Showing papers in "Analytical Chemistry in 2004"

A Model for Random Sampling and Estimation of Relative Protein Abundance in Shotgun Proteomics

Abstract: Proteomic analysis of complex protein mixtures using proteolytic digestion and liquid chromatography in combination with tandem mass spectrometry is a standard approach in biological studies. Data-dependent acquisition is used to automatically acquire tandem mass spectra of peptides eluting into the mass spectrometer. In more complicated mixtures, for example, whole cell lysates, data-dependent acquisition incompletely samples among the peptide ions present rather than acquiring tandem mass spectra for all ions available. We analyzed the sampling process and developed a statistical model to accurately predict the level of sampling expected for mixtures of a specific complexity. The model also predicts how many analyses are required for saturated sampling of a complex protein mixture. For a yeast-soluble cell lysate 10 analyses are required to reach a 95% saturation level on protein identifications based on our model. The statistical model also suggests a relationship between the level of sampling observed for a protein and the relative abundance of the protein in the mixture. We demonstrate a linear dynamic range over 2 orders of magnitude by using the number of spectra (spectral sampling) acquired for each protein.

Electrochemical biosensing platforms using platinum nanoparticles and carbon nanotubes.

Abstract: Platinum nanoparticles with a diameter of 2-3 nm were prepared and used in combination with single-wall carbon nanotubes (SWCNTs) for fabricating electrochemical sensors with remarkably improved sensitivity toward hydrogen peroxide. Nafion, a perfluorosulfonated polymer, was used to solubilize SWCNTs and also displayed strong interactions with Pt nanoparticles to form a network that connected Pt nanoparticles to the electrode surface. TEM and AFM micrographs illustrated the deposition of Pt nanoparticles on carbon nanotubes whereas cyclic voltammetry confirmed an electrical contact through SWCNTs between Pt nanoparticles and the glassy carbon (GC) or carbon fiber backing. With glucose oxidase (GOx) as an enzyme model, we constructed a GC or carbon fiber microelectrode-based biosensor that responds even more sensitively to glucose than the GC/GOx electrode modified by Pt nanoparticles or CNTs alone. The response time and detection limit (S/N = 3) of this biosensor was determined to be 3 s and 0.5 microM, respectively.

Selective Isolation at the Femtomole Level of Phosphopeptides from Proteolytic Digests Using 2D-NanoLC-ESI-MS/MS and Titanium Oxide Precolumns

Abstract: Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel automated method for the enrichment of phosphopeptides from complex mixtures. The method employs a two-dimensional column setup, with titanium oxide-based solid-phase material (Titansphere) as the first dimension and reversed-phase material as the second dimension. Phosphopeptides are separated from nonphosphorylated peptides by trapping them under acidic conditions on a TiO2 precolumn. Nonphosphorylated peptides break through and are trapped on a reversed-phase precolumn after which they are analyzed by nanoflow LC-ESI-MS/MS. Subsequently, phosphopeptides are desorbed from the TiO2 column under alkaline conditions, reconcentrated onto the reversed-phase precolumn, and analyzed by nanoflow LC-ESI-MS/MS. The selectivity and practica...

Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection.

Abstract: A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possibl...

Pinched Flow Fractionation: Continuous Size Separation of Particles Utilizing a Laminar Flow Profile in a Pinched Microchannel

Abstract: A concept of “pinched flow fractionation” for the continuous size separation and analysis of particles in microfabricated devices has been proposed and demonstrated. In this method, particles suspended in liquid were continuously introduced into a microchannel having a pinched segment and were aligned to one sidewall in the pinched segment by another liquid flow without particles. The particles were then separated perpendicularly to the flow direction according to their sizes by the spreading flow profile inside the microchannel. Polymer microbeads were successfully separated, and the effects of the flow rate and channel shapes on the separation performance were examined. Also, separated particles were collected independently by making branches at the end of the pinched segment. Since this method utilizes only the laminar flow profile inside a microchannel, complicated outer field control could be eliminated, which is usually required for other kinds of particle separation methods such as field flow fract...

