Simultaneous Detection Of Multiple Analytes Research Articles

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective. Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8 This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

Hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective. Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin. Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements. This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH, which are important metabolic indicators of disease states (e.g., cancer proliferation) and exercise-induced acidosis.

Abstract 1237: TGF-β signaling pathway analysis using MILLIPLEX MAP TGF-β multiplex panel

Abstract Transforming growth factor beta (TGF-β) is a secreted protein that controls cellular proliferation, differentiation, apoptosis, migration, adhesion, immune response, and other functions in most cells. The binding of TGF-β to its receptor, TGFβRII, can lead to activation of both the SMAD-dependent canonical pathway as well as the SMAD-independent non-canonical pathways (such as PI3K/Akt, ERK, JNK, and p38). The TGF-β signaling pathway is coordinately regulated by phosphorylation and understanding the role of this pathway in normal physiological processes and in diseases such as cancer and diabetes requires the ability to simultaneously measure phosphorylation status of multiple protein targets. In recent years, bead-based assays, such as those using Luminex xMAP technology, have enabled the measurement of phosphorylation levels of up to 12 proteins simultaneously. Using the TGF-β Signaling Pathway multiplex panel, we observed increased levels of phosphorylated Smad2 and Smad3, but not phosphorylated ERK or Akt in TGF-β treated cells when compared to their unstimulated counterparts. Modifications in phosphorylation levels were confirmed by the treatment of cells with specific inhibitors against TGFβRI, which blocked phosphorylation of Smad2 and Smad3 in TGF-β treated HepG2 cells. Furthermore, significant increase in phosphorylated Smad3 was observed in human breast cancer versus matched breast normal tissues. The TGF-β Signaling Pathway multiplex panel allowed for simultaneous detection of multiple analytes in both the SMAD-dependent canonical pathway as well as the SMAD-independent non-canonical pathways in a specific, sensitive, and reproducible manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1237. doi:1538-7445.AM2012-1237

Analysis of phosphorodiamidate morpholino oligomer chemisrty CpG effects in murine and human cells (158.12)

Abstract Many RNA-based therapeutic oligomer backbone chemistries will elicit undesirable immunological response through a number of pathways. Additionally, it has been shown that certain sequences can also elicit cellular pro-inflammatory responses in the form cytokine and chemokines production. For example, non-methylated CpG DNA motifs are considered pathogen-associated molecular patterns (PAMPS) due to their abundance in microbial genomes and their recognition by TLR-9 receptors. Some synthetic oligonucleotides, containing modified backbone or other synthetic nucleotide motifs, will still retain potent immunostimulatory activity which may be inappropriate within the desired therapeutic effect. The purpose of this study was to look in vitro for immune activation resulting from exposure to optimized CpG sequences incorporated into the phosphorodiamidate morpholino oligomer (PMO) chemistry. Human and murine dendritic cells (DCs) were treated with varying concentration of PMO and culture media was assayed by multiplex cytokine and chemokine detection platform for simultaneous detection of multiple analytes. There was no evidence for a sequence- or chemistry-specific activation of the DCs indicating that PMO do not provide an innate stimulus to the immune system.

Single-walled carbon nanotubes as optical materials for biosensing

In this review, we summarize recent progress in the development of single-walled carbon nanotubes (SWNTs) as optical materials for biosensing applications. First, as optical labels, we discuss the use of SWNTs in Raman-based protein detection. Strong and simple resonance Raman spectroscopy of SWNTs opens up a method of protein microarray with detection sensitivity down to femtomolar range. Also, tunable isotopic SWNT-Raman signature enables the simultaneous detection of multiple analytes in complex fluids. Second, the photoluminescence properties of SWNTs are also explored. We examine fluorescence biosensors that integrate the quenching property of SWNTs and the recognition property of functional nucleic acids. Particularly, SWNTs are established as an efficient signal transduction substrate in different biosensing systems, including the detection of specific proteins and DNA sequences, regulation of singlet oxygen generation and label-free fluorescence assays, and all have exhibited very high selectivity and sensitivity.

