Supramolecular host-guest interaction for labeling and detection of cellular biomarkers

Be my guest: A supramolecular host-guest interaction is utilized for highly efficient bioorthogonal labeling of cellular targets. Antibodies labeled with a cyclodextrin host molecule bind to adamantane-labeled magnetofluorescent nanoparticles (see picture) and provide an amplifiable strategy for biomarker detection that can be adapted to different diagnostic techniques such as molecular profiling or magnetic cell sorting.     

Fluorescent probes for cellular assays

A fluorescent probe is a fluorophore designed to localize within a specific region of a biological specimen or to respond to a specific stimulus. Fluorescent probes have been used for nearly a century to study cellular processes due to their exquisite sensitivity and selectivity. Fluorescent probes have also gained in popularity as safety and environmental concerns over the use of radioactive probes have grown. At the same time, cellular assays are being more widely used now than ever before. This review will give a broad overview of types of fluorescent probes, types of fluorescent assays, and their application in cellular assays for a number of pharmaceutically relevant target classes.     

Carbohydrate biomarkers for future disease detection and treatment

Carbohydrates are considered as one of the most important classes of biomarkers for cell types,disease states,protein functions,and developmental states.Carbohydratebindersthat can specifically recognize a carbohydrate biomarker can be used for developing novel types of site specific delivery methods and imaging agents.In this review,we present selected examples of important carbohydrate biomarkers and how they can be targeted for the development of therapeutic and diagnostic agents.Examples are arranged ba...     

Use of quantum dots in the development of assays for cancer biomarkers

Biomarker assays may be useful for screening and diagnosis of cancer if a set of molecular markers can be quantified and statistically differentiated between cancerous cells and healthy cells. Markers of disease are often present at very low concentrations, so methods capable of low detection limits are required. Quantum dots (QDs) are nanoparticles that are emerging as promising probes for ultrasensitive detection of cancer biomarkers. QDs attached to antibodies, aptamers, oligonucleotides, or peptides can be used to target cancer markers. Their fluorescent properties have enabled QDs to be used as labels for in-vitro assays to quantify biomarkers, and they have been investigated as in-vivo imaging agents. QDs can be used as donors in assays involving fluorescence resonance energy transfer (FRET), or as acceptors in bioluminescence resonance energy transfer (BRET). The nanoparticles are also capable of electrochemical detection and are potentially useful for “lab-on-a-chip” applications. Recent developments in silicon QDs, non-blinking QDs, and QDs with reduced-size and controlled-valence further make these QDs bioanalytically attractive because of their low toxicity, biocompatibility, high quantum yields, and diverse surface modification flexibility. The potential of multiplexed sensing using QDs with different wavelengths of emission is promising for simultaneous detection of multiple biomarkers of disease.

Optofluidic detection for cellular phenotyping

Quantitative analysis of the output of processes and molecular interactions within a single cell is highly critical to the advancement of accurate disease screening and personalized medicine. Optical detection is one of the most broadly adapted measurement methods in biological and clinical assays and serves cellular phenotyping. Recently, microfluidics has obtained increasing attention due to several advantages, such as small sample and reagent volumes, very high throughput, and accurate flow control in the spatial and temporal domains. Optofluidics, which is the attempt to integrate optics with microfluidics, shows great promise to enable on-chip phenotypic measurements with high precision, sensitivity, specificity, and simplicity. This paper reviews the most recent developments of optofluidic technologies for cellular phenotyping optical detection.     

Bead injection for biomolecular assays: Affinity chromatography enhanced by bead injection spectroscopy

Selective capture of target biomolecules by ligands immobilized on a solid support is a cornerstone of two seemingly unrelated techniques: micro-Affinity Chromatography (microAC) and micro-Bead Injection Spectroscopy (microBIS). This work shows, for the first time, how these techniques can be carried out using the same instrument and how the data obtained this way complement each other, yielding complete information on retention and elution of target biomolecules. Biomolecular association and dissociation were investigated by microAC and microBIS, using computer-controlled programmable flow and the same instrument for automated bead transport, packing of a micro-column, assay of the analyte, and bead disposal. The absorbance of the analyte was monitored within the fiber optic flow cell configured either for monitoring directly on the beads or post-column after elution. The separation, binding, and elution of immunoglobulins (human IgG, rabbit IgG, and horse IgG) on protein G-coated Sepharose beads were studied as model systems. The limit of detection of the microAC technique was determined to be 5 ng microL(-1) IgG, and that of the microBIS technique was 50 ng microL(-1) IgG.     

Polymerization-assisted signal amplification for electrochemical detection of biomarkers

We present a novel immunosensor by using polymerization-assisted signal amplification strategy coupled with electrochemical detection. A sandwich immunoassay process was used to immobilize a polymerization reaction center, the initiator-conjugated polyclonal prostate specific antigen (PSA) or polyclonal carcinoembryonic antigen (CEA) antibodies on the surface of the electrode. Activator generated electron transfer for atom transfer radical polymerization (AGET ATRP) subsequently triggered the local accumulation of glycidyl methacrylate (GMA) monomers. Growth of long chain polymers provided excess epoxy groups for electrochemical tags aminoferrocene (FcNH(2)) coupling, which in turn significantly increased the loading of the signal molecules and enhanced the electrochemical readouts. The detection limit was ～0.14 pg mL(-1) for PSA and ～0.10 pg mL(-1) for CEA in PBS buffers. The proposed immunosensor was highly sensitive, selective and has a good match to the clinical electrochemiluminescent method. This suggested that the polymerization-assisted immunosensing strategy could be used as an effective method to significantly enhance signal output of the sandwich immunoassays and acted as a promising platform for the clinical screening of cancer biomarkers.     