Carbon Nanotube−Chitosan System for Electrochemical Sensing Based on Dehydrogenase Enzymes

Abstract: Multiwalled carbon nanotubes (CNT) were solubilized in aqueous solutions of a biopolymer chitosan (CHIT). The CHIT-induced solubilization of CNT facilitated their manipulations, including the modification of electrode surfaces for sensor and biosensor development. The colloidal solutions of CNT−CHIT were placed on the surface of glassy carbon (GC) electrodes to form robust CNT−CHIT films, which facilitated the electrooxidation of NADH. The GC/CNT−CHIT sensor for NADH required ∼0.3 V less overpotential than the GC electrode. The susceptibility of CHIT to chemical modifications was explored in order to covalently immobilize glucose dehydrogenase (GDH) in the CNT−CHIT films using glutaric dialdehyde (GDI). The stability and sensitivity of the GC/CNT−CHIT−GDI−GDH biosensor allowed for the interference-free determination of glucose in the physiological matrix (urine). In pH 7.40 phosphate buffer solutions, linear least-squares calibration plots over the range 5−300 μM glucose (10 points) had slopes 80 mA M-1 c...

Multiplexed toxin analysis using four colors of quantum dot fluororeagents.

Abstract: Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, we prepared bioinorganic conjugates made with highly luminescent semiconductor nanocrystals (CdSe−ZnS core−shell QDs) and antibodies to perform multiplexed fluoroimmunoassays. Sandwich immunoassays for the detection of cholera toxin, ricin, shiga-like toxin 1, and staphylococcal enterotoxin B were performed simultaneously in single wells of a microtiter plate. Initially the assay performance for the detection of each toxin was examined. We then demonstrated the simultaneous detection of the four toxins from a single sample probed with a mixture of all four QD−antibody reagents. Using a simple linear equation-based algorithm, it was possible to deconvolute the signal from mixed toxin samples, which allowed quantitation of all four toxins simultaneously.

Discrimination of Bacteria Using Surface-Enhanced Raman Spectroscopy

Abstract: Raman spectroscopy has recently been shown to be a potentially powerful whole-organism fingerprinting technique and is attracting interest within microbial systematics for the rapid identification ...

Parametric time warping.

Abstract: A parametric model is proposed for the warping function when aligning chromatograms. A very fast and stable algorithm results that consumes little memory and avoids the artifacts of dynamic time warping. The parameters of the warping function are useful for quality control. They also are easily interpolated, allowing alignment of batches of chromatograms based on warping functions for a limited number of calibration samples.

Label-Free Biosensing by Surface Plasmon Resonance of Nanoparticles on Glass: Optimization of Nanoparticle Size

Abstract: The unique optical properties of noble metal nanoparticles have been used to design a label-free biosensor in a chip format. In this paper, we demonstrate that the size of gold nanoparticles significantly affects the sensitivity of the biosensor. Gold nanoparticles with diameters in the range of 12−48 nm were synthesized in solution and sensor chips were fabricated by chemisorption of these nanoparticles on amine-functionalized glass. Sensors fabricated from 39-nm-diameter gold nanoparticles exhibited maximum sensitivity to the change of the bulk refractive index and the largest “analytical volume”, defined as the region around the nanoparticle within which a change in refractive index causes a change in the optical properties of the immobilized nanoparticles. The detection limit for streptavidin−biotin binding of a sensor fabricated from 39-nm-diameter nanoparticles was 20-fold better than a previously reported sensor fabricated from 13-nm-diameter gold nanoparticles. We also discuss several other factor...

Magnetite-Containing Spherical Silica Nanoparticles for Biocatalysis and Bioseparations

Abstract: The simultaneous entrapment of biological macromolecules and nanostructured silica-coated magnetite in sol−gel materials using a reverse-micelle technique leads to a bioactive, mechanically stable, nanometer-sized, and magnetically separable particles. These spherical particles have a typical diameter of 53 ± 4 nm, a large surface area of 330 m2/g, an average pore diameter of 1.5 nm, a total pore volume of 1.427 cm3/g and a saturated magnetization (MS) of 3.2 emu/g. Peroxidase entrapped in these particles shows Michaelis−Mentan kinetics and high activity. The catalytic reaction will take place immediately after adding these particles to the reaction solution. These enzyme entrapping particles catalysts can be easily separated from the reaction mixture by simply using an external magnetic field. Experiments have proved that these catalysts have a long-term stability toward temperature and pH change, as compared to free enzyme molecules. To further prove the application of this novel magnetic biomaterial in...

