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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.

We applied surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) with HgTe nanostructures as the matrix for the detection of single- and double-stranded oligodeoxynucleotides (ss-ODNs and ds-ODNs). The concentrations of surfactant and additives (metal ions, an amine) and the pH and ionic strength of the sample matrix played significantly different roles in the detection of ss- and ds-ODNs with various sequences. In the presence of Brij 76 (1.5 %), Hg(2+) (7.5 μM), and cadaverine (10 μM) at pH 5.0, this SALDI-MS approach allowed the simultaneous detection of T15, T20, T33, and T40, with limits of detection at the femtomole-to-picomole level and sample-to-sample intensity variation23 %. In the presence of Ag(+) (1 μM) and cadaverine (10 μM) at pH 7.0, this technique allowed the detection of randomly sequenced ss- and ds-ODNs at concentrations down to the femtomole level. To the best of our knowledge, this paper is the first to report the detection of ss-ODNs (up to 50-mer) and ds-ODNs (up to 30 base pairs) through the combination of SALDI-MS with HgTe nanostructures as matrices. We demonstrated the practicality of this approach through analysis of a single nucleotide polymorphism that determines the fate of the valine residue in the β-globin of sickle cell megaloblasts.

Glycoconjugates are ubiquitously present and play a critical role in various biological processes. Due to their low stability and incredibly high degree of structural diversity, the structural characterization of glycan generally requires chemical derivatization and sophisticated instrumentation. Herein, we report a method for complicated glycan characterization in a single assay by employing 2,5-dihydroxybenzoic acid functionalized mercury telluride nanoparticles (HgTe@DHB NPs) as a dual ionization–dissociation element in matrix-assisted laser desorption/ionization mass spectrometry. Using a linear glycan, HgTe@DHB NPs promote laser-induced extensive and intense dissociation of the glycan, superior to HgTe microparticles and other inorganic nanoparticles (TiO2, ZnO, and Mn2O3 NPs). Abundant generation of diagnostic glycosidic (Y-, and B-type ions) and cross-ring cleavage (A-type ions) ions permits unambiguous determination of the composition, sequence, branching, and linkage of labile sialylated glycans. The general utility of this approach was demonstrated by the characterization of labile sialylated glycans and two sets of complicated isomeric glycans. This phenomenon was delineated further by investigating the NP's physico-chemical characteristics, revealing that their nanoscale-dependent thermodynamic properties, including UV absorption, photoelectron release dynamics and thermal energy, were the key to levitate temperature synergistically, thus inducing spontaneous glycan decomposition during the nanoparticle-assisted laser desorption–ionization process. Our results show that this “pseudo-MS/MS” obtained by HgTe@DHB can be beneficial for the analysis of biologically relevant and more complicated carbohydrates, without the need for chemical pre-derivatization and conventional tandem mass spectrometry.

Cysteine sulfinic acid decarboxylase (Csad) is the rate-limiting enzyme in the de novo biosynthesis of taurine. There are a number of physiological roles of taurine, such as bile salt synthesis, osmoregulation, lipid metabolism, and oxidative stress inhibition. To investigate the role of de novo synthesis of taurine during embryonic development, zebrafish csad was cloned and functionally analyzed. Semi-quantitative RT-PCR showed that csad transcripts are maternally deposited, while whole-mount in situ hybridization demonstrated that csad is expressed in yolk syncytial layer and various embryonic tissues such as notochord, brain, retina, pronephric duct, liver, and pancreas. Knockdown of csad significantly reduced the embryonic taurine level, and the affected embryos had increased early mortality and cardiac anomalies. mRNA coinjection and taurine supplementation rescued the cardiac phenotypes suggesting that taurine originating from the de novo synthesis pathway plays a role in cardiac development. Our findings indicated that the de novo synthesis pathway via Csad plays a critical role in taurine homeostasis and cardiac development in zebrafish early embryos.

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.

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.

