Ming Shi

Chemiluminescence resonance energy transfer-based detection for microchip electrophoresis.

Since the channels in micro- and nanofluidic devices are extremely small, a sensitive detection is required following microchip electrophoresis (MCE). This work describes a highly sensitive and yet universal detection scheme based on chemiluminescence resonance energy transfer (CRET) for MCE. It was found that an efficient CRET occurred between a luminol donor and a CdTe quantum dot (QD) acceptor in the luminol-NaBrO-QD system and that it was sensitively suppressed by the presence of certain organic compounds of biological interest including biogenic amines and thiols, amino acids, organic acids, and steroids. These findings allowed developing sensitive MCE-CL assays for the tested compounds. The proposed MCE-CL methods showed desired analytical figures of merit such as a wide concentration range of linear response. Detection limits obtained were approximately 10(-9) M for biogenic amines including dopamine and epinephrine and approximately 10(-8) M for biogenic thiols (e.g., glutathione and acetylcysteine), organic acids (i.e., ascorbic acid and uric acid), estrogens, and native amino acids. These were 10-1000 times more sensitive than those of previously reported MCE-based methods with chemiluminescence, electrochemical, or laser-induced fluorescence detection for quantifying corresponding compounds. To evaluate the applicability of the present MCE-CL method for analyzing real biological samples, it was used to determine amino acids in individual human red blood cells. Nine amino acids, including Lys, Ser, Ala, Glu, Trp, etc., were detected. The contents ranged from 3 to 31 amol/cell. The assay proved to be simple, quick, reproducible, and very sensitive.

Quantification of biogenic amines by microchip electrophoresis with chemiluminescence detection

A highly sensitive microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of biogenic amines including agmatine (Agm), epinephrine (E), dopamine (DA), tyramine, and histamine in human urine samples. To achieve a high assay sensitivity, the targeted analytes were pre-column labeled by a CL tagging reagent, N-(4-aminobutyl)-N-ethylisoluminol (ABEI). ABEI-tagged biogenic amines after MCE separation reacted with hydrogen peroxide in the presence of horseradish peroxidase (HRP), producing CL emission. Since no CL reagent was added to the running buffer, the background of the CL detection was extremely low, resulting in a significant improvement in detection sensitivity. Detection limits (S/N=3) were in the range from 5.9x10(-8) to 7.7x10(-8) M for the biogenic amines tested, which were at least 10 times lower than those of the MCE-CL methods previously reported. Separation of a urine sample on a 7 cm glass/poly(dimethylsiloxane) (PDMS) microchip channel was completed within 3 min. Analysis of human urine samples found that the levels of Agm, E and DA were in the ranges of 2.61x10(-7) to 4.30x10(-7) M, 0.81x10(-7) to 1.12x10(-7) M, and 8.76x10(-7) to 11.21x10(-7) M (n=4), respectively.

Intermolecular and Intramolecular Quencher Based Quantum Dot Nanoprobes for Multiplexed Detection of Endonuclease Activity and Inhibition

DNA cleavage by endonucleases plays an important role in many biological events such as DNA replication, recombination, and repair and is used as a powerful tool in medicinal chemistry. However, conventional methods for assaying endonuclease activity and inhibition by gel electrophoresis and chromatography techniques are time-consuming, laborious, not sensitive, or costly. Herein, we combine the high specificity of DNA cleavage reactions with the benefits of quantum dots (QDs) and ultrahigh quenching abilities of inter- and intramolecular quenchers to develop highly sensitive and specific nanoprobes for multiplexed detection of endonucleases. The nanoprobe was prepared by conjugating two sets of DNA substrates carrying quenchers onto the surface of aminated QDs through direct assembly and DNA hybridization. With this new design, the background fluorescence was significantly suppressed by introducing inter- and intramolecular quenchers. When these nanoprobes are exposed to the targeted endonucleases, specific DNA cleavages occur and pieces of DNA fragments are released from the QD surface along with the quenchers, resulting in fluorescence recovery. The endonuclease activity was quantified by monitoring the change in the fluorescence intensity. The detection was accomplished with a single excitation light. Multiplexed detection was demonstrated by simultaneously assaying EcoRI and BamHI (as model analytes) using two different emissions of QDs. The limits of detection were 4.0 × 10(-4) U/mL for EcoRI and 8.0 × 10(-4) U/mL for BamHI, which were at least 100 times more sensitive than traditional gel electrophoresis and chromatography assays. Moreover, the potential application of the proposed method for screening endonuclease inhibitors has also been demonstrated. The assay protocol presented here proved to be simple, sensitive, effective, and easy to carry out.

