Automated purification of dye terminator sequencing reactions: an approach to high-throughput capillary electrophoresis sequencing of large templates

Demands for higher quantity and quality of sequence data during genome sequencing projects have led to a need for completely automated reagent systems designed to isolate, process, and analyze DNA samples. While much attention has been given to methodologies aimed at increasing the throughput of sample preparation and reaction setup, purification of the products of sequencing reactions has received less scrutiny despite the profound influence that purification has on sequence quality. Commonly used and commercially available sequencing reaction cleanup methods are not optimal for purifying sequencing reactions generated from larger templates, including bacterial artificial chromosomes (BACs) and those generated by rolling circle amplification. Theoretically, these methods would not remove the original template since they only exclude small molecules and retain large molecules in the sample. If the large template remains in the purified sample, it could understandably interfere with electrokinetic injection and capillary performance. We demonstrate that the use of MagneSil^® paramagnetic particles (PMPs) to purify ABI PRISM^® BigDye^® sequencing reactions increases the quality and read length of sequences from large templates. The high-quality sequence data obtained by our procedure is independent of the size of template DNA used and can be completely automated on a variety of automated platforms.     

Automated Purification of Transfection-Grade Plasmid DNA Using Wizard MagneSil Tfx System

We describe a reagent system and robotic protocol for the isolation of highly purified plasmid DNA from cultured cells. The method is based on the Wizard® MagneSil™ Plasmid Purification System, which purifies sequencing-grade plasmid DNA. Two modifications to the sequencing-grade system were made to create the Wizard MagneSil Tfx™ System. This system provides lower endotoxin and other contaminant levels, giving higher quality plasmid for transfection applications. The Wizard MagneSil Tfx™ System uses MagneSil™ Paramagnetic Particles (PMPs) to clear lysate and bind plasmid, eliminating the need for filtration devices. The endotoxin removal step uses MagneSil™ (PMPs) and a guanidine/isopropanol wash to remove RNA and protein. One 96-well plate may be processed in 45 minutes on the Beckman Biomek® FX robotic workstation. We provide data showing DNA yield, contaminant levels, and transfection efficiency for 5 commonly used cell lines. Comparisons with other systems are also shown.     

Automated Purification of Plasmid DNA Using Paramagnetic Particles

We describe a reagent system and robotic method for purifying plasmid DNA for restriction digestion, PCR, and fluorescent sequencing applications. The method uses two types of Wizard® MagneSilTM paramagnetic particles. Following lysis and neutralization procedures, the first particle type binds and removes cell debris; the second type is then used to bind plasmid DNA from the cleared lysate. The particles are then washed to eliminate unwanted contaminants. Purified plasmid DNA is then eluted from the particles with nuclease free water. When using a cell mass of approximately 4 O.D.₆₀₀, the yield is 10–12μg of DNA when using high copy number plasmid. When used in BigDye® terminator sequencing, these DNA templates typically yield read lengths greater than 700 bases and Phred 20 scores of 600 to 750 bases. This purification method has been adapted for use on several robotic platforms in a 96-well format.     

IQ technology: an automated, high-throughput screening assay for kinase, phosphatase, and protease targets

IQ^® Technology, a homogeneous, universal-detection platform, originally designed for high-throughput screening (HTS) of kinases and phosphatases, has now been applied to protease screening. Representative enzymes from the major classes of proteases have been assayed in the IQ^® format. Enzyme activity and compound inhibition data are presented for such enzymes as Trypsin, Matrix Metalloproteinase 3 (MMP-3) and Calpain 1. The technology has been tested in 96- to 384- to 1536-well microplate formats and is universally suited for automated screening. IQ^® Technology is a direct, noncompetitive assay that does not require antibodies or radioisotopes. Fluorophore-labeled peptides are used as enzyme substrates. Kinase or phosphatase activity is quantified by direct measurement of the phosphorylation state of the substrate. For protease activity, cleavage is quantified with a peptide substrate containing a phospho-residue distal to the fluorphore. Cleavage of the substrate liberates the fluorphore-labeled terminus from the terminus containing the phospho-residue. Protease activity is measured by the change in fluorescence intensity that occurs when a proprietary compound binds specifically to phosphoryl groups on peptides and quenches the fluorescence. IQ^® Technology can be used with any peptide sequence and is insensitive to high concentrations of ATP and substrate. The IQ^® Technology has been validated against a large number of detergents, organics, and other reagents found in reaction mixtures and has been optimized for HTS applications exhibiting representative Z' values of 0.7.     