Multiplexed Detection of Small Analytes by Structure-Switching Aptamer-Based Capillary Electrophoresis

Affinity probe capillary electrophoresis (APCE) assays, combining the separation power of CE with the specificity of interactions occurring between a target and a molecular recognition element (MRE), have become important analytical tools in many application fields. In this report, a rationalized strategy, derived from the structure-switching aptamer concept, is described for the design of a novel APCE mode dedicated to small molecule detection. Two assay configurations were reported. The first one, developed for the single-analyte determination, was based on the use of a cholesteryl-tagged aptamer (Chol-Apt) as the MRE and its fluorescein-labeled complementary strand (CS*) as the tracer (laser-induced fluorescence detection). Under micellar electrokinetic chromatography (MEKC) conditions, free CS* and the hybrid formed with Chol-Apt (duplex*) were efficiently separated (and then quantified) through the specific shift of the electrophoretic mobility of the cholesteryl-tagged species in the presence of a neutral micellar phase. When the target was introduced into the preincubated sample, the hybridized form was destabilized, resulting in a decrease in the duplex* peak area and a concomitant increase in the free CS* peak area. The second format, especially designed for multianalyte sensing, employed dually cholesteryl- and fluorescein-labeled complementary strands (Chol-CS*) of different lengths and unmodified aptamers (Apt). The size-dependent electrophoretic separation of different Chol-CS* forms from each other and from their corresponding duplexes* was also accomplished under MEKC conditions. The simultaneous detection of multiple analytes in a single capillary was performed by monitoring accurately each target-induced duplex-to-complex change. This method could expand significantly the potential of small solute APCE analysis in terms of simplicity, adaptability, generalizability, and high-throughput analysis capability.

Single Quantum Dot-Based Nanosensor for Multiple DNA Detection

Owing to their unique optical properties, quantum dots (QDs) with different colors have been applied for simultaneous detection of multiple analytes. However, the use of single QD for multiplex detection of analytes with single-molecule detection has not been explored. Here we report a single QD-based nanosensor for multiplex detection of HIV-1 and HIV-2 at single-molecule level in a homogeneous format. In this single QD-based nanosensor, the QD functions not only as a fluorescence pair for coincidence detection and as a fluorescence-resonance-energy-transfer (FRET) donor for FRET detection but also as a local nanoconcentrator which significantly amplifies the coincidence-related fluorescence signals and the FRET signals. This single-QD-based nanosensor takes advantage of a simple 'mix and detection' assay with extremely low sample consumption, high sensitivity, and short analysis time and has the potential to be applied for rapid point-of-care testing, gene expression studies, high-throughput screening, and clinical diagnostics.

Fluorescent-Dye-Doped Sol−Gel Sensor for Highly Sensitive Carbon Dioxide Gas Detection below Atmospheric Concentrations

Optical fluorescence sol-gel sensors have been developed for the detection of carbon dioxide gas in the 0.03-30% range with a detection limit of 0.008% (or 80 ppm) and a quantitation limit of 0.02% (or 200 ppm) CO(2). Sol-gels were spin-coated on glass slides to create an organically modified silica-doped matrix with the 1-hydroxypyrene-3,6,8-trisulfonate (HPTS) fluorescent indicator. The luminescence intensity of the HPTS indicator (513 nm) is quenched by CO(2), which protonates the anionic form of HPTS. An ion pair technique was used to incorporate the lipophilic dye into the hydrophilic sol-gel matrix. TiO(2) particles (<5 microm diameter) were added to induce Mie scattering and increase the incident light interaction with the sensing film, thus increasing the signal-to-noise ratio. Moisture-proof overcoatings have been used to maintain a constant level of water inside the sensor films. The optical sensors are inexpensive to prepare and can be easily coupled to fiber optics for remote sensing capabilities. A fiber-optic bundle was used for the gas detection and shown to work as part of a multianalyte platform for simultaneous detection of multiple analytes. The studies reported here resulted in the development of sol-gel optical fluorescent sensors for CO(2) gas with sensitivity below that in the atmosphere (ca. 387 ppm). These sensors are a complementary approach to current FT-IR measurements for real-time carbon dioxide detection in environmental applications.

