Qingling Li

A New Nanobiosensor for Glucose with High Sensitivity and Selectivity in Serum Based on Fluorescence Resonance Energy Transfer (FRET) between CdTe Quantum Dots and Au Nanoparticles

A novel assembled nanobiosensor QDs-ConA-beta-CDs-AuNPs was designed for the direct determination of glucose in serum with high sensitivity and selectivity. The sensing approach is based on fluorescence resonance energy transfer (FRET) between CdTe quantum dots (QDs) as an energy donor and gold nanoparticles (AuNPs) as an energy acceptor. The specific combination of concanavalin A (ConA)-conjugated QDs and thiolated beta-cyclodextrins (beta-SH-CDs)-modified AuNPs assembles a hyperefficient FRET nanobiosensor. In the presence of glucose, the AuNPs-beta-CDs segment of the nanobiosensor is displaced by glucose which competes with beta-CDs on the binding sites of ConA, resulting in the fluorescence recovery of the quenched QDs. Experimental results show that the increase in fluorescence intensity is proportional to the concentration of glucose within the range of 0.10-50 muM under the optimized experimental conditions. In addition, the nanobiosensor has high sensitivity with a detection limit as low as 50 nM, and has excellent selectivity for glucose over other sugars and most biological species present in serum. The nanobiosensor was applied directly to determine glucose in normal adult human serum, and the recovery and precision of the method were satisfactory. The unique combination of high sensitivity and good selectivity of this biosensor indicates its potential for the clinical determination of glucose directly and simply in serum, and provides the possibility to detect low levels of glucose in single cells or bacterial cultures. Moreover, the designed nanobiosensor achieves direct detection in biological samples, suggesting the use of nanobiotechnology-based assembled sensors for direct analytical applications in vivo or in vitro.

Simultaneous Determination of Superoxide and Hydrogen Peroxide in Macrophage RAW 264.7 Cell Extracts by Microchip Electrophoresis with Laser-Induced Fluorescence Detection

A method for the first time to simultaneously determine superoxide and hydrogen peroxide in macrophage RAW 264.7 cell extracts by microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF) was developed. 2-Chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and bis(p-methylbenzenesulfonyl) dichlorofluorescein (FS), two probes that can be specifically derivatized by superoxide and hydrogen peroxide, respectively, were synthesized and used. Parameters influencing the derivatization and on-chip separation were optimized. With the use of a HEPES (20 mM, pH 7.4) running buffer, a 50 mm long separation channel, and a separation voltage of 1800 V, baseline separation was achieved within 48 s for the two derivatization products, DBZTC-oxide (DBO) and 2,7-dichlorofluorescein (DCF). The linearity ranges of the method were 0.08-5.0 and 0.02-5.0 microM with detection limits (signal-to-noise ratio = 3) of 10 nM (1.36 amol) and 5.6 nM (0.76 amol) for superoxide and hydrogen peroxide, respectively. The relative standard deviations (RSDs) of migration time and peak area were less than 2.0% and 5.0%, respectively. The recoveries of the cell extract samples spiked with 1.0 microM standard solutions were 96.1% and 93.0% for superoxide and hydrogen peroxide, respectively. With the use of this method, superoxide and hydrogen peroxide in phorbol myristate acetate (PMA)-stimulated macrophage RAW 264.7 cell extracts were found to be 0.78 and 1.14 microM, respectively. The method has paved a way for simultaneously determining two or more reactive oxygen species (ROS) in a biological system with high resolution.

Potent method for the simultaneous determination of glutathione and hydrogen peroxide in mitochondrial compartments of apoptotic cells with microchip electrophoresis-laser induced fluorescence.

The first application of microchip electrophoresis with laser-induced fluorescence (MCE-LIF) detection to simultaneously determine glutathione (GSH) and hydrogen peroxide (H(2)O(2)) in mitochondria was described. Organoselenium probe Rh-Se-2 and bis(p-methylbenzenesulfonate)dichlorofluorescein (FS) synthesized in our laboratory were utilized as fluorescent probes for GSH and H(2)O(2), respectively. Rh-Se-2, which is nonfluorescent, reacts with GSH to produce rhodamine 110 (Rh110) with high quantum yield. Similarly, nonfluorescent FS reacts with H(2)O(2) and produces dichlorofluorescein (DCF) accompanied by drastic fluorescence enhancement. Both probes exhibit good sensitivity toward their respective target molecule determination. Fast, simple, and sensitive determination of GSH and H(2)O(2) was realized within 37 s using a running buffer of 50 mM mannitol, 40 mM HEPES (pH 7.4), and an electric field of 360 V/cm for separation. The linear ranges of the method were 3.3 x 10(-9)-1.0 x 10(-7) M/2.9 x 10(-7)-1.0 x 10(-4) M and 2.7 x 10(-9)-4.0 x 10(-7) M with detection limits (signal-to-noise ratio = 3) of 1.3 nM (0.16 amol) and 1.0 nM (0.12 amol) for GSH and H(2)O(2), respectively. The relative standard deviations (RSDs) of migration time and peak area were less than 1.0% and 4.0%, respectively. The MCE-LIF assay was utilized to investigate the levels of GSH and H(2)O(2) in mitochondria isolated from HepG2 cells and were found to be 2.01 +/- 0.21 mM and 5.36 +/- 0.45 microM, respectively. The method was further extended to observe situations of the two species in mitochondria of HepG2 cells experiencing cell apoptosis that were induced by doxorubicin and photodynamic therapy (PDT).

