Encoded metal nanoparticle-based molecular beacons for multiplexed detection of DNA

In this paper we describe a molecular beacon format assay in which encoded nanowire particles are used to achieve multiplexing. We demonstrate this principle with the detection of five viral pathogens; Hepatitis A virus, Hepatitis C virus, West Nile Virus, Human Immune Deficiency virus and Severe Acute Respiratory Syndrome virus. Oligonucleotides are designed complementary to a target sequence of interest containing a 3′ universal fluorescence dye. A 5′ thiol causes the oligonucleotides to self-assemble onto the metal nanowire. The single-stranded oligonucleotide contains a self-complementary hairpin stem sequence of 10 bases that forces the 3′ fluorophore to come into contact with the metallic nanowire surface, thereby quenching the fluorescence. Upon addition of target DNA, there is hybridization with the complementary oligonucleotides. The resulting DNA hybrid is rigid, unfolds the hairpin structure, and causes the fluorophore to be moved away from the surface such that it is no longer quenched. By using differently encoded nanowires, each conjugated with a different oligonucleotide sequence, multiplexed DNA assays are possible using a single fluorophore, from a multiplexed RT-PCR reaction.     

Multiplexed detection of cardiac biomarkers in serum with nanowire arrays using readout ASIC

Early detection of cardiac biomarkers for diagnosis of heart attack is the key to saving lives. Conventional method of detection like the enzyme-linked immunosorbent assay (ELISA) is time consuming and low in sensitivity. Here, we present a label-free detection system consisting of an array of silicon nanowire sensors and an interface readout application specific integrated circuit (ASIC). This system provides a rapid solution that is highly sensitive and is able to perform direct simultaneous-multiplexed detection of cardiac biomarkers in serum. Nanowire sensor arrays were demonstrated to have the required selectivity and sensitivity to perform multiplexed detection of 100 fg/ml troponin T, creatine kinase MM, and creatine kinase MB in serum. A good correlation between measurements from a probe station and the readout ASIC was obtained. Our detection system is expected to address the existing limitations in cardiac health management that are currently imposed by the conventional testing platform, and opens up possibilities in the development of a miniaturized device for point-of-care diagnostic applications.     

Multiplexed detection and differentiation of the DNA strains for influenza A (H1N1 2009) using a silicon-based microfluidic system

Pandemic influenza by the swine-origin influenza virus (H1N1 2009) has attracted considerable concern worldwide. A convenient and accurate diagnostic approach that can be deployed at the point of care, such as in a doctor's office or at an airport, is critical for disease control. Here we report the development of a silicon-based microfluidic system for subtype differentiation of the novel H1N1 2009 strain vs. the seasonal influenza A (FluA) strain. The proposed system included two functional modules: a multiplexed PCR module for amplification of nucleic acid targets and a multiplexed silicon nanowire (SiNW) module for sequence determination. The PCR module consisted of a microfluidic PCR chamber and an electrical controller to perform a multiplexed protocol that simultaneously enriched specific segments of both H1N1 and FluA strains (if present), with 10(4)-10(5) amplification efficiency. The PCR amplicon was subsequently denatured and transferred to the SiNW sensing module for a label-free, multiplexed detection. A control SiNW was implemented, for the first time, in order to eliminate background interference. The detection module demonstrated a 10× change in the magnitude of differential current when the target DNA was injected. Overall, the system achieved a sensitivity of 20-30 fg/μl for H1N1 and seasonal FluA nucleic acids in a 10 μl sample. The low sample consumption, high sensitivity and high specificity render it a potential point-of-care (POC) platform to help doctors reach a yes/no decision for infectious diseases.     

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.     