On-chip free-flow magnetophoresis: continuous flow separation of magnetic particles and agglomerates.

Abstract: The separation of magnetic microparticles was achieved by on-chip free-flow magnetophoresis. In continuous flow, magnetic particles were deflected from the direction of laminar flow by a perpendicular magnetic field depending on their magnetic susceptibility and size and on the flow rate. Magnetic particles could thus be separated from each other and from nonmagnetic materials. Magnetic and nonmagnetic particles were introduced into a microfluidic separation chamber, and their deflection was studied under the microscope. The magnetic particles were 2.0 and 4.5 microm in diameter with magnetic susceptibilities of 1.12 x 10(-4) and 1.6 x 10(-4) m(3) kg(-1), respectively. The 4.5-microm particles with the larger susceptibility were deflected further from the direction of laminar flow than the 2.0-microm magnetic particles. Nonmagnetic 6-microm polystyrene beads, however, were not deflected at all. Furthermore, agglomerates of magnetic particles were found to be deflected to a larger extent than single magnetic particles. The applied flow rate and the strength and gradient of the applied magnetic field were the key parameters in controlling the deflection. This separation method has a wide applicability since magnetic particles are commonly used in bioanalysis as a solid support material for antigens, antibodies, DNA, and even cells. Free-flow magnetophoretic separations could be hyphenated with other microfluidic devices for reaction and analysis steps to form a micro total analysis system.

Dielectrophoretic Concentration and Separation of Live and Dead Bacteria in an Array of Insulators

Abstract: Insulator-based (electrodeless) dielectrophoresis (iDEP) is an innovative approach in which the nonuniform electric field needed to drive DEP is produced by insulators, avoiding problems associated with the use of electrodes. Live and dead Escherichia coli were concentrated and selectively released by applying stepped DC voltages across a microchannel containing an array of insulating posts etched in glass. The only electrodes present were two platinum wires placed in the inlet and outlet reservoirs, producing mean electric fields of up to 200 V/mm across the insulators. The cells were labeled with Syto 9 and propidium iodide and imaged through a fluorescent microscope. Cell trapping and release were controlled by modifying the relative responses of electrokinesis and DEP by adjusting the magnitude of the applied voltage. Dead cells were observed to have significantly lower dielectrophoretic mobility than live cells, whereas the electrokinetic mobilities of live and dead cells were indistinguishable. The ...

Serum Peptide Profiling by Magnetic Particle-Assisted, Automated Sample Processing and MALDI-TOF Mass Spectrometry

Abstract: Human serum contains a complex array of proteolytically derived peptides (serum peptidome) that may provide a correlate of biological events occurring in the entire organism; for instance, as a diagnostic for solid tumors (Petricoin, E. F.; Ardekani, A. M.; Hitt, B. A.; Levine, P. J.; Fusaro, V. A.; Steinberg, S. M.; Mills, G. B.; Simone, C.; Fishman, D. A.; Kohn, E. C.; Liotta, L. Lancet 2002, 359, 572−577). Here, we describe a novel, automated technology platform for the simultaneous measurement of serum peptides that is simple, scalable, and generates highly reproducible patterns. Peptides are captured and concentrated using reversed-phase (RP) batch processing in a magnetic particle-based format, automated on a liquid handling robot, and followed by a MALDI TOF mass spectrometric readout. The protocol is based on a detailed investigation of serum handling, RP ligand and eluant selection, small-volume robotics design, an optimized spectral acquisition program, and consistent peak extraction plus binnin...

Peer Reviewed: Top-Down Proteomics

Abstract: A growing number of labs are using many types of mass spectrometers to directly analyze intact proteins and to improve conversion of MS data into biological knowledge

Basal plane pyrolytic graphite modified electrodes: comparison of carbon nanotubes and graphite powder as electrocatalysts.