Automated genomic and proteomic applications on the Biomek NX laboratory automation workstation

This technical paper describes the utilization of a new automated liquid handler from Beckman Coulter, Inc., the Biomek^® NX Laboratory Automation Workstation, for genomic and proteomic applications. For genomic applications, methodology for plasmid DNA purification using Promega Wizard^® SV 96 reagents was developed for the Biomek NX. A single plate of bacterial pellets can be processed to purified plasmid DNA without user interaction after initial setup. DNA quantity and quality were assessed by spectrophotometric analysis, restriction digestion, PCR (The PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman La Roche, Ltd.), and capillary sequencing. Additionally, the plasmid preparation method was used to purify plasmid DNA from bacterial clones isolated in a bacterial two-hybrid screening procedure. In this case, the system quickly and efficiently prepared clones for rapid identification of target sequences. For proteomic applications, His-tag proteins were purified from bacterial cultures in a 96-well plate format. Following purification, a Bradford assay was used to determine the quantitative yields of the His-tag protein products in each of the aliquots from the purified samples. The AD 340 Automated Labware Positioner (ALP), an integrated absorbance reader, was used for absorbance measurements in the Bradford assay. Given the placement of this ALP on the deck of the Biomek NX, the entire process of protein purification and quantitation was performed in a complete walk-away automated format. Results obtained when purifying proteins, from both uninduced and induced bacterial cultures, on the worksurface of the Biomek NX will be described.     

A Fully Automated Process Using a Magnetic Particle Based Kit for Removal of Dye Terminators from Sequencing Reactions

Prolinx,® Inc. of Bothell, WA has developed the RapXtract™ 384 Dye Terminator Removal Kit for full automation of DNA sequencing reaction purification. The RapXtract product line is based upon proprietary superparamagnetic particle technology that eliminates the need for centrifugation, vacuum filtration, or modified primers to achieve purification of sequencing reactions. The kit described here is pre-dispensed in a 384-well microtiter plate and run on the TECAN GENESIS Workstation 150 (Tecan U.S. Inc., Research Triangle Park, NC). This system enables rapid purification of up to 384 sequencing reactions in a single run.As the completion of the Human Genome Project nears, it is imperative for biotechnology and pharmaceutical companies to increase throughput of DNA sequencing in order to be competitive in the drug discovery and validation process. The “race to market” requires a shift from standard DNA sequencing processes-including DNA sequencing reaction purification-towards complete walk-away automation for all steps.Existing sequencing reaction purification methods (Table 1) require considerable resources including: plastic and other laboratory consumables; specialized equipment, such as high-speed centrifuges or vacuum filtration apparatus; and labor-intensive protocols requiring large amounts of technician time. As a result, walk-away automation of standard purification methods is difficult and expensive.     

Corning® 384 Well Low Volume Microplate Performance in Miniaturized Assays

In response to the ever-increasing demand for microplates that facilitate assay miniaturization without compromising assay integrity, Corning's new 384 well Low Volume (384 LV) microplates were evaluated using a homogenous, fluorescence polarization protease assay. Fluorescently labeled casein substrate was incubated over time with dilutions of protease at working volumes that range from 1 to 20 μL and reagent concentrations that range from 0.125 to 50 pg/μL. Four types of plates were evaluated:

• Corning 384 well LV black medium bind

• Corning 384 well LV black non-binding surface (NBS™)

• Corning 1536 well black medium bind

• Competitor 384 well small volume

Protease activity was detected as a decrease in polarized fluorescence resulting from substrate degradation over time. Following assay miniaturization to 20 μL working volumes and protease concentration reduction from 25 to 0.25 pg/μL, Corning 384 well LV medium bind and NBS coated assay plates maintained superior signal to noise ratios compared to competitor's 384 well small volume assay plates. At even lower 10 μL working volumes, Corning 384 well LV medium bind assay plates maintained signal to noise ratios comparable to the competitor's assay plates. However as the reagent concentrations were further reduced to 2.5 pg/μL, Corning 384 well LV medium bind and NBS coated assay plates had significantly greater signal to noise ratios compared to the competitor's assay plates. As working volumes were reduced even further to 5 μL and 1 μL, Corning 384 well LV medium bind and NBS coated assay plates had significantly greater signal to noise ratios than the competitor's 384 well small volume assay plates. Furthermore, at 1 μL working volumes, Corning 384 well LV NBS coated assay plates had significantly greater signal to noise ratios than the competitor's assay plates and standard 1536 well assay plates.These results suggest that Corning 384 well LV medium bind and NBS coated assay plates facilitate assay miniaturization while maintaining assay integrity. Furthermore, as working volumes were significantly reduced to those typically found in 1536 deep well and standard 1536 well assay plates (5 and 1 μL, respectively), Corning's 384 well LV NBS coated assay plates provided superior assay performance.     