Multiplexed electrical detection of cancer markers with nanowire sensor arrays

We describe highly sensitive, label-free, multiplexed electrical detection of cancer markers using silicon-nanowire field-effect devices in which distinct nanowires and surface receptors are incorporated into arrays. Protein markers were routinely detected at femtomolar concentrations with high selectivity, and simultaneous incorporation of control nanowires enabled discrimination against false positives. Nanowire arrays allowed highly selective and sensitive multiplexed detection of prostate specific antigen (PSA), PSA-alpha1-antichymotrypsin, carcinoembryonic antigen and mucin-1, including detection to at least 0.9 pg/ml in undiluted serum samples. In addition, nucleic acid receptors enabled real-time assays of the binding, activity and small-molecule inhibition of telomerase using unamplified extracts from as few as ten tumor cells. The capability for multiplexed real-time monitoring of protein markers and telomerase activity with high sensitivity and selectivity in clinically relevant samples opens up substantial possibilities for diagnosis and treatment of cancer and other complex diseases.     

Fabrication of silicon nanowire devices for ultrasensitive, label-free, real-time detection of biological and chemical species

Detection and quantification of biological and chemical species are central to many areas of healthcare and the life sciences, ranging from diagnosing disease to discovery and screening of new drug molecules. Semiconductor nanowires configured as electronic devices have emerged as a general platform for ultra-sensitive direct electrical detection of biological and chemical species. Here we describe a detailed protocol for realizing nanowire electronic sensors. First, the growth of uniform, single crystal silicon nanowires, and subsequent isolation of the nanowires as stable suspensions are outlined. Second, fabrication of addressable nanowire device arrays is described. Third, covalent modification of the nanowire device surfaces with receptors is described. Fourth, an example modification and measurements of the electrical response from devices are detailed. The silicon nanowire (SiNW) devices have demonstrated applications for label-free, ultrasensitive and highly-selective real-time detection of a wide range of biological and chemical species, including proteins, nucleic acids, small molecules and viruses.     

Detection technologies in proteome analysis

Common strategies employed for general protein detection include organic dye, silver stain, radiolabeling, reverse stain, fluorescent stain, chemiluminescent stain and mass spectrometry-based approaches. Fluorescence-based protein detection methods have recently surpassed conventional technologies such as colloidal Coomassie blue and silver staining in terms of quantitative accuracy, detection sensitivity, and compatibility with modern downstream protein identification and characterization procedures, such as mass spectrometry. Additionally, specific detection methods suitable for revealing protein post-translational modifications have been devised over the years. These include methods for the detection of glycoproteins, phosphoproteins, proteolytic modifications, S-nitrosylation, arginine methylation and ADP-ribosylation. Methods for the detection of a range of reporter enzymes and epitope tags are now available as well, including those for visualizing beta-glucuronidase, beta-galactosidase, oligohistidine tags and green fluorescent protein. Fluorescence-based and mass spectrometry-based methodologies are just beginning to offer unparalleled new capabilities in the field of proteomics through the performance of multiplexed quantitative analysis. The primary objective of differential display proteomics is to increase the information content and throughput of proteomics studies through multiplexed analysis. Currently, three principal approaches to differential display proteomics are being actively pursued, difference gel electrophoresis (DIGE), multiplexed proteomics (MP) and isotope-coded affinity tagging (ICAT). New multiplexing capabilities should greatly enhance the applicability of the two-dimensional gel electrophoresis technique with respect to addressing fundamental questions related to proteome-wide changes in protein expression and post-translational modification.     

Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes

Rapid, multiplexed, sensitive and specific molecular detection is of great demand in gene profiling, drug screening, clinical diagnostics and environmental analysis. One of the major challenges in multiplexed analysis is to identify each specific reaction with a distinct label or 'code'. Two encoding strategies are currently used: positional encoding, in which every potential reaction is preassigned a particular position on a solid-phase support such as a DNA microarray, and reaction encoding, where every possible reaction is uniquely tagged with a code that is most often optical or particle based. The micrometer size, polydispersity, complex fabrication process and nonbiocompatibility of current codes limit their usability. Here we demonstrate the synthesis of dendrimer-like DNA-based, fluorescence-intensity-coded nanobarcodes, which contain a built-in code and a probe for molecular recognition. Their application to multiplexed detection of the DNA of several pathogens is first shown using fluorescence microscopy and dot blotting, and further demonstrated using flow cytometry that resulted in detection that was sensitive (attomole) and rapid.     