FRET-Based Biofriendly Apo-GOx-Modified Gold Nanoprobe for Specific and Sensitive Glucose Sensing and Cellular Imaging

In this paper, we have developed a biofriendly and high sensitive apo-GOx (inactive form of glucose oxidase)-modified gold nanoprobe for quantitative analysis of glucose and imaging of glucose consumption in living cells. This detection system is based on fluorescence resonance energy transfer between apo-GOx modified AuNPs (Au nanoparticles) and dextran-FITC (dextran labeled with fluorescein isothiocyanate). Once glucose is present, quenched fluorescence of FITC recovers due to the higher affinity of apo-GOx for glucose over dextran. The nanoprobe shows excellent selectivity toward glucose over other monosaccharides and most biological species present in living cells. A detection limit as low as 5 nM demonstrates the high sensitivity of the nanoprobe. Introduction of apo-GOx, instead of GOx, can avoid the consumption of O2 and production of H2O2 during the interaction with glucose, which may exert effects on normal physiological events in living cells and even lead to cellular damage. Due to the low toxicity of this detection system and reliable cellular uptake ability of AuNPs, imaging of intracellular glucose consumption was successfully realized in cancer cells.

Simultaneous Quantitation of Na+ and K+ in Single Normal and Cancer Cells Using a New Near-Infrared Fluorescent Probe

Considering the important functions of cellular Na(+) and K(+) together with their cooperative efforts on various biological processes, it is significant to simultaneously detect Na(+) and K(+) at a single-cell level. Here, we present a novel method to discriminate and quantify simultaneously Na(+) and K(+) in single cells using a new near-infrared fluorescent probe associated with microchip electrophoresis. The fluorescent probe selectively responds to both Na(+) and K(+). The microchip electrophoresis allows accurate single-cell manipulation and effective distinction of Na(+) and K(+). Based on the method, the concentration of Na(+) and K(+) in single normal and cancer cells was compared, and the variation of Na(+) and K(+) in single cancer cells during the early stage of apoptotic volume decrease was monitored, which would help us to better understand the critical roles of Na(+) and K(+) in malignant cells and apoptosis. This method has paved a new way for the research of the synergistic function of Na(+) and K(+) in the regulation of various biological processes at a single-cell level.

Electrokinetic gated injection-based microfluidic system for quantitative analysis of hydrogen peroxide in individual HepG2 cells.

A microfluidic system to determine hydrogen peroxide (H(2)O(2)) in individual HepG2 cells based on the electrokinetic gated injection was developed for the first time. A home-synthesized fluorescent probe, bis(p-methylbenzenesulfonate)dichlorofluorescein (FS), was employed to label intracellular H(2)O(2) in the intact cells. On a simple cross microchip, multiple single-cell operations, including single cell injection, cytolysis, electrophoresis separation and detection of H(2)O(2), were automatically carried out within 60 s using the electrokinetic gated injection and laser-induced fluorescence detection (LIFD). The performance of the method was evaluated under the optimal conditions. The linear calibration curve was over a range of 4.39-610 amol (R(2)=0.9994). The detection limit was 0.55 amol or 9.0×10(-10) M (S/N=3). The relative standard deviations (RSDs, n=6) of migration time and peak area were 1.4% and 4.8%, respectively. With the use of this method, the average content of H(2)O(2) in single HepG2 cells was found to be 16.09±9.84 amol (n=15). Separation efficiencies in excess of 17,000 theoretical plates for the cells were achieved. These results demonstrated that the efficient integration and automation of these single-cell operations enabled the sensitive, reproducible, and quantitative examination of intracellular H(2)O(2) at single-cell level. Owing to the advantages of simple microchip structure, controllable single-cell manipulation and ease in building, this platform provides a universal way to automatically determine other intracellular constituents within single cells.

Reduction of the impedance of a contactless conductivity detector for microchip capillary electrophoresis: compensation of the electrode impedance by addition of a series inductance from a piezoelectric quartz crystal.