Evaluation of phenolic assays for the detection of nitrite

A series of generic nitrite assay systems based on the single step nitrosation of phenol derivatives are presented. The chemical reactivity offered by the C-nitroso compounds provides an opportunity to pursue a number of analytical strategies of which three spectroscopic (UV/Vis) and two electrochemical options (linear sweep voltammetry/differential pulse voltammetry) were evaluated. The capacity for multiple detection options from a single analyte species without significant sample manipulation is a major advantage with each assay system providing sub ppm detection limits with linear ranges up to milli-molar concentrations of nitrite. The influence of common interferents such as nitrate, ascorbate and paracetamol was investigated. The applicability of the assay procedures to the analysis of authentic biological samples (saliva and urine samples) was assessed with the analytical accuracy independently corroborated with a standard Griess protocol. In addition, a brief comparison with alternative nitrite detection strategies is also presented.     

High-throughput ribozyme-based assays for detection of viral nucleic acids

Many reports have suggested that target-activated ribozymes hold potential value as detection reagents. We show that a "half"-ribozyme ligase is activated similarly by three unstructured oligoribonucleotides representing the major sequence variants of a hepatitis C virus 5'-untranslated region (5'-UTR) target and by a structured RNA corresponding to the entire 5'-UTR. Half-ribozyme ligation product was detected both in an ELISA-like assay and in an optical immunoassay through the use of hapten-carrying substrate RNAs. Both assay formats afford a limit of detection of approximately 1 x 10(6) HCV molecules (1.6 attomol, 330 fM), a sensitivity which compares favorably to that provided by standard immunoassays. These data suggest that target-activated ribozyme systems are a viable approach for the sensitive detection of viral nucleic acids using high-throughput platforms.     

Functional cellular assays with multiparametric silicon sensor chips

Multiparametric silicon sensor chips mounted into biocompatible cell culture units have been used for investigations on cellular microphysiological patterns. Potentiometric, amperometric and impedimetric microsensors are combined on a common cell culture surface on the chip with an area of approximately 29 mm2. Extracellular acidification rates (with pH-sensitive field effect transistors, ISFETs), cellular oxygen consumption rates (with amperometric electrode structures) and cell morphological alterations (with impedimetric electrode structures, IDES) are monitored on single chips simultaneously for up to several days. The corresponding test device accommodates six of such sensor chips in parallel, provides electronic circuitry and maintains the required cell culture conditions (temperature, fluid perfusion system). Sensor data are transformed into quantitative information about microphysiologic conditions. The outcome of this transformation as well as reliability and sensitivity in detection of drug effects is discussed. This is the first report on multiparametric cell based assays with data obtained solely with integrated sensors on silicon chips. Those assays are required in different fields of application such as pharmaceutical drug screening, tumor chemosensitivity tests and environmental monitoring.     

HPLC with UV-VIS spectrophotometric detection for blood and tissue drug assays

Summary Experience in blood and tissue drug assays by means of HPLC with UV-VIS detection is reported. More than 20 drugs or their analogues, which belong to many different therapeutic classes, are analyzed with a limited number of columns and of eluents (isocratic mode). A simple device to warm a reverse-phase column has also been tested. The guidelines applied here will be of interest to people who want to simplify the analysis of a wide range of drugs.     

Microchip-based electrochemical detection for monitoring cellular systems

The use of microchip devices to study cellular systems is a rapidly growing research area. There are numerous advantages of using on-chip integrated electrodes to monitor various cellular processes. The purpose of this review is to give examples of advancements in microchip-based cellular analysis, specifically where electrochemistry is used for the detection scheme. These examples include on-chip detection of single-cell quantal exocytosis, electrochemical analysis of intracellular contents, the ability to integrate cell culture/immobilization with electrochemistry, and the use of integrated electrodes to ensure cell confluency in longer-term cell culture experiments. A perspective on future trends in this area is also given.     

Microchip-based multiplex electro-immunosensing system for the detection of cancer biomarkers

Microfluidic-based microchips have become the focus of research interest for immunoassays and biomarker diagnostics. This is due to their aptitude for high-throughput processing, small sample volume, and short analysis times. In this paper, we describe the development of a microchip-based multiplex electro-immunosensing system for simultaneous detection of cancer biomarkers using gold nanoparticles and silver enhancer. Our microchip is composed of biocompatible poly(PDMS) and glass substrates. To fix the antibody-immobilized microbeads, we used pillar-type microfilters within a reaction chamber. An immunogold silver staining (IGSS) method was used to amplify the electrical signal that corresponded to the immune complex. To demonstrate this approach, we simultaneously assayed three cancer biomarkers, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate-specific antigen (PSA) on the microchip. The