Abstract: The oxidations of NADH, epinephrine, and norepinephrine are studied using carbon nanotube and graphite powder-modified basal plane pyrolytic graphite electrodes. Immobilization is achieved in two ways: first, via abrasive attachment of multiwall carbon nanotubes or graphite powder by gently rubbing the electrode surface on a fine quality paper supporting the desired material; second, via “film” modification from dispersing either graphite powder or nanotubes in acetonitrile and pipeting a small volume onto the electrode surface and allowing the solvent to volatilize. While electrocatalytic behavior of both types of nanotube-modified electrodes is shown, with enhanced currents and reduced peak-to-peak separations in the voltammetry in comparison with naked basal plane pyrolytic graphite, similar catalytic behavior is also seen at the graphite powder-modified electrodes. Caution is, therefore, suggested in assigning unique catalytic properties to carbon nanotubes.

Interdigitated Array microelectrode-based electrochemical impedance immunosensor for detection of Escherichia coli O157:H7.

Abstract: A label-free electrochemical impedance immunosensor for rapid detection of Escherichia coli O157:H7 was developed by immobilizing anti-E. coli antibodies onto an indium−tin oxide interdigitated array (IDA) microelectrode. Based on the general electronic equivalent model of an electrochemical cell and the behavior of the IDA microelectrode, an equivalent circuit, consisting of an ohmic resistor of the electrolyte between two electrodes and a double layer capacitor, an electron-transfer resistor, and a Warburg impedance around each electrode, was introduced for interpretation of the impedance components of the IDA microelectrode system. The results showed that the immobilization of antibodies and the binding of E. coli cells to the IDA microelectrode surface increased the electron-transfer resistance, which was directly measured with electrochemical impedance spectroscopy in the presence of [Fe(CN)6]3-/4- as a redox probe. The electron-transfer resistance was correlated with the concentration of E. coli cel...

Anodic stripping voltammetry of arsenic(III) using gold nanoparticle-modified electrodes.

Abstract: A novel method for the detection of arsenic(III) in 1 M HCl at a gold nanoparticle-modified glassy carbon electrode has been developed. The gold nanoparticles were electrodeposited onto the glassy carbon electrode via a potential step from +1.055 to −0.045 V vs SCE for 15 s from 0.5 M H2SO4 containing 0.1 mM HAuCl4. The resulting electrode surfaces were characterized with both AFM and cyclic voltammetry. Anodic stripping voltammetry of arsenic(III) on the modified electrode was performed. After optimization, a LOD of 0.0096 ppb was obtained with LSV.

Microfluidic Shear Devices for Quantitative Analysis of Cell Adhesion

Abstract: We describe the design, construction, and characterization of microfluidic devices for studying cell adhesion and cell mechanics. The method offers multiple advantages over previous approaches, including a wide range of distractive forces, high-throughput performance, simplicity in experimental setup and control, and potential for integration with other microanalytic modules. By manipulating the geometry and surface chemistry of the microdevices, we are able to vary the shear force and the biochemistry during an experiment. The dynamics of cell detachment under different conditions can be captured simultaneously using time-lapse videomicroscopy. We demonstrate assessment of cell adhesion to fibronectin-coated substrates as a function of the shear stress or fibronectin concentration in microchannels. Furthermore, a combined perfusion-shear device is designed to maintain cell viability for long-term culture as well as to introduce exogenous reagents for biochemical studies of cell adhesion regulation. In ag...

Design of a Compact Disk-like Microfluidic Platform for Enzyme-Linked Immunosorbent Assay

Abstract: This paper presents an integrated microfluidic device on a compact disk (CD) that performs an enzyme-linked immunosorbent assay (ELISA) for rat IgG from a hybridoma cell culture. Centrifugal and capillary forces were used to control the flow sequence of different solutions involved in the ELISA process. The microfluidic device was fabricated on a plastic CD. Each step of the ELISA process was carried out automatically by controlling the rotation speed of the CD. The work on analysis of rat IgG from hybridoma culture showed that the microchip-based ELISA has the same detection range as the conventional method on the 96-well microtiter plate but has advantages such as less reagent consumption and shorter assay time over the conventional method.

A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry.