A gold nanoparticle-enhanced fluorescence polarization biosensor for amplified detection of T4 polynucleotide kinase activity and inhibition

A novel fluorescence polarization (FP) nanosensor based on λ exonuclease cleavage reaction and FP enhancement effect of gold nanoparticles (AuNPs) has been developed for the detection of T4 polynucleotide kinase activity and inhibition. This FP sensor exhibits higher detection sensitivity over traditional fluorescence sensors by two orders of magnitude.

Carbon nanotube signal amplification for ultrasensitive fluorescence polarization detection of DNA methyltransferase activity and inhibition.

A versatile sensing platform based on multiwalled carbon nanotube (MWCNT) signal amplification and fluorescence polarization (FP) is developed for the simple and ultrasensitive monitoring of DNA methyltransferase (MTase) activity and inhibition in homogeneous solution. This method uses a dye-labeled DNA probe that possess a doubled-stranded DNA (dsDNA) part for Mtase and its corresponding restriction endonuclease recognition, and a single-stranded DNA part for binding MWCNTs. In the absence of MTase, the dye-labeled DNA is cleaved by restriction endonuclease, and releases very short DNA carrying the dye that cannot bind to MWCNTs, which has relatively small FP value. However, in the presence of MTase, the specific recognition sequence in the dye-labeled DNA probe is methylated and not cleaved by restriction endonuclease. Thus, the dye-labeled methylated DNA product is adsorbed onto MWCNTs via strong π-π stacking interactions, which leads to a significant increase in the FP value due to the enlargement of the molecular volume of the dye-labeled methylated DNA/MWCNTs complex. This provides the basic of a quantitative measurement of MTase activity. By using the MWCNT signal amplification approach, the detection sensitivity can be significantly improved by two orders of magnitude over the previously reported methods. Moreover, this method also has high specificity and a wide dynamic range of over five orders of magnitude. Additionally, the suitability of this sensing platform for MTase inhibitor screening has also been demonstrated. This approach may serve as a general detection platform for sensitive assay of a variety of DNA MTases and screening potential drugs.

A Nonenzymatic Chemiluminescent Reaction Enabling Chemiluminescence Resonance Energy Transfer to Quantum Dots

Resonance energy transfer (RET)-based measurement approaches are very useful for innovative studies such as that of protein-protein interactions in living cells with spatial and /or temporal resolution [1]. RET involves non-radiative (dipole-dipole) energy transfer between a donor and an acceptor that are in close proximity (normally <10 nm). RET that occurs between two fluorophores is known as fluorescence RET (FRET), whereas RET that occurs between a light-emitting donor enzyme (e.g. luciferase) and a fluorophore is known as bioluminescence RET (BRET). Numerous publications have been seen on FRET being used in various areas such as structural elucidation of biological macromolecules, their interactions, in vitro assays, in vivo monitoring, and signal transduction in living cells [2, 3]. BRET is also well documented as a technique useful for these studies [4, 5]. Major disadvantages of BRET include the requirement of at least one carefully designed protein fusion and the low emission intensity that compromises the spatial and /or temporal resolutions in BRET measurements. Chemiluminescence RET (CRET) involves nonradiative transfer of energy from a chemiluminescent (CL) donor (instead of a bioluminescent enzyme donor as in BRET) to a fluorophore acceptor [6]. CRET occurs by the oxidation of a CL compound that then excites the fluorescent acceptor. Since no external light source is used for excitation in CRET approaches, nonspecific signals caused by external light excitation as often observed in FRET measurements can be minimized. Compared with BRET, a CRET-based approach involves no protein fusion. Both the CL donor and fluorescent acceptor can be conjugated to antibodies, promising a widespread application. However, little study has been so far reported on CRET [6-8]. A major difficulty is to identify an effective CL donor or reaction that can excite a fluorescent acceptor by energy transfer. In all of the previous CRET works reported, the luminol-H2O2 CL reaction catalyzed by horseradish peroxidase (HRP) was used. Unfortunately, involving an exogenous enzyme (i.e. HRP) limits the applicability of the CRET system. In many cases, it complicates the assay by, for example, disturbing the biological interactions under study.

