Tai-Chia Chiu

Aptamer-Functionalized Nano-Biosensors

Nanomaterials have become one of the most interesting sensing materials because of their unique size- and shape-dependent optical properties, high surface energy and surface-to-volume ratio, and tunable surface properties. Aptamers are oligonucleotides that can bind their target ligands with high affinity. The use of nanomaterials that are bioconjugated with aptamers for selective and sensitive detection of analytes such as small molecules, metal ions, proteins, and cells has been demonstrated. This review focuses on recent progress in the development of biosensors by integrating functional aptamers with different types of nanomaterials, including quantum dots, magnetic nanoparticles (NPs), metallic NPs, and carbon nanotubes. Colorimetry, fluorescence, electrochemistry, surface plasmon resonance, surface-enhanced Raman scattering, and magnetic resonance imaging are common detection modes for a broad range of analytes with high sensitivity and selectivity when using aptamer bioconjugated nanomaterials (Apt-NMs). We highlight the important roles that the size and concentration of nanomaterials, the secondary structure and density of aptamers, and the multivalent interactions play in determining the specificity and sensitivity of the nanosensors towards analytes. Advantages and disadvantages of the Apt-NMs for bioapplications are focused.

Determining estrogens using surface-assisted laser desorption/ionization mass spectrometry with silver nanoparticles as the matrix

We describe the application of silver nanoparticles (Ag NPs) as matrices for the determination of three estrogens using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). Because Ag NPs have extremely high absorption coefficients (1.2 × 108 M−1 cm−1) at 337 nm, they are effective SALDI matrices when using a nitrogen laser. Three tested estrogens—estrone (E1), estradiol (E2), and estriol (E3)—adsorb weakly onto the surfaces of the Ag NPs, through van der Waals forces. After centrifugation, the concentrated analytes adsorbed on the Ag NPs were subjected directly to SALDI-MS analyses, with the limits of detection for E1, E2, and E3 being 2.23, 0.23, and 2.11 µM, respectively. The shot-to-shot and batch-to-batch variations for the three analytes were less than 9% and 13%, respectively. We validated the practicality of this present approach through the quantitation of E2 in human urine. Using this approach, we determined the concentration of E2 in a sample of a pregnant woman’s urine to be 0.16 ± 0.05 µM (n = 10).

Laser-induced fluorescence technique for DNA and proteins separated by capillary electrophoresis

Recent developments in capillary electrophoresis (CE) in conjunction with laser-induced fluorescence (LIF) using long-wavelength (maximum excitation wavelength>500 nm) dyes are reviewed. These dyes are particularly of interest when conducting the analyses of biopolymers by CE-LIF using He-Ne lasers. These systems are benefited from low background, low costs, easy maintenance, and compactness. Derivatizations of DNA and proteins with fluorescent or nonfluorescent chemicals can be carried out prior to, during, or after separations. With the advantages of sensitivity, rapidity, and high efficiency, the applications of CE-LIF to the analysis of polymerase chain reaction products, DNA sequencing, trace analysis of proteins, and single cell analysis have been presented.

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 aminothiols through surface-assisted laser desorption/ionization mass spectrometry using mixed gold nanoparticles

We have employed mixtures of two differently sized (average diameters: 3.5 and 14 nm) gold nanoparticles (Au NPs) as selective probes and matrices for the determination of aminothiols using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). When using 38 and 150 pM solutions of the 3.5- and 14-nm Au NPs, respectively, as the probe and matrix, SALDI-MS provided limits of detection (signal-to-noise ratio = 3) of 2, 20, and 44 nM for 1.0 mL solutions of glutathione (GSH), cysteine (Cys), and homocysteine, respectively. The signal intensities of these analytes varied by less than 20% for SALDI-MS analyses recorded over 50 sample spots; in contrast, they varied by as much as 60% when using a conventional matrix (2,5-dihydroxybenzoic acid). We validated the practicality of this approach - with its advantages of sensitivity, reproducibility, rapidity, and simplicity - through the analysis of GSH in MCF-7 cell lysates and Cys in plasma.