A system for dispensing sub-milligram doses of active pharmaceutical powders for early stage solubility assays

At early stages of drug development, the solubility of compounds is an important screening criterion. However, because scientists lack the automated tools needed to perform comprehensive early stage solubility studies, they are only able to perform a small number of experiments by hand, thus exploring only a fraction of the potential formulation design space. To allow a larger formulation design space to be explored at relatively early stages of pharmaceutical development (when 100 mg of a prototype compound is available), TransForm has created the AquaSFinX^™ and SFinX^™ micro-solubility platforms. In this article, a novel solid deposition system is described which is an enabling component of TransForm's solubility platforms. Given 100 mg of a starting material, the deposition system can dispense over six hundred 50-μg plugs of powder into 384-well or 1,536-well plates with well openings as small as 1.5 mm. Currently no commercial powder deposition system can provide this functionality. Including the time required to characterize and run the system, the dispense times range from around 2 minutes per dose for small runs involving 100 mg of starting material, to 18 seconds per dose for larger runs involving a gram or more of starting material.     

Involution codes: with application to DNA coded languages

For an involution θ : Σ^* → Σ^* over a finite alphabet Σ we consider involution codes: θ-infix, θ-comma-free, θ-k -codes and θ-subword-k-codes. These codes arise from questions on DNA strand design. We investigate conditions under which both X and X⁺ are same type of involution codes. General methods for generating such involution codes are given. The information capacity of these codes show to be optimized in most cases. A specific set of these codes was chosen for experimental testing and the results of these experiments are presented.     

Quantitative small molecule bioanalysis using chip-based nanoESI-MS/MS

A fully automated chip-based nanoelectrospray (nanoESI) system, NanoMate^® 100 (Advion BioSciences, Inc., Ithaca, NY), was evaluated for its application on quantitative bioanalysis of small molecules in support of exploratory pharmacokinetic (PK) studies. The NanoMate^® 100 was compared with the conventional autosampler coupled with liquid chromatography-electrospray (LC-ESI) interface. An API^® 3000 triple quadrupole mass spectrometer (Applied Biosystems, Inc., Foster City, CA) was used for the evaluation. The results show that the NanoMate^® 100 performs comparably to LC-ESI in terms of standard curve fitting, low limit of quantitation (LLOQ), dynamic range, accuracy, and precision. Parallel analyses of exploratory PK study samples show high correlation (R² = 0.971) between the NanoMate^® 100 and the LC-ESI. The NanoMate^® 100 exhibits advantages in carryover, sample consumption, sample cycle time, and the ability to be full automated. Despite these advantages, the necessarily rigorous sample preparation process limits the application of the NanoMate^® 100 for quantitative analysis in areas such as exploratory PK studies, which often involve multiple compounds in one study and require rapid turnaround. However, the NanoMate^® 100 has great potential in qualitative work (e.g., metabolite identification) as well as in high-throughput quantitative analysis of compound in the development stage (i.e., a single analyte with a well-established sample extraction method).     

Completeness of Continuation Models for λμ-Calculus

We show that a certain simple call-by-name continuation semantics of Parigot's λ_μ-calculus is complete. More precisely, for every λ_μ-theory we construct a cartesian closed category such that the ensuing continuation-style interpretation of λ_μ, which maps terms to functions sending abstract continuations to responses, is full and faithful. Thus, any λ_μ-category in the sense of L. Ong (1996, in “Proceedings of LICS '96,” IEEE Press, New York) is isomorphic to a continuation model (Y. Lafont, B. Reus, and T. Streicher, “Continuous Semantics or Expressing Implication by Negation,” Technical Report 93-21, University of Munich) derived from a cartesian-closed category of continuations. We also extend this result to a later call-by-value version of λ_μ developed by C.-H. L. Ong and C. A. Stewart (1997, in “Proceedings of ACM SIGPLAN-SIGACT Symposium on Principles of Programming Languages, Paris, January 1997,” Assoc. Comput. Mach. Press, New York).     