We have constructed a dark-field light scattering microscope using a very low-cost digital camera to investigate the adsorption of gold nanoparticles (AuNPs) on four different substrates at various pH values. The substrates used are glass, polycarbonate (PC), poly(dimethylsiloxane) (PDMS), and poly(methyl methacrylate) (PMMA). The coverage of AuNPs on hydrophobic substrates such as PDMS is greater than that on hydrophilic substrates like glass. The adsorption and aggregation of AuNPs on a particular substrate increased upon decreasing the pH (from 9.0 to 4.0). A greater coverage percentage of AuNPs, but less aggregation, occurs on glass treated with poly(diallyldimethylammonium) (PDDA) than on bare glass. The scattering intensity increases upon increasing the number of layers of adsorbed AuNPs on glass that was treated sequentially with AuNPs and PDDA. When compared to UV-Vis absorption, dark-field microscope provides greater sensitivity and qualitative surface information.

The separation of DNA by capillary electrophoresis using poly(ethylene oxide) (PEO) containing gold nanoparticles (GNPs) is presented. The impacts of PEO, GNPs, ethidium bromide (EtBr), and pH on the separation of double-stranded DNA have been carefully explored. Using a capillary dynamically coated with 5.0% poly(vinylpyrrolidone) and filled with 0.2% PEO containing 0.3 x GNPs (the viscosity less than 15 cP), we have demonstrated the separation of DNA markers V and VI within 5 min at pH 8.0 and 9.0. In terms of resolution and reproducibility, GNPs have a greater impact on the separation of DNA at pH 9.0. Resolution improvements for large DNA fragments (> 300 base pairs, bp) are greater than those for small ones in the presence of GNPs. It is important to point out that reproducibility is excellent (relative standard deviations for the migration times less than 0.5%) and thus no further dynamic coating is required in at least 20 consecutive runs in the presence of GNPs. Using 0.2% PEO (pH 9.0) containing 0.3 x GNPs, the separation of DNA fragments ranging in size from 21 to 23,130 bp was accomplished in 7 min. The results presented in this study show the advantage of PEO containing GNPs for DNA separation, including rapidity, high resolving power, excellent reproducibility, and ease of filling capillaries.

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 ...

Separations of phiX-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 microg/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/ Bg/l digest and pBR 328/Hinfl digest) has been achieved in 1.5% PEO.

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 containi...

In this article, we describe the analysis of aptamers for Hg(2+) 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 - T(33), T(5)C(28), T(5)C(5)T(23), and T(15)C(5)T(13) - could not be separated through CE-LIF in the absence of Hg(2+). At 0.3 mM Hg(2+), 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-Hg(2+)-T bonding. In addition, we found that this CE approach provided different electropherograms patterns for T(7), T(15), and T(33) in the absence and presence of Hg(2+), indicating various interactions of the DNA strands with Hg(2+). 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 T(33). 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.

We have demonstrated the analysis of aristolochic acids (AAs) that are naturally occurring nephrotoxin and carcinogen by capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF). Owing to lack of intrinsic fluorescence characteristics of oxidized AAs (OAAs), reduction of the analytes by iron powder in 10.0 mM HCl is required prior to CE analysis. The reduced AAs (RAAs) exhibit fluorescence at 477 nm when excited at 405 nm using a solid-state blue laser. By using 50.0 mM sodium tetraborate (pH 9.0) containing 10.0 mM SDS, the determination of AA-I and AA-II by CE-LIF has been achieved within 12 min. The CE-LIF provides the LODs of 8.2 and 5.4 nM for AA-I and AA-II, respectively. The simple CE-LIF method has been validated by the analysis of 61 Chinese herbal samples. Prior to CE analysis, OAAs were extracted by using 5.0 mL MeOH, and then the extracts were subjected to centrifugation at 3,000 rpm for 5 min. After reduction, extraction, and centrifugation, the supernatants were collected and subjected to CE analysis. Of the 61 samples, 14 samples contain AA-I and AA-II, as well as 10 samples contain either AAI or AAII. The relative standard deviation (RSD) values of the migration times for AA-I and AA-II are less than 2.5% and 2.1% for three consecutive measurements of each sample. The RSD values for the peak heights corresponding to AA-I and AA-II in most samples are about 8.0% and 10.0%, respectively. The result shows that the present CE-LIF approach is sensitive, simple, efficient, and accurate for the determination of AAs in real samples.

Capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) using polymer solutions are two of the most powerful techniques for the analysis of DNA. Problems, such as the difficulty of filling polymer solution to small separation channels, recovering DNA, and narrow separation size ranges, have put a pressure on developing new techniques for DNA analysis. In this review, we deal with DNA separation using chip-based nanostructures and nanomaterials in CE and MCE. On the basis of the dependence of the mobility of DNA molecules on the size and shape of nanostructures, several unique chip-based devices have been developed for the separation of DNA, particularly for long DNA molecules. Unlike conventional CE and MCE methods, sieving matrices are not required when using nanostructures. Filling extremely low-viscosity nanomaterials in the presence and absence of polymer solutions to small separation channels is an alternative for the separations of DNA from several base pairs (bp) to tens kbp. The advantages and shortages of the use of nanostructured devices and nanomaterials for DNA separation are carefully addressed with respect to speed, resolution, reproducibility, costs, and operation.

We present the first example of the analysis of long double-stranded (ds) DNA molecules by nanoparticle-filled capillary electrophoresis (NFCE). To avoid aggregation of the gold nanoparticles (GNPs) and to allow strong interactions with the DNA molecules, the gold nanoparticles were modified with poly(ethylene oxide) (PEO) via noncovalent bonding to form gold nanoparticle/polymer composites (GNPPs). The neutral GNPPs are heavy (approximately 2.0 x 10(8) g/mol for the 32-nm GNP) and thus slow the DNA molecules that they encounter during the electrophoretic process. Compared to linear polymer solutions, such as hydroxyethyl cellulose and PEO, the GNPPs provide greater efficiency and require significantly shorter times to separate long dsDNA. The separation of lambda-DNA (0.12-23.1 kbp) by NFCE at -250 V/cm was accomplished in 3 min. The ability to separate high molecular weight DNA markers (8.27-48.5 kbp) with plate numbers greater than 10(6) suggests that this novel method may hold great promise for the analysis of long-stranded DNA molecules such as chromosomes. Moreover, this method is simple and affordable when compared to those that use micro- and nanofabricated devices for separating long DNA molecules.

The association between vinyl chloride monomer (VCM) exposure and DNA damage has been established. However, the relationship between individual exposure and DNA single strand breaks was limited. Since environmental monitoring may not reflect the actual exposure, a useful marker of exposure is needed to assess the individual exposure. In our previous study, we have found a high correlation between air VCM level and urinary thiodiglycolic acid (TdGA) at the commencement of the next shift. Here, we further used comet assay to evaluate the relationship between urinary TdGA levels and DNA single strand breaks in polyvinyl chloride monomer (PVC) workers. Urinary TdGA levels (n=26) at the commencement of the following shift were analyzed. Ten of the 26 workers also had personal air sampling for air VCM exposure. Questionnaires were administered to obtain epidemiological information including detailed history of occupation and lifestyles. Workers experiencing air VCM level greater than 5 ppm had higher tail moment and tail intensity (%) than those experiencing VCM exposure between 1 and 5, or

The analysis of double-stranded (ds) DNA fragments by capillary electrophoresis (CE) using poly(ethylene oxide) (PEO) solution containing gold nanoparticles (GNPs) is presented, focusing on evaluating size dependence of the GNPs and PEO on resolution and speed. To prevent the interaction of the capillary wall with DNA, the capillary was dynamically coated with polyvinylpyrrolidone. Using different PEO solutions containing GNPs ranging in diameter from 3.5 to 56 nm, we have achieved reproducible, rapid, and high-resolution DNA separations. The results indicate that the sizes of PEO and GNPs as well as the concentration of PEO affect resolution. The separation of DNA ranging in size from 8 to 2176 base pairs (bp) was accomplished in 5 min using 0.2% PEO (8 MDa) containing 56 nm GNPs. We have also demonstrated the separations of the DNA fragments ranging from 5 to 40 kbp using 0.05% PEO (2 MDa) containing 13 nm GNPs or 0.05% PEO (4 MDa) containing 32 nm GNPs. With very low viscosity (< 15 cP), automatic replacement of the sieving matrices is easy, indicating a great potential for high-throughput DNA analysis using capillary array electrophoresis systems.

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.

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.

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-microM to microM. 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+/-0.05 mM (n = 5) for C14 and C16 acids, respectively, in a tablet of marine lipid concentrate sample.