Fluorescence Lifetime Multiplexing with Nanocrystals and Organic Labels

The potential of semiconducting nanocrystals or so-called quantum dots (QDs) for lifetime multiplexing has not been investigated yet, despite the increasing use of QDs in (bio)analytical detection, biosensing, and fluorescence imaging and the obvious need for simple and cost-effective tools and strategies for the simultaneous detection of multiple analytes or events. This is most likely related to their multiexponential decay behavior as for multiplex chromophores, typically monoexponential decay kinetics are requested. The fluorescence decay kinetics of various mixtures of a long-lived, multiexponentially decaying CdSe QD and a short-lived organic dye were analyzed, and a model was developed for the quantification of these labels from the measured complex decay kinetics as a first proof-of-concept for the huge potential of these labels for lifetime multiplexing. In a second step, we evaluated the potential of mixtures of two types of QDs, varying in constituent material to realize distinguishable, yet multiexponential decay kinetics and similar absorption and emission spectra. Strategies for lifetime multiplexing with nanocrystalline labels were derived on the basis of these measurements.

Simultaneous visual detection of single-nucleotide variations in tuna DNA using DNA/RNA chimeric probes and ribonuclease A

The need for detection of single-nucleotide polymorphisms (SNPs) is rapidly increasing for molecular diagnostics, species authentication, and food traceability. In many detection technologies, fluorescence probes have the advantage of simultaneous detection of multiple analytes using multiple color fluorescence dyes. In addition, an adequate concentration of fluorescence can be observed by the naked eye. We conducted a visual ribonuclease protection assay using multicolor fluorescence probes for the simultaneous detection of multiple tuna species. The assay includes amplification of a target RNA sequence by in vitro transcription, hybridization with DNA/RNA chimeric fluorescence probes, and cleavage of mismatched RNA bases by ribonuclease A. Fragmented dye-labeled oligonucleotides were easily removed by centrifugal gel filtration. Using three fluorescent probes, fluorescence signals related to the target SNP were simply found by the visual observation of eluates. Moreover, the dual fluorescence system was used for obtaining the mixing ratio of two tuna species. This technique appeared to be convenient for detection of interest species in food products.

Multianalyte sensor array based on an organic light emitting diode platform

A compact photoluminescence (PL)-based sensor array, utilizing pulsed organic light emitting diode (OLED) pixels as the excitation sources, for sequential or simultaneous detection of multiple analytes in a single sample, is described. The utility and potential advantages of the structurally integrated OLED-based platform for multianalyte detection are demonstrated for oxygen, glucose, lactate, and ethanol. The detection of glucose, lactate, and ethanol is based on monitoring the concentration of dissolved oxygen (DO) at the completion of the enzymatic oxidation reactions of these analytes in sealed cells. The monitoring in sealed cells and the ready access of the enzyme, when in solution, to the analyte enable a limit of detection of ∼0.02 mM, which is better than that obtained with enzymes embedded in sol–gel films. The DO concentration is determined via its effect on the PL decay time of the oxygen-sensitive dye Pt octaethylporphyrin embedded in a polystyrene film. A modified Stern–Volmer equation is derived to generate a linear calibration. The 2 mm × 2 mm OLED pixels and the sensor films are fabricated on glass substrates that are attached back-to-back, generating a compact module devoid of any optical couplers. Two individually addressable OLED pixels are associated with the detection of each analyte. This configuration enables consecutive detection of all analytes within a few minutes utilizing a single photodetector (PD). Simultaneous detection is achieved by using an array of small-size Si photodiode PDs compatible with the OLED pixel array. The OLED-based sensing array is unique in its ease of fabrication and integration with the sensing component, while its performance attributes are comparable to those obtained for detection of a single analyte using any excitation source.

Automated analytical microarrays: a critical review.

