Application of polymer quantum dot-enzyme hybrids in the biosensor development and test paper fabrication.

Both glutathione capped CdTe quantum dots (QDs) and enzymes were encapsulated with poly(diallyldimethylammonium chloride) (PDDA) via electrostatic attraction to form hybrid films. The obtained PDDA QD-enzyme hybrids feature both high fluorescence and biorecognition. In the obtained hybrid materials, the fluorescence emission of the QDs was stable for at least 3 months, and the structure and activity of the enzyme was also well maintained as the Michaelis constant of tyrosinase was determined to be 0.90 mmol/L, which is just 2 times higher than that of free enzyme. This hybrid material was then utilized as a platform for the development of biosensors based on the quenching effects of the enzymatic products on the emission of the QDs with a kind of phenol (catechol) and glucose as example analytes. The detection limits of catechol and glucose were 1.0 × 10(-5) and 5.0 × 10(-6) mol/L, respectively. Moreover, this hybrid material was applied to the fabrication of test paper for these two analytes. The test paper was very stable with respect to the fluorescence of the QDs and the activity of the enzyme maintained for at least 1 month.

Organic-soluble fluorescent Au8 clusters generated from heterophase ligand-exchange induced etching of gold nanoparticles and their electrochemiluminescence.

A unique heterophase ligand exchange induced etching process was used to transform gold nanoparticles into organic-soluble fluorescent gold clusters which were assigned to Au(8) by optical spectroscopy and MALDI-TOF mass spectrometry. Both the annihilation electrochemiluminescence of fluorescent Au(8) clusters in organic solution and the coreactant electrochemiluminescence of Au(8) cluster film in aqueous solution were studied.

Oligonucleotide stabilized silver nanoclusters as fluorescence probe for drug-DNA interaction investigation.

In this work, oligonucleotide stabilized silver nanoclusters as novel fluorescent probes were successfully utilized for the drug-DNA interaction study. Silver nanoclusters were proved to be sensitive probes for the drugs investigated (including of two kinds of intercalators, daunorubicin and quinacrine, as well as a non-intercalating binder bisBenzimide H 33258), as the detection limits at 10(-8) mol L(-1) level of studied drugs can be achieved. The interactions of drugs and calf thymus DNA were investigated using non-linear fit analysis, and the binding constants as well as binding site sizes were obtained. As biocompatible materials, silver nanoclusters are promising in the chemical especially biochemical analysis fields.

Strand exchange reaction modulated fluorescence "off-on" switching of hybridized DNA duplex stabilized silver nanoclusters.

The fluorescence (FL)"off-on" switching of designed DNA duplex stabilized silver nanoclusters can be accomplished through the control of DNA strand exchange reaction. The successful sequential control of the FL emission of silver nanoclusters in "off-on" switching cycles confirms that the DNA duplex stabilized silver nanoclusters can work as a new kind of DNA FL switch.

Highly sequence-dependent formation of fluorescent silver nanoclusters in hybridized DNA duplexes for single nucleotide mutation identification.

Fluorescent silver nanoclusters were successfully synthesized using hybridized DNA duplexes as capping scaffolds. The formation of these emitters was highly sequence-dependent and could specifically identify a single nucleotide mutation, the sickle cell anemia gene mutation. Furthermore, the identification of single-nucleotide differences using this strategy was extended to more general types of single-nucleotide mismatches.

Oligonucleotide-stabilized Ag nanoclusters as novel fluorescence probes for the highly selective and sensitive detection of the Hg2+ ion.

Fluorescent oligonucleotide-stabilized Ag nanoclusters are demonstrated as novel and environmentally-friendly fluorescence probes for the determination of Hg(2+) ions with a low detection limit and high selectivity.

Sensitive and selective sensor for biothiols in the cell based on the recovered fluorescence of the CdTe quantum dots-Hg(II) system.