Multiplexing to the max

Researchers demonstrate how microfluidics can be used to both synthesize millions of differentially encoded particles for multiplexed in vitro biological detection assays, as well as to decode their identities.     

Protein microarrays with carbon nanotubes as multicolor Raman labels

The current sensitivity of standard fluorescence-based protein detection limits the use of protein arrays in research and clinical diagnosis. Here, we use functionalized, macromolecular single-walled carbon nanotubes (SWNTs) as multicolor Raman labels for highly sensitive, multiplexed protein detection in an arrayed format. Unlike fluorescence methods, Raman detection benefits from the sharp scattering peaks of SWNTs with minimal background interference, affording a high signal-to-noise ratio needed for ultra-sensitive detection. When combined with surface-enhanced Raman scattering substrates, the strong Raman intensity of SWNT tags affords protein detection sensitivity in sandwich assays down to 1 fM--a three-order-of-magnitude improvement over most reports of fluorescence-based detection. We use SWNT Raman tags to detect human autoantibodies against proteinase 3, a biomarker for the autoimmune disease Wegener's granulomatosis, diluted up to 10(7)-fold in 1% human serum. SWNT Raman tags are not subject to photobleaching or quenching. By conjugating different antibodies to pure (12)C and (13)C SWNT isotopes, we demonstrate multiplexed two-color SWNT Raman-based protein detection.     

Molecular analysis for medicine: a new technological platform based on nanopatterning and label free optical direction

In this contribution, we show that by biopatterning probe molecules at the nanoscale using soft-lithography, we can produce protein biochips at a cost sufficiently small for their use as a systematic method of molecular analysis for medical diagnosis purposes. The combination of multiplexed nanoscale Micro-Contact printing and label-free optical detection using the principle of light diffraction, is implemented for generating engineered glass slides of analysis and a dedicated diffractive scanner for reading the multiplexed results of an assay.     

Monoclonal antibody selection for interleukin-4 quantification using suspension arrays and forward-phase protein microarrays

A recombinant mouse interleukin-4 (IL-4) and three different purified rat antimouse IL-4 monoclonal antibodies (Mab) with different clonalities were employed as a model system. This system was used to examine monoclonal antibody effectiveness using both conventional and high-throughput measurement techniques to select antibodies for attaining the most sensitive detection of the recombinant IL-4 through the "sandwich-type" immunoassays. Surface plasmon resonance (SPR) measurements and two high-throughput methods, suspension arrays (also called multiplexed bead arrays) and forward-phase protein microarrays, predicted the same capture (BVD4-1D11) and detection (BVD6-24G2) antibody pair for the most sensitive detection of the recombinant cytokine. By using this antibody pair, we were able to detect as low as 2 pg/mL of IL-4 in buffer solution and 13.5 pg/mL of IL-4 spiked in 100% normal mouse serum with the multiplexed bead arrays. Due to the large amount of material required for SPR measurements, the study suggests that the multiplexed bead arrays and protein microarrays are both suited for the selection of numerous antibodies against the same analyte of interest to meet the need in the areas of systems biology and reproducible clinical diagnostics for better patient care.     

Bar-coded hydrogel microparticles for protein detection: synthesis, assay and scanning

This protocol describes the core methodology for the fabrication of bar-coded hydrogel microparticles, the capture and labeling of protein targets and the rapid microfluidic scanning of particles for multiplexed detection. Multifunctional hydrogel particles made from poly(ethylene glycol) serve as a sensitive, nonfouling and bio-inert suspension array for the multiplexed measurement of proteins. Each particle type bears a distinctive graphical code consisting of unpolymerized holes in the wafer structure of the microparticle; this code serves to identify the antibody probe covalently incorporated throughout a separate probe region of the particle. The protocol for protein detection can be separated into three steps: (i) synthesis of particles via microfluidic flow lithography at a rate of 16,000 particles per hour; (ii) a 3-4-h assay in which protein targets are captured and labeled within particles using an antibody sandwich technique; and (iii) a flow scanning procedure to detect bar codes and quantify corresponding targets at rates of 25 particles per s. By using the techniques described, single- or multiple-probe particles can be reproducibly synthesized and used in customizable multiplexed panels to measure protein targets over a three-log range and at concentrations as low as 1 pg ml(-1).     

