser induced fluorescence (CE-LIF) detection has been utilized to investigate the metastable states of DNA bulge loops during strand displacement reactions. DNA samples consisting of one of the oligonucleotides with sequences of X(CAG)nY (n = 4–8, X = CACGAGCACGC and Y = CACACCGAAGC) and a 22-mer oligonucleotide with a sequence of Y′X′ (Y′ = GCGTGCTCGTG and X′ = GCTTCGGTGTG) were separated within 7 min by CE-LIF using 2.7% poly(ethylene oxide) (PEO) in the presence of EOF. Our CE data allowed calculation of the equilibrium constant (K) of the ds-DNA of 34-mer X(CAG)4Y and 22-mer Y′X′ at 25 °C to be 1.3 (±0.1) × 109 M−1, with a rate constant (k) of 3.7 (±0.4) × 105 M−1 s−1. We have further applied this CE-LIF technique to separately study the strand displacement of a ds-DNA of the 34-mer X(CAG)4Y and 22-mer Y′X′ bulge loop DNA with a 22-mer XY and that of a ds-DNA of 22-mer XY and Y′X′ with the 34-mer X(CAG)4Y. Our study reveals that the metastable DNA bulge loops and unbinding oligonucleotides were coexistent at room temperature, while they underwent displacement during a heating/cooling cycle. Our study reveals that this CE-LIF approach has great potential for the investigation of DNA related biological functions.