Herein, a sensitive and selective sensor for biothiols based on the recovered fluorescence of the CdTe quantum dots (QDs)-Hg(II) system is reported. Fluorescence of QDs could be quenched greatly by Hg(II). In the presence of biothiols, such as glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), however, Hg(II) preferred to react with them to form the Hg(II)-S bond because of the strong affinity with the thiols of biothiols rather than quenching the fluorescence of the QDs. Thus, the fluorescence of CdTe QDs was recovered. The restoration ability followed the order GSH > Hcy > Cys due to the decreased steric hindrance effect. A good linear relationship was obtained from 0.6 to 20.0 micromol L(-1) for GSH and from 2.0 to 20.0 micromol L(-1) for Cys, respectively. The detection limits of GSH and Cys were 0.1 and 0.6 micromol L(-1), respectively. In addition, the method showed a high selectivity for Cys among the other 19 amino acids. Furthermore, it succeeded in detecting biothiols in the Hela cell.

Anticancer drug-DNA interactions measured using a photoinduced electron-transfer mechanism based on luminescent quantum dots.

A sensing system based on the photoinduced electron transfer of quantum dots (QDs) was designed to measure the interaction of anticancer drug and DNA, taking mitoxantrone (MTX) as a model drug. MTX adsorbed on the surface of QDs can quench the photoluminescence (PL) of QDs through the photoinduced electron-transfer process; and then the addition of DNA will bring the restoration of QDs PL intensity, as DNA can bind with MTX and remove it from QDs. Sensitive detection of MTX with the detection limit of 10 nmol L(-1) and a linear detection range from 10 nmol L(-1) to 4.5 micromol L(-1) was achieved. The dependence of PL intensity on DNA amount was successfully utilized to investigate the interactions between MTX and DNA. Both the binding constants and the sizes of binding site of MTX-DNA interactions were calculated based on the equations deduced for the PL recovery process. The binding constant obtained in our experiment was generally consistent with previous reports. The sensitive and speedy detection of MTX as well as the avoidance of modification or immobilization process made this system suitable and promising in the drug-DNA interaction studies.

Glutathione-capped CdTe quantum dots for the sensitive detection of glucose.

A simple and sensitive assay system for glucose based on the glutathione (GSH)-capped CdTe quantum dots (QDs) was developed. GSH-capped CdTe QDs exhibit higher sensitivity to H(2)O(2) produced from the glucose oxidase catalyzed oxidation of glucose, and are also more biocompatible than other thiols-capped QDs. Based on the quenching of H(2)O(2) on GSH-capped QDs, glucose can be detected. The detection conditions containing reaction time, the concentration of glucose oxidase and the sizes of QDs were optimized and the detection limits for glucose was determined to be 0.1 microM; two detection ranges of glucose from 1.0 microM to 0.5mM and from 1.0 mM to 20 mM, respectively were obtained. The detection limit was almost a 1000 times lower than other QDs-based optical glucose sensing systems. The developed glucose detection system was simple and facile with no need of complicated enzyme immobilization and modification of QDs.

Investigation of some critical parameters of buffer conditions for the development of quantum dots-based optical sensors.

The unique surface-sensitive properties make quantum dots (QDs) great potential in the development of sensors for various analytes. However, quantum dots are not only sensitive to a certain analyte, but also to the surrounding conditions. The controlled response to analyte may be the first step in the designing of functional quantum dots sensors. In this study, taking the quenching effect of benzoquinone (BQ) on CdTe QDs as model, several critical parameters of buffer solution conditions with potential effect on the sensors were investigated. The pH value and the concentration of sodium citrate in the buffer solution critically influenced the quenching effects of BQ. Dozens folds elevation of the quenching extents were observed with the increase of concentrations of H(+) and sodium citrate, and the quenching mechanisms were also fundamentally different with the changes of the surrounding buffer solutions. The quenching models were proposed and analyzed at different buffer conditions. Taking pH values for example, QDs quenching obeyed the sphere of effective quenching model with the sphere radii of 8.29 nm at pH 8.0, the linear Stern-Volmer equation with Stern-Volmer constant of 2.0 x 10(3)mol(-1)L at pH 7.0, and the two binding site static quenching model at basic conditions. The elucidation of parameters for assay performance was important in the development of QDs-based optical sensors.

Nanoscale-enhanced Ru(bpy)3(2+) electrochemiluminescence labels and related aptamer-based biosensing system.