APDS: the autonomous pathogen detection system

We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system was developed to provide early warning to civilians in the event of a bioterrorism incident and can be used at high profile events for short-term, intensive monitoring or in major public buildings or transportation nodes for long-term monitoring. The APDS is completely automated, offering continuous aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and nucleic acid-based polymerase chain reaction (PCR) amplification and detection. Highly multiplexed antibody-based and duplex nucleic acid-based assays are combined to reduce false positives to a very low level, lower reagent costs, and significantly expand the detection capabilities of this biosensor. This article provides an overview of the current design and operation of the APDS. Certain sub-components of the ADPS are described in detail, including the aerosol collector, the automated sample preparation module that performs multiplexed immunoassays with confirmatory PCR, and the data monitoring and communications system. Data obtained from an APDS that operated continuously for 7 days in a major U.S. transportation hub is reported.     

Automated methods for multiplexed pathogen detection

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides "live vs. dead" capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.     

Development of a Multiplexed Bead-Based Suspension Array for the Detection and Discrimination of Pospiviroid Plant Pathogens

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies.     

Dendrimer-like DNA-based fluorescence nanobarcodes

A major challenge in clinical diagnostics and environmental analysis is the difficulty in rapid and sensitive detection of multiple target molecules simultaneously (i.e., multiplexed detections). Our group has designed and synthesized a dendrimer-like DNA (DL-DNA) that is multivalent and anisotropic; using this unique DNA structure, we have developed a fluorescence-tagged nanobarcode system for multiplex detection. This nanobarcode system allows the rapid and sensitive detection of multiple pathogens simultaneously using the ratios of two different fluorescent dyes, green and red, with which different DL-DNAs are labeled. The key step of our nanobarcode model lies in the monodisperse preparation of DL-DNA. Two methods, solution phase and solid phase, are presented here. With slight modifications, this platform technology can also be extended to the multiplexed detection of RNA and proteins. This protocol can be completed in 2-5 d.     

Multiplexed Biomolecular Detection Based on Single Nanoparticles Immobilized on Pneumatically Controlled Microfluidic Chip

Plasmonics - A microfluidic chip integrated with pneumatically controlled valves was developed for multiplexed biomolecular detection via localized surface plasmonic resonance (LSPR) of single gold...     

Multiplexed mass spectrometric immunoassay in biomarker research: a novel approach to the determination of a myocardial infarct

Reported here is the development of a multiplexed mass spectrometric immunoassay (MSIA) for the detection of myocardial infarction (MI). The assay is the product of a study that systematically progresses from biomarker discovery--to identification and verification--to assay design, data analysis, and statistical challenge. During targeted population proteomics investigations, two novel biomarkers, serum amyloid A1alpha and S-sulfated transthyretin, were found to be responsive to MI. These putative markers were subsequently screened in larger cohorts of individuals to verify their responsiveness toward MI. Upon verification, a multiplexed assay was designed that was capable of simultaneously monitoring the new markers plus a previously established MI-marker (myoglobin). The multiplexed MSIA was applied to two 96-sample sets comprised of 48-MI/48-healthy and 19-MI/77-healthy, which served as training and case cohorts, respectively. Data evaluation using either preset reference levels or multivariate analysis exhibited sensitivities and specificities of >97%. These findings illustrate the importance of using systematic approaches in clinical proteomics to discover biomarkers and produce high-performance assays relevant to disease.     

Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma

The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 μl of plasma. Assay reproducibility was acceptable for verification studies, with median intra- and interlaboratory coefficients of variance above the limit of quantification of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope-labeled protein as an internal standard instead of stable isotope-labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-MRM-MS can be made reproducible across independent laboratories and has the potential to be adopted widely for assaying proteins in matrices as complex as plasma.