Nanomaterial Based Affinity Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Biomolecules and Pathogenic Bacteria

This paper describes mass spectrometry (MS) using nanoparticles (NPs) for the analysis of biomolecules such as aminothiol compounds, drugs, peptides, proteins, and bacteria. Papers and patents dealing with preparation and use of several NPs in MS have been briefly reviewed, including carbon nanotubes, gold NPs, and magnetic NPs. The NPs or bioconjugated NPs were used for selective concentration and/or assisted matrices for desorption and ionization of analytes of interest. When compared to conventional organic matrixes, the NPs provide low MS background in low-mass region and low shot-to-shot variation. MS techniques using NPs and bioconjugated NPs for the analysis of disease-associated biomarkers and bacteria in real samples such as blood and urine are highlighted, showing the advantages of high sensitivity, reproducibility, and simplicity.

Migration behavior and separation of s-triazines in micellar electrokinetic capillary chromatography using a cationic surfactant

Abstract The migration behavior and separation of various s-triazines, including five chloro-, three methoxy- and five alkylthio-s-triazines, were investigated in micellar electrokinetic chromatography (MEKC) using a cationic surfactant. In this study, tetradecyltrimethyl ammonium bromide (TTAB) was selected as a cationic surfactant. The results indicate that the selectivity of neutral species of s-triazines in each class is not significantly influenced by buffer pH and micelle concentration, but the overall selectivity is considerably affected by these two separation parameters when charged solutes are present, particularly, at buffer pH below 5.0. Complete separation of thirteen s-triazines was optimally achieved within 6 min on addition of TTAB (15 mM) to a phosphate buffer (70 mM) at pH 4.75 or 3.8. Based on a model that describes the relationship of the effective electrophoretic mobility of a neutral solute and micelle concentration in MEKC, the migration behavior of chloro-s-triazines at pH 6.0 is predicted and the binding constants of s-triazines to TTAB micelles are evaluated. The correlation between the binding constants and Pow (the partition coefficient of a solute between 1-octanol and water) reveals that the migration order of s-triazines in each class is primarily determined by the hydrophobicity of the solutes.

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.

Maximization of injection volumes for DNA analysis in capillary electrophoresis

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris‐borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large‐volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 νm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 νL DNA samples (ϕX 174 RF DNA‐HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/BglI, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400‐fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 νL PCR products amplified after 17 cycles without any sample pretreatment.

Quantitation of branched‐chain amino acids in ascites by capillary electrophoresis with light‐emitting diode‐induced fluorescence detection

Branched‐chain amino acids (BCAAs) are one of the important biomarkers for monitoring liver disease such as hepatitis or hepatoma. In this communication, we present the determination of the concentrations of BCAA in ascites by CE light‐emitted diode‐induced fluorescence (LEDIF) using 1.5% m/v poly(ethylene oxide) (average Mv: ∼8 000 000 g/mol) that was prepared in 10 mM sodium tetraborate solution (pH 9.3). Naphthalene‐2,3‐dicarboxaldehyde was used to derivatize 15 amino acids (AAs) to form naphthalene‐2,3‐dicarboxaldehyde (NDA)‐AA derivatives prior to CE analysis. The separation of 15 NDA‐AA derivatives was accomplished within 15 min, with RSD values of <5.8% (within‐day) and 7.4% (between‐days) with respect to their migration times. The limits of detection for the tested BCAAs ranged from 10.6 to 10.9 nM. We determined the concentrations of three BCAAs – leucine, isoleucine and valine – in ascites by applying a standard addition method, with recovery percentages ranging from 93.9 to 111%. The results obtained from this CE‐LEDIF method is in good agreement with those by a gold standard method using an AA analyzer. We have found that the concentrations of the three BCAAs in ascites obtained from patients suffering from liver diseases were lower than those from healthy individuals. Our approach is highly efficient, sensitive, and cost‐effective, which holds great potential for the diagnosis of liver diseases.