A novel microreactor with 3D rotating flow to boost fluid reaction and mixing of viscous fluids

Based on a concept encompassing splitting and recombination (SAR) and chaotic advection, an efficient microreactor, called a SAR μ-reactor, has been designed to mix fluids at Reynolds numbers from 0.01 to 100 and to be suitable for mixing fluids with viscosity over a wide range (μ = 0.000855–0.186 kg m⁻¹ s⁻¹). This SAR μ-reactor was compared, numerically and experimentally, with a slanted-groove micromixer (SGM) for reaction or mixing of fluids. Results of simulations characterized the designed structure with inducing a 3D rotating flow involving a strong lateral component to stretch intensely the contact interface in the SAR μ-reactor. Chemical colorimetry of two kinds – involving reactions of phenolphthalein with sodium hydroxide and of ascorbic acid with diiodine – revealed that the SAR μ-reactor provided a smaller mixing length, reaction length and period than the SGM; the mixing performance of the SAR μ-reactor was much better than that of the SGM. We assessed the mixing behavior of fluorescent proteins (C-phycocyanin and R-phycoerythrin) in viscous fluids with a confocal microscope. Experimental results and simulations showed that the effect of fluid viscosity on the mixing efficiency of the SAR μ-reactor is less than for the SGM; the SAR mechanism effectively augmented the contact interface even though the intrinsic diffusivity of fluids was diminished.     

Characterization of carbon paste electrodes modified with manganese based perovskites-type oxides from the amperometric determination of hydrogen peroxide

This work proposes the amperometric determination of hydrogen peroxide reduction and oxidation as a tool for the characterization of La_1−xA_xMnO₃ perovskites dispersed in a graphite composite electrode (carbon paste electrode, CPE). The catalytic activity of perovskites towards the oxidation and reduction of hydrogen peroxide is highly dependent on the nature of the A cation and on the temperature and time of calcination employed during the synthesis. Therefore, the selection of the optimal synthesis conditions to obtain the best catalytic activity towards hydrogen peroxide can be performed from amperometric determinations.We also report the analytical application of the perovskite modified CPE through the quantification of hydrogen peroxide in two real samples. Some preliminary results about the usefulness of La_0.66Sr_0.33MnO₃–CPE to develop a glucose biosensor by incorporation of the enzyme glucose oxidase (GOx) within the electrode are also reported. The difference in sensitivity to glucose at CPE–GOx and CPE–La_0.66Sr_0.33MnO₃–GOx (11.9 μA mol⁻¹ L and 158.1 μA mol⁻¹ L, respectively), clearly demonstrate the advantages of the association of the biocatalytic activity of GOx and the catalytic activity of perovskites towards hydrogen peroxide oxidation/reduction, and opens the doors to the development of new sensors for other important bioanalytes.     

Amperometric phenol biosensor based on polyaniline

The catechol biosensor is constructed by cross-linking between polyphenol oxidase (PPO) and polyaniline (PANI) using glutaraldehyde as a cross-linking agent. The PANI, which is electrochemically synthesized in a solution containing ionic liquid, 1-ethyl-3-methylimidazolium ethyl sulfate, possesses good electroactivity and high conductivity above pH 6. In the presence of catechol as a substrate, the biosensor exhibits a linear range from 0.2 to 80 μmol dm⁻³. The maximum response current (I_max) and the Michaelis–Menten constant (k^′_m) are 9.44 μA and 117 μmol dm⁻³, respectively. The effects of pH and operating potential are also explored to optimize measurement conditions. The activation energy (E_a) of the PPO catalytic reaction is 30.23 kJ mol⁻¹ in the B–R buffer. Electrochemical impedance spectroscopy (EIS), UV–vis and SEM are used to characterize the PANI–PPO biosensor. The biosensor exhibits good long-term stability.     