A low-impedance capacitively coupled contactless conductivity detector (LIC (4)D) for microchip capillary electrophoresis was reported. The LIC (4)D was the series combination of a piezoelectric quartz crystal (PQC) resonator with a capacitively coupled contactless conductivity detector (C (4)D) outside on the microchip lid. The electrode impedance in the LIC (4)D was reduced because the capacitive impedance from the wall capacitance was compensated by the inductive impedance from the PQC. The operation frequency of the LIC (4)D was set at the resonant frequency of the series combination of a PQC with a C (4)D, wherein a minimum in the total impedance was obtained. It was shown that the sensitivity of LIC (4)D was much higher than that of C (4)D itself, especially in the microchip with a thick lid. Under the experimental conditions, the signal-to-noise ratios of the LIC (4)D were improved by approximately 20-50 times over those of the C (4)D. Reproducible separations of a mixture of inorganic cations (K (+), Na (+), Li (+)) were demonstrated. After a digital filter treatment by the fast Fourier transform algorithm, the detection limits were 0.38, 0.49, and 1.6 microM for K (+) in the LI C (4)D with the microchip lid thickness of 0.20, 0.40, and 1.0 mm, respectively.

Consecutive Gated Injection-Based Microchip Electrophoresis for Simultaneous Quantitation of Superoxide Anion and Nitric Oxide in Single PC-12 Cells.

As important reactive oxygen species (ROS) and reactive nitrogen species (RNS), cellular superoxide anion (O2(•-)) and nitric oxide (NO) play significant roles in numerous physiological and pathological processes. Cellular O2(•-) and NO also have a close relationship and always interact with each other. Thus, the simultaneous detection of intracellular O2(•-) and NO, especially at the single-cell level, is important. In this paper, we present a novel method to simultaneously detect and quantify O2(•-) and NO in single cells using microchip electrophoresis based on a new consecutive gated injection method. This novel injection method achieved consecutive manipulation of single cells, guaranteeing an almost constant volumetric flow rate and thus good quantitative reproducibility. After cellular content separation by microchip electrophoresis and detection by laser-induced fluorescence (MCE-LIF), O2(•-) and NO in single PC-12 cells were simultaneously quantified in an automated fashion. This is the first report of consecutive absolute quantitation at the single-cell level. The quantitative results obtained from single cells is beneficial for deep understanding of the biological roles of cellular O2(•-) and NO. This new method constitutes a consecutive, accurate way to study the synergistic function of O2(•-) and NO and other biomolecules in various biological events at the single-cell level.

Multicolor Fluorescence Detection-Based Microfluidic Device for Single-Cell Metabolomics: Simultaneous Quantitation of Multiple Small Molecules in Primary Liver Cells

Single-cell metabolomics can be used to study cell diversity and how cells respond to environment. There is an urgent need to develop effective detection methods for single-cell metabolomics. Microchip electrophoresis with laser-induced fluorescence detection (MCE-LIFD) is a powerful tool to detect metabolites at the single-cell level. However, the existing one-laser excitation and one-color fluorescence collection in MCE-LIFD is not sufficient for the simultaneous detection of multiple small molecules with wide variations in their fluorescence excitation and emission spectra. In this manuscript, we describe a multicolor fluorescence detection-based microfluidic device (MFD-MD) for single-cell metabolomics research. We selected primary liver cells from acute ethanol-stimulated mice as the model cells and hydrogen peroxide (H2O2), glutathione (GSH), and cysteine (Cys) as representative small-molecule metabolites for single-cell analysis. The microfluidic chip enabled accurate single-cell manipulation and effective electrophoresis separation. The new multicolor fluorescence detection permitted simultaneous analysis of H2O2, GSH, and Cys. Ethanol exposure induced an increase in H2O2 and a decrease in GSH and Cys. Obvious cell heterogeneity was observed. These results provide insights regarding the intracellular oxidative/antioxidative molecular mechanism in response to external stimuli. The MFD-MD provides a new opportunity for simultaneous single-cell analysis of multiple metabolites.

A new route for simple and rapid determination of hydrogen peroxide in RAW264.7 macrophages by microchip electrophoresis

A new method using MCE with LIF detection was developed for the determination of hydrogen peroxide (H2O2). Bis(p‐methylbenzenesulfonyl)dichlorofluorescein, a new fluorogenic reagent synthesized by our laboratory was employed as a labeling reagent, the derivatization reaction was performed in 0.10 M HEPES buffer (pH 7.4) for 30 min at 37°C. The detection of H2O2 was accomplished in 55 s, using a 40 mM HEPES buffer, 20% mannitol, pH 7.4, on a glass microchip. The RSDs of migration time and peak area were 1.8 and 3.7%, respectively. Method validation showed the linear response ranging from 0.50 to 50 μM with an LOD (S/N=3) of 0.20 μM (19.1 amol). The proposed method was applied to determine H2O2 in phorbol myristate acetate‐stimulated RAW264.7 macrophages, the concentration of H2O2 was found to be 1.86±0.05 μM; recoveries for macrophage samples were from 96.7 to 97.8%, within‐days and between‐days accuracies were 4.5% (n=5) and 6.8% (n=5), respectively.