A unique multilabeling at a single-site protocol of the Ru(bpy)(3)(2+) electrochemiluminescence (ECL) system is proposed. Nanoparticles (NPs) were used as assembly substrates to enrich ECL co-reactants of Ru(bpy)(3)(2+) to construct nanoscale-enhanced ECL labels. Two different kinds of NP substrates [including semiconductor NPs (CdTe) and noble metal NPs (gold)] capped with 2-(dimethylamino)ethanethiol (DMAET) [a tertiary amine derivative which is believed to be one of the most efficient of co-reactants of the Ru(bpy)(3)(2+) system] were synthesized through a simple one-pot synthesis method in aqueous media. Although both CdTe and gold NPs realized the enrichment of ECL co-reactants, they presented entirely different ECL performances as nanoscale ECL co-reactants of Ru(bpy)(3)(2+). The different effects of these two NPs on the ECL of Ru(bpy)(3)(2+) were studied. DMAET-capped CdTe NPs showed enormous signal amplification of Ru(bpy)(3)(2+) ECL, whereas DMAET-capped gold NPs showed a slight quenching effect of the ECL signal. DMAET-capped CdTe NPs can be considered to be excellent nanoscale ECL labels of the Ru(bpy)(3)(2+) system, as even a NP solution sample of 10(-18) M was still detectable after an electrostatic self-assembly concentration process. DMAET-capped CdTe NPs were further applied in the construction of aptamer-based biosensing system for proteins and encouraging results were obtained.

Kinetic study of prolidase activity in erythrocytes against different substrates using capillary electrophoresis with electrochemiluminescence detection.

Capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection was used to explore the kinetics of the enzymatic reaction. The different effects of reaction conditions including the concentration of Mn(2+), incubation temperature and pH on prolidase (PLD, EC 3.4.13.9) activity in erythrocyte lysates against three different substrates, Gly-Pro, Val-Pro and Leu-Pro were investigated. Also, the effects of colchicine which can prevent or delay cancer of liver on the PLD activity were studied. For Gly-Pro and Leu-Pro, colchicine enhanced PLD activity, while for the other substrate Val-Pro, it had a slight inhibiting effect on prolidase activity. Kinetic study of colchicine on prolidase activity in erythrocytes indicated different effects for different substrates with the addition of colchicine. Compared with some other methods, CE coupled with electrochemiluminescence detection showed its merits of simplicity and efficiency on the bioanalysis.

Quantum dots-bienzyme hybrid system for the sensitive determination of glucose.

In this paper, we attempt to develop a sensitive detection method for glucose with the combination of the unique optical property of quantum dots and the specificity of enzymatic reactions. With glucose and hydroquinone as substrates, benzoquinone that intensively quenches the photoluminescence of quantum dots can be produced via the catalysis of bienzyme (glucose oxidase and horseradish peroxidase) system. A relatively low detection limit of 1.0x10(-8)mol/L can be achieved. Two linear ranges from 1.0x10(-6) to 1.5x10(-4)M and from 1.5x10(-4) to 1.0x10(-3)M were obtained. For the detection of 1.0x10(-4)M glucose, six replicative measurements showed the reproducibility (R.S.D.) of 4.43%. The quantum dots-enzymes system possesses advantages of simple procedure without modification of quantum dots or immobilization of enzymes, low cost, high sensitivity and short detection times within several minutes.

Utilizing a CdTe quantum dots-enzyme hybrid system for the determination of both phenolic compounds and hydrogen peroxide.

In this paper, we attempt to construct a simple and sensitive detection method for both phenolic compounds and hydrogen peroxide, with the successful combination of the unique property of quantum dots and the specificity of enzymatic reactions. In the presence of H2O2 and horseradish peroxidase, phenolic compounds can quench quantum dots' photoluminescence efficiently, and the extent of quenching is severalfold to more than 100-fold increase. Quinone intermediates produced from the enzymatic catalyzed oxidation of phenolic compounds were believed to play the main role in the photoluminescence quenching. Using a quantum dots-enzyme system, the detection limits for phenolic compounds and hydrogen peroxide were detected to be approximately 10(-7) mol L(-1). The coupling of efficient quenching of quantum dot photoluminescence by quinone and the effective enzymatic reactions make this a simple and sensitive method for phenolic compound detection and great potential in the development of H2O2 biosensors for various analytes.