Simultaneous measurement of Pb, Cd and Zn using differential pulse anodic stripping voltammetry at a bismuth/poly(p-aminobenzene sulfonic acid) film electrode

A novel sensor was developed for simultaneous detection of Pb, Cd and Zn, based on the differential pulse anodic stripping response at a bismuth/poly(p-aminobenzene sulfonic acid) (Bi/poly(p-ABSA)) film electrode. This electrode was generated in situ by depositing simultaneously bismuth and the metals by reduction at −1.40 V on the poly(p-ABSA) modified electrode. Compared with the bismuth film electrode, the Bi/poly(p-ABSA) film electrode can yield a larger stripping signal for Pb, Cd and Zn. Under the optimum conditions, a linear response was observed for Cd and Zn in the range from 1.00 to 110.00 μg L⁻¹ and for Pb in the range from 1.00 to 130.00 μg L⁻¹. The detection limits of Pb(II), Cd(II) and Zn(II) were 0.80, 0.63 and 0.62 μg L⁻¹, respectively. Finally this sensor had been applied to the simultaneous determination of Pb(II), Cd(II) and Zn(II) in river water samples and the results were quite corresponding to the value obtained by atomic absorption spectrometry.     

A Novel Stochastic Game Via the Quantitative μ-calculus

The quantitative μ-calculus qMμ extends the applicability of Kozen's standard μ-calculus [D. Kozen, Results on the propositional μ-calculus, Theoretical Computer Science 27 (1983) 333–354] to probabilistic systems. Subsequent to its introduction [C. Morgan, and A. McIver, A probabilistic temporal calculus based on expectations, in: L. Groves and S. Reeves, editors, Proc. Formal Methods Pacific '97 (1997), available at [PSG, Probabilistic Systems Group: Collected reports, http://web.comlab.ox.ac.uk/oucl/research/areas/probs/bibliography.html]; also appears at [A. McIver, and C. Morgan, “Abstraction, Refinement and Proof for Probabilistic Systems,” Technical Monographs in Computer Science, Springer, New York, 2005, Chap. 9], M. Huth, and M. Kwiatkowska, Quantitative analysis and model checking, in: Proceedings of 12th annual IEEE Symposium on Logic in Computer Science, 1997] it has been developed by us [A. McIver, and C. Morgan, Games, probability and the quantitative μ-calculus qMu, in: Proc. LPAR, LNAI 2514 (2002), pp. 292–310, revised and expanded at [A. McIver, and C. Morgan, Results on the quantitative μ-calculus qMμ (2005), to appear in ACM TOCL]; also appears at [A. McIver, and C. Morgan, “Abstraction, Refinement and Proof for Probabilistic Systems,” Technical Monographs in Computer Science, Springer, New York, 2005, Chap. 11], A. McIver, and C. Morgan, “Abstraction, Refinement and Proof for Probabilistic Systems,” Technical Monographs in Computer Science, Springer, New York, 2005, A. McIver, and C. Morgan, Results on the quantitative μ-calculus qMμ (2005), to appear in ACM TOCL] and by others [L. de Alfaro, and R. Majumdar, Quantitative solution of omega-regular games, Journal of Computer and System Sciences 68 (2004) 374–397]. Beyond its natural application to define probabilistic temporal logic [C. Morgan, and A. McIver, An expectation-based model for probabilistic temporal logic, Logic Journal of the IGPL 7 (1999), pp. 779–804, also appears at [A. McIver, and C. Morgan, “Abstraction, Refinement and Proof for Probabilistic Systems,” Technical Monographs in Computer Science, Springer, New York, 2005, Chap.10]], there are a number of other areas that benefit from its use.One application is stochastic two-player games, and the contribution of this paper is to depart from the usual notion of “absolute winning conditions” and to introduce a novel game in which players can “draw”.The extension is motivated by examples based on economic games: we propose an extension to qMμ so that they can be specified; we show that the extension can be expressed via a reduction to the original logic; and, via that reduction, we prove that the players can play optimally in the extended game using memoryless strategies.     

A Study of a High-Throughput Plasmid DNA Purification System

An automated process that incorporates Millipore's Plasmid Miniprep₉₆ Montáge™ Kit with the Apogent Discoveries PlateMate Plus® and Tango™ automated high-throughput dispensing systems has been developed for purifying plasmid DNA. To test the efficacy of this process, parameters such as the reproducibility and consistency of the purified DNA quantity and quality as well as the purification speed were analyzed. The purification time for two plates of the Plasmid Miniprep₉₆ Kit (192 samples) was approximately 60 minutes using a PlateMate Plus equipped with 96 disposable tips and the Tango system equipped with 96 RB (resin bead) syringes. High uniformity and consistency in DNA yields (determined by spectrophotometric analysis) and quality (determined by gel electrophoresis analysis) among the different wells were observed. The purified plasmid DNA samples sequenced at an exceptional level with an average PHRED Q > 20 of 819 ± 25.*Millipore and Montage are the trademarks of Millipore Corporation     