Quantification of D‐Asp and D‐Glu in rat brain and human cerebrospinal fluid by microchip electrophoresis

A microchip electrophoresis (MCE) method with LIF detection was presented for quantification of D-aspartic acid (D-Asp) and D-glutamate (D-Glu) in biological samples. D-Asp and D-Glu were determined after precolumn derivatization with FITC. The chiral separation was performed on a glass/PDMS hybrid microfluidic chip using gamma-CD as chiral selector in the running buffer. High sensitive detection was obtained by the LIF detection. The LODs (S/N = 3) for D-Asp and D-Glu were 6.0x10(-8) and 4.0x10(-8) M, respectively. Using this method, the levels of D-Asp and D-Glu in rat brain and human cerebrospinal fluid (CSF) were determined.

Noncompetitive immunoassay for carcinoembryonic antigen in human serum by microchip electrophoresis for cancer diagnosis

BACKGROUND Carcinoembryonic antigen (CEA) as one of the most widely used tumor markers is used in the clinical diagnosis of colorectal, pancreatic, gastric, and cervical carcinomas. We describe a microchip electrophoresis (MCE)-based noncompetitive immunoassay technique for assaying CEA in human serum for cancer diagnosis. METHODS Based on a noncompetitive immunoassay format, CEA reacts first with an excess amount of monoclonal antibody (Ab(1)), then free Ab(1) and the bound CEA-Ab(1) complex react with an excess amount of fluorescein isothiocyanate (FITC)-labeled secondary antibody (Ab(2)*) to form CEA-Ab(1)-Ab(2)* and Ab(1)-Ab(2)* complexes. Finally, the free Ab(2)* and bound CEA-Ab(1)-Ab(2)*, Ab(1)-Ab(2)* complexes were separated and detected by MCE coupling laser-induced fluorescence (LIF), and CEA was quantified by measuring the fluorescence intensity of CEA-Ab(1)-Ab(2)*. RESULTS The linear range for CEA was 60pg/ml-8ng/ml with a correlation coefficient of 0.9992 and a detection limit of 45.7pg/ml. The immunocomplex including CEA-Ab(1)-Ab(2)*, Ab(1)-Ab(2)*and free Ab(2)* was separated within 80s. The amounts of CEA in normal person serum were found to be in the range of 2.9-5.1microg/l, and seven cancer patients serum were analyzed, and CEA levels range from 79.6-270.1ng/ml. CONCLUSIONS This method may become a useful tool for rapid analysis of CEA and other tumor markers in biomedical analysis and clinical diagnosis.

Chemiluminescent immunoassay of thyroxine enhanced by microchip electrophoresis.

A homogeneous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophoresis separation has been developed. The method deployed the competitive immunoreaction of T4 and horseradish peroxidase (HRP)-labeled T4 (HRP-T4) with anti-T4 mouse monoclonal antibody (Ab). HRP-T4 and the HRP-T4-Ab complex were separated and quantified by using microchip electrophoresis (MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol-H(2)O(2) reaction. Due to the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. Under the selected assay conditions, the MCE separation was accomplished within 60s. The linear range for T4 was 5-250 nM with a detection limit of 2.2 nM (signal/noise ratio=3). The current method was successfully applied for the quantification of T4 in human serum samples. It was demonstrated that the current MCE-CL-enhanced competitive immunoassay was quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of T4 to assist in the diagnosis of thyroid gland functions.

Quantification of carnosine- related peptides by microchip electrophoresis with chemiluminescence detection

A microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of carnosine-related peptides, including carnosine, homocarnosine, and anserine, in biological samples. A simple integrated MCE-CL system was built to perform the assays. The highly sensitive CL detection was achieved by means of the CL reaction between hydrogen peroxide and N-(4-aminobutyl)-N-ethylisoluminol-tagged peptides in the presence of adenine as a CL enhancer and Co(2+) as a catalyst. Experimental conditions for analyte labeling, MCE separation, and CL detection were studied. MCE separation of the above-mentioned three peptides took less than 120 s. Detection limits (signal/noise ratio [S/N]=3) of 3.0x10(-8), 2.8x10(-8), and 3.4x10(-8) M were obtained for carnosine, anserine, and homocarnosine, respectively. The current MCE-CL method was applied for the determination of carnosine, anserine, and homocarnosine in human cerebrospinal fluid (CSF) and canine plasma. Homocarnosine was detected at the micromolar (microM) level in the CSF samples analyzed, whereas the levels of carnosine and anserine in these samples were below the detection limit of the assay. Interestingly, both carnosine and anserine were detected in the canine plasma samples, whereas homocarnosine was not.