Characterization of procaine metabolism as probe for the butyrylcholinesterase enzyme investigation by simultaneous determination of procaine and its metabolite using capillary electrophoresis with electrochemiluminescence detection.

Capillary electrophoresis with electrochemiluminescene detection was used to characterize procaine hydrolysis as a probe for butyrylcholinesterase by in vitro procaine metabolism in plasma with butyrylcholinesterase acting as bioscavenger. Procaine and its metabolite N,N-diethylethanolamine were separated at 16 kV and then detected at 1.25 V in the presence of 5.0 mM Ru(bpy)(3)2+, with the detection limits of 2.4x10(-7) and 2.0x10(-8) mol/L (S/N=3), respectively. The Michaelis constant Km value was 1.73x10(-4) mol/L and the maximum velocity Vmax was 1.62x10(-6) mol/L/min. Acetylcholine bromide and choline chloride presented inhibition effects on the enzymatic cleavage of procaine, with the 50% inhibition concentration (IC50) of 6.24x10(-3) and 2.94x10(-4) mol/L.

Capillary electrophoresis with electrochemiluminescence detection for measurement of aspartate aminotransferase and alanine aminotransferase activities in biofluids.

A new sensitive assay for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in biofluids was developed, based on the separation and detection of alanine, glutamate, and aspartate using capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. The three amino acids were separated in 5 mM phosphate of pH 2.1 as background electrolyte, and detected on a 500 microm platinum disk electrode at 1.2V (versus Ag/AgCl) in the presence of 10 mM tris(2,2'-bipyridyl)ruthenium(II) dissolved in 80 mM phosphate of pH 10.5. A mass detection limit of 37.3 fmol (or 81.5 fmol) for glutamate, corresponding to the product in the enzyme reaction catalyzed by 1.24 x 10(-9)U AST (or 2.72 x 10(-9)U ALT) in a 30 min reaction period, was achieved. This assay was applied to investigate the cytotoxicity effect of ethanol on HepG2 cells and differentiating nonalcoholic steatohepatitis (NASH) from alcoholic liver disease, indicating that the technique is promising for the application in the cell biological and clinical fields.

Kinetic study of paracetamol on prolidase activity in erythrocytes by capillary electrophoresis with Ru(bpy)(3) (2+) electrochemiluminescence detection.

We explored the CE with (bpy) (2+) (3) electrochemiluminescence detection for the kinetic study of drug-enzyme interaction. Effects of four nonsteroidal anti-inflammatory drugs including aspirin, paracetamol, sodium salicylate and phenacetin on prolidase (PLD) activity in erythrocytes were investigated. Aspirin enhanced PLD activity whereas the other three had inhibiting effects. This may reveal their different effects on the collagen biosynthesis and catabolism that influence tumor invasiveness. Kinetic study of paracetamol on PLD showed that the value of Michaelis constant K(m) for PLD was 1.23 mM. The mechanism of PLD inhibition by paracetamol is noncompetitive inhibition, and the inhibitor constant K(i) value obtained in our research was 9.73 x 10(3) microg/L.

Characterization of prolidase activity using capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection and application to evaluate collagen degradation in diabetes mellitus.

A new method for prolidase (PLD, EC 3.4.13.9) activity assay was developed based on the determination of proline produced from enzymatic reaction through capillary electrophoresis (CE) with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)3(2+)] electrochemiluminescence detection (ECL). A detection limit of 12.2 fmol (S/N = 3) for proline, corresponding to 1.22 x 10(-8) units of prolidase catalyzing for 1 min was achieved. PLD activity determined by CE-ECL method was in agreement with that obtained from the classical Chinard's one. CE-ECL showed its powerful resolving ability and selectivity as no sample pretreatment was needed and no interference existed. The clinical utility of this method was successfully demonstrated by its application to assay PLD activity in the serum of diabetic patients in order to evaluate collagen degradation in diabetes mellitus (DM). The results indicated that enhanced collagen degradation occurred in DM.