Abstract: Recent advances in phosphopeptide enrichment prior to mass spectrometric analysis show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissociation (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MSn, for phosphopeptide ion dissociation in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simulataneously stored, the method generates a “composite” spectrum containing fragments derived from multiple precursors. In comparison to analysis by conventional MS/MS (CAD), Ps...

Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis.

Abstract: To quantify proteins on a global level from mammalian tissue, a method was developed to metabolically introduce 15N stable isotopes into the proteins of Rattus norvegicus for use as internal standards. The long-term metabolic labeling of rats with a diet enriched in 15N did not result in adverse health consequences. The average 15N amino acid enrichments reflected the relative turnover rates in the different tissues and ranged from 74.3 mpe in brain to 92.2 mpe in plasma. Using the 15N-enriched liver as a quantitative internal standard, changes in individual protein levels in response to cycloheximide treatment were measured for 310 proteins. These measurements revealed 127 proteins with altered protein level (p < 0.05). Most proteins with altered level have previously reported functions involving xenobiotic metabolism and protein-folding machinery of the endoplasmic reticulum. This approach is a powerful tool for the global quantitation of proteins, is capable of measuring proteome-wide changes in response to a drug, and will be useful for studying animal models of disease.

A glucose biosensor based on surface-enhanced Raman scattering: improved partition layer, temporal stability, reversibility, and resistance to serum protein interference.

Abstract: This work updates the recent progress made toward fabricating a real-time, quantitative, and biocompatible glucose sensor based on surface-enhanced Raman scattering (SERS). The sensor design relies on an alkanethiolate tri(ethylene glycol) monolayer that acts as a partition layer, preconcentrating glucose near a SERS-active surface. Chemometric analysis of the captured SERS spectra demonstrates that glucose is quantitatively detected in the physiological concentration range (0-450 mg/dL, 0-25 mM). In fact, 94% of the predicted glucose concentrations fall within regions A and B of the Clarke error grid, making acceptable predictions in a clinically relevant range. The data presented herein also demonstrate that the glucose sensor provides stable SERS spectra for at least 3 days, making the SERS substrate a candidate for implantable sensing. Glucose sensor reversibility and reusability is evaluated as the sensor is alternately exposed to glucose and saline solutions; after each cycle, difference spectra reveal that the partitioning process is largely reversible. Finally, the SERS glucose sensor successfully partitions glucose even when challenged with bovine serum albumin, a serum protein mimic.

Raman microscopic analysis of single microbial cells

Abstract: We demonstrate the utility of the Raman confocal microscope to generate a spectral profile from a single microbial cell and the use of this approach to differentiate bacterial species. In general, profiles from different bacterial taxa shared similar peaks, but the relative abundances of these components varied between different species. The use of multivariate methods subsequently allowed taxa discrimination. Further investigations revealed that the single-cell spectra could be used to differentiate between growth phases of a single species, but these differences did not obscure the overall interspecies discrimination. Finally, we tested the efficacy of the method as a means to identify cells responsible for the uptake of a specific substrate. A single strain was grown in media containing incrementally varying ratios of 13C6 to 12C6 glucose, and it was found that 13C incorporation shifted characteristic peaks to lower wavenumbers. These findings suggest that Raman microscopy has significant potential for...

Formation of Droplets of Alternating Composition in Microfluidic Channels and Applications to Indexing of Concentrations in Droplet-Based Assays

Abstract: For screening the conditions for a reaction by using droplets (or plugs) as microreactors, the composition of the droplets must be indexed Indexing here refers to measuring the concentration of a solute by addition of a marker, either internal or external Indexing may be performed by forming droplet pairs, where in each pair the first droplet is used to conduct the reaction, and the second droplet is used to index the composition of the first droplet This paper characterizes a method for creating droplet pairs by generating alternating droplets, of two sets of aqueous solutions in a flow of immiscible carrier fluid within PDMS and glass microfluidic channels The paper also demonstrates that the technique can be used to index the composition of the droplets, and this application is illustrated by screening conditions of protein crystallization The fluid properties required to form the steady flow of the alternating droplets in a microchannel were characterized as a function of the capillary number Ca and water fraction Four regimes were observed At the lowest values of Ca, the droplets of the two streams coalesced; at intermediate values of Ca the alternating droplets formed reliably At even higher values of Ca, shear forces dominated and caused formation of droplets that were smaller than the cross-sectional dimension of the channel; at the highest values of Ca, coflowing laminar streams of the two immiscible fluids formed In addition to screening of protein crystallization conditions, understanding of the fluid flow in this system may extend this indexing approach to other chemical and biological assays performed on a microfluidic chip

Separation of long DNA molecules by quartz nanopillar chips under a direct current electric field.

Abstract: We have established the nanofabrication technique for constructing nanopillars with high aspect ratio (100−500 nm diameter and 500−5000 nm tall) inside a microchannel on a quartz chip. The size of pillars and the spacing between pillars are designed as a DNA sieving matrix for optimal analysis of large DNA fragments over a few kilobase pairs (kbp). A chip with nanopillar channel and simple cross injector was developed based on the optimal design and applied to the separation of DNA fragments (1−38 kbp) and large DNA fragments (λ DNA, 48.5 kbp; T4 DNA, 165.6 kbp) that are difficult to separate on conventional gel electrophoresis and capillary electrophoresis without a pulsed-field technique. DNA fragments ranging from 1 to 38 kbp were separated as clear bands, and furthermore, the mixture of λ DNA and T4 DNA was successfully separated by a 380-μm-long nanopillar channel within only 10 s even under a direct current (dc) electric field. Theoretical plate number N of the channel (380−1450 μm long) was 1000−30...

Use of Room Temperature Ionic Liquids in Gas Sensor Design

Abstract: The attainable steady-state limiting currents and time responses of membrane-covered and membrane-independent gas sensors incorporating different electrode and electrolyte materials have been compared. A new design comprising a membrane-free microelectrode modified with a thin layer of a room temperature ionic liquid is considered. While the use of ionic liquid as electrolyte eliminates the need for a membrane and added supporting electrolyte, the slower diffusion of analyte within the more viscous medium results in slower time responses. Such sensors do, however, have potential application in more extreme operating conditions, such as high temperature and pressure, where traditional solvents would volatise.

Adenosine-dependent assembly of aptazyme-functionalized gold nanoparticles and its application as a colorimetric biosensor.

Abstract: Previous work has shown that DNAzyme-directed assembly of gold nanoparticles can be utilized to make effective colorimetric biosensors. However, the method is restricted to analytes that are directly involved in phosphodiester cleavage. To expand the methodology to a broader range of analytes, a colorimetric adenosine biosensor based on the aptazyme-directed assembly of gold nanoparticles is reported here. The aptazyme is based on the 8−17 DNAzyme with an adenosine aptamer motif that can modulate the DNAzyme activity through allosteric interactions depending on the presence of adenosine. In the absence of adenosine, the aptazyme is inactive and the substrate strands can serve as linkers to assemble DNA-functionalized 13-nm-diameter gold nanoparticles, resulting in a blue color. However, the presence of adenosine activates the aptazyme, which cleaves the substrate strand, disrupting the formation of nanoparticle aggregates. A red color of separated gold nanoparticles is observed. Concentrations of adenosin...

Amplified chemiluminescence surface detection of DNA and telomerase activity using catalytic nucleic acid labels.

Abstract: A G-rich nucleic acid sequence binds hemin and yields a biocatalytic complex (DNAzyme) of peroxidase activity, namely, the biocatalyzed generation of chemiluminescence in the presence of H2O2 and luminol. The DNAzyme is used as a label for the amplified detection of DNA, or for the analysis of telomerase activity in cancer cells, using chemiluminescence as an output signal. In one configuration, the analyzed DNA is hybridized with a primer nucleic acid that is associated with a Au surface, and the DNAzyme label is hybridized with the surface-confined analyte DNA. The DNA is analyzed with a detection limit of ∼1 × 10-9 M. In the second system, telomerase from HeLa cancer cells induces telomerization of a primer associated with a Au surface and the complementary DNAzyme units are hybridized with the telomere to yield the chemiluminescence. The detection limit of the system corresponds to 1000 HeLa cells in the analyzed sample.