Gold nanowire array electrode for non-enzymatic voltammetric and amperometric glucose detection

The non-enzymatic voltammetric and amperometric detection of glucose using a gold nanowire array electrode is described. The voltammetric detection of glucose was performed by cyclic and differential-pulse voltammetry. The detection of glucose by partial and direct oxidation of glucose during the anodic and cathodic potential sweeps was shown in cyclic voltammetry. An unusual decrease in overpotential for partial oxidation of glucose on a Au NW array electrode was observed. A linear differential-pulse voltammetric response for partial oxidation of glucose was observed up to a glucose concentration of at least 20 mM with a sensitivity of 41.9 μA mM⁻¹ cm⁻² and detection limit below 30 μM (signal-to-noise ratio of 3) for glucose oxidation at low potentials, where the influence of possible intermediates can be avoided. The amperometric response was also linear up to a glucose concentration of 10 mM with a sensitivity of 309.0 μA mM⁻¹ cm⁻². The wide dynamic range and high sensitivity, selectivity and stability, as well as good biocompatibility of the Au NW electrode make it promising for the fabrication of non-enzymatic glucose sensors.     

The μFill: A New 96-/384- Well Microplate Reagent Dispenser for HTS and Drug Discovery

Today's biomedical research requires instrumentation that is both functional and versatile. While high throughput screening (HTS) and drug discovery laboratories require instrumentation that can be automated, pilot assay laboratories may not necessarily need total automation. Towards that end, Bio-Tek has developed the μFill, a 96-/384- well microplate reagent dispenser capable of running stand-alone or computer-controlled as part of a robotics system. The μFill is compatible with both 96- and 384-well microplates, and using a specially designed adaptor can also dispense to deep-well microplates. It is capable of dispensing from 10-3000 μl for 96- well plates and 5-1500 μl for 384-well plates in 1-μl increments. The μFill can dispense 20 μl into a 96-well plate in four seconds and into a 384-well microplate in 12 seconds. The microprocessor-controlled syringe pump is based on a tested, low-maintenance design that requires no calibration, yet provides a high degree of accuracy and precision. The accuracy with an 80 μl dispense is within 1 μl with a percentage CV of less than 2%, and with a dispense volume of 20 μl, the percentage CV is still less than 5%. For those needing to dispense organic solvents or sterile aqueous solutions, a model that is autoclavable and has increased solvent resistance is also available. The programming allows for the control of flow rates from 225 μl/well/sec, for dispensing to cell cultures, and to 1000 μl/well/sec for rapid and vigorous reagent dispensing. The flexible software provides complete programming capabilities from the keypad. For more complete automation, robotics interfaces can be developed using ActiveX® software commands. The μFill's size, with a 14 x 14-inch footprint and a height of seven inches, allows it to be used almost anywhere.     

Single quantum dot-micelles coated with gemini surfactant for selective recognition of a cation and an anion in aqueous solutions

The synthesis of quantum dot coated with cetyltrimethyl ammonium bromide (CTAB) and gemini surfactant [C₁₂H₂₅N⁺(CH₃)₂(CH₂)₄(CH₃)₂N⁺C₁₂H₂₅]·2Br⁻ (C_12-4-12) in aqueous solution have been described. It is characterized by photoluminescent spectroscopy, UV–vis spectroscopy and transmission electron microscopy (TEM), etc. In comparison with CTAB-coated QDs, the QDs coated with C_12-4-12 respond selectively to both transition metal ion copper and fluoride ion in aqueous media. When Cu²⁺ is bound to C_12-4-12-coated QD micelles, the fluorescence intensity is quenched. Linear relationships are found between the relative fluorescence intensity and the concentration of Cu²⁺ in the range 0–500 μM, which is best described by a Stern–Volmer-type equation. Meanwhile, it is found that F⁻ enhanced the luminescence of the C_12-4-12-coated QD micelles in a concentration dependence that is described by a Langmuir binding isotherm equation in the range 0–300 μM. The limits of detection of Cu²⁺ and F⁻ are 1.1 and 0.68 μM, respectively. The possible mechanism is discussed.