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple analytes in a single experiment. The specific affinity reaction of nucleic acids (hybridization) and antibodies towards antigens is the most common bioanalytical method for generating multiplexed quantitative results. Nucleic acid-based analysis is restricted to the detection of cells and viruses. Antibodies are more universal biomolecular receptors that selectively bind small molecules such as pesticides, small toxins, and pharmaceuticals and to biopolymers (e.g. toxins, allergens) and complex biological structures like bacterial cells and viruses. By producing an appropriate antibody, the corresponding antigenic analyte can be detected on a multiplexed immunoanalytical microarray. Food and water analysis along with clinical diagnostics constitute potential application fields for multiplexed analysis. Diverse fluorescence, chemiluminescence, electrochemical, and label-free microarray readout systems have been developed in the last decade. Some of them are constructed as flow-through microarrays by combination with a fluidic system. Microarrays have the potential to become widely accepted as a system for analytical applications, provided that robust and validated results on fully automated platforms are successfully generated. This review gives an overview of the current research on microarrays with the focus on automated systems and quantitative multiplexed applications. FigureMCR 3: A fully automated chemiluminescence microarray reader for analytical microarrays

A nanoparticle based chromogenic chemosensor for the simultaneous detection of multiple analytes

Quantum Dot-Schiff base conjugate displays selectivity for Cu(2+) and Fe(3+) enabling the simultaneous detection of these ions in semi-aqueous solution; in contrast, the Schiff base itself displayed no selectivity.

Laser desorption/ionization mass spectrometry on porous silicon for metabolome analyses: influence of surface oxidation

Laser desorption/ionization mass spectrometry (LDI-MS) on porous silicon is a promising analytical strategy for the rapid detection of metabolites in biological matrices. We show that both oxidized and unoxidized porous silicon surfaces are useful in detecting protonated/deprotonated molecules from compounds when analyzed in mixtures. We demonstrate the feasibility of using this technique for the simultaneous detection of multiple analytes using a synthetic cocktail of 30 compounds commonly associated with prokaryotic and eukaryotic primary metabolism. The predominantly detected species were the protonated molecules or their sodium/potassium adducts in the positive-ion mode and the deprotonated molecules in the negative-ion mode, as opposed to fragments or other adducts. Surface oxidation appears to influence mass spectral responses; in particular, in the mixture we studied, the signal intensities of the hydrophobic amino acids were noticeably reduced. We show that whilst quantitative changes in individual analytes can be detected, ion suppression effects interfere when analyte levels are altered significantly. However, the response of most analytes was relatively unaffected by changes in the concentration of one of the analytes, so long as it was not allowed to dominate the mixture, which may limit the dynamic range of this approach. The differences in the response of the analytes when analyzed in mixtures could not be accounted for by considering their gas-phase and aqueous basicities alone. The implications of these findings in using the technique for metabolome analyses are discussed.

Microfluidic Immunoassay Platform Using Antibody-immobilized Glass Beads and Its Application for Detection of Escherichia coli O157:H7

We developed a microfluidic immunoassay platform for the detection of various analytes such as bacterial pathogen by packing antibody-immobilized glass beads in spatially-isolated microchambers on a microfluidic device. Primary amines of antibody were covalently conjugated to carboxyl-terminated glass beads previously treated with aminosilane followed by glutaraldehyde. Through this covalent binding, up to 905 <TEX>$\mu$</TEX>g immunoglobulin G (IgG) per gram of glass beads was immobilized. For application, glass beads attaching antibody specific to Escherichia coli O157:H7, a foodborne pathogen, were packed into a microfluidic device and used for the detection of the serotype. This prototype immunoassay device can be used for the simultaneous detection of multiple analytes by sequentially packing different-sized glass beads attaching different antibody in discrete microchambers on a single microfluidic device.

DNA-Directed Protein Immobilization for Simultaneous Detection of Multiple Analytes by Surface Plasmon Resonance Biosensor

A versatile multichannel biosensor surface is prepared by site-directed immobilization of single-stranded DNA-protein conjugates onto a patterned self-assembled monolayer composed of ssDNA thiols and oligo(ethylene glycol)-terminated thiols. The conjugates each consist of an antibody chemically linked to a unique ssDNA target with a sequence complementary to the surface-bound ssDNA probes and are immobilized on the surface via sequence-specific hybridization. The exceptional specificity of DNA hybridization combined with the diversity of available sequences makes this platform perfect for multichannel sensors. Once the surface is patterned with the appropriate probe sequences, sequence-specific hybridization sorts out the target conjugates and directs them to the appropriate spots on the surface. Previously (Boozer, C. L.; Ladd, J.; Chen, S.; Yu, Q.; Homola, J.; Jiang, S. Anal. Chem. 2004, 76, 6967-6972), we performed proof-of-concept experiments demonstrating the feasibililty of using DNA-directed protein immobilization to produce a single channel biosensor. In this work, we extend this technique and employ DNA-directed protein immobilization to functionalize a multichannel biosensor, which was used for the simultaneous detection of a set of three fertility hormones: human chorionic gonadotropin, human luteinizing hormone, and follicle stimulating hormone by surface plasmon resonance sensor.

Multi-analyte surface plasmon resonance biosensing

Surface plasmon resonance (SPR) biosensors are affinity sensing devices exploiting a special mode of electromagnetic field—surface plasmon-polariton—to detect the binding of analyte molecules from a liquid sample to biomolecular recognition elements immobilized on the surface of the sensor. In this paper, we review advances of SPR biosensor technology towards detection systems for the simultaneous detection of multiple analytes (multi-analyte detection). In addition, we report application of a recently developed multichannel SPR sensor based on spectroscopy of surface plasmons and wavelength division multiplexing of sensing channels to multi-analyte detection.

Development of a High-Throughput Automated Analyzer Using Biochip Array Technology

Use of protein array technology over conventional assay methods has advantages that include simultaneous detection of multiple analytes, reduction in sample and reagent volumes, and high output of test results. The susceptibility of ligands to denaturation, however, has impeded production of a stable, reproducible biochip platform, limiting most array assays to manual or, at most, semiautomated processing techniques. Such limitations may be overcome by novel biochip fabrication procedures. After selection of a suitable biochip substrate, biochip surfaces were chemically modified and assessed to enable optimization of biochip fabrication procedures for different test panels. The assay procedure was then automated on a dedicated instrument, and assay performance was determined for a panel of cytokine markers. Assay results were then compared with a commercial method for measurement of cytokine markers. Secondary ion mass spectrometry and x-ray photoelectron spectroscopy demonstrated appropriate and reproducible modification of the biochip surface. Contact-angle studies also confirmed generation of hydrophobic surfaces that enabled containment of droplets for fabrication of discrete test regions. Automation of the biochip assays on a dedicated instrument produced excellent cytokine marker performance with intra- and interassay imprecision <10% for most analytes. Comparison studies showed good agreement with other methods (r = 0.95-0.99) for cytokines. Performance data from this automated biochip array analyzer provide evidence that it is now possible to produce stable and reproducible biochips for output of more than 2000 test results per hour.

Bioluminescence resonance energy transfer from aequorin to a fluorophore: an artificial jellyfish for applications in multianalyte detection

In nature, the green light emission observed in the jellyfish Aequorea victoria is a result of a non-radiative energy transfer from the excited-state aequorin to the green fluorescent protein. In this work, we have modified the photoprotein aequorin by attaching selected fluorophores at a unique site on the protein. This will allow for in vitro transfer of bioluminescent energy from aequorin to the fluorophore thus creating an "artificial jellyfish". The fluorophores are selected such that the excitation spectrum of the fluorophore overlaps with the emission spectrum of aequorin. By modifying aequorin with different fluorophores, bioluminescent labels with different emission maxima are produced, which will allow for the simultaneous detection of multiple analytes. By examining the X-ray crystal structure of the protein, four different sites for introduction of the unique cysteine residue were evaluated. Two fluorophores with differing emission maxima were attached individually to the mutants through the sulfhydryl group of the cysteine molecule. Two of the fluorophore-labeled mutants showed a peak corresponding to fluorophore emission thus indicating resonance energy transfer from aequorin to the fluorophore.

Multichannel surface plasmon resonance biosensor with wavelength division multiplexing

A novel multichannel surface plasmon resonance (SPR) biosensor with spectral interrogation and wavelength division multiplexing of sensing channels is presented. A special design of SPR prism element is used to enable wavelength division multiplexing of serial sensing channels. In addition to increasing the number of sensing channels, this approach allows discrimination of the sensor response due to analyte–receptor binding from interfering background refractive index fluctuations. A novel eight-channel SPR sensor combining the wavelength division multiplexing of serial sensing channels with the conventional parallel channel architecture is described. Application of this multichannel SPR sensor for simultaneous detection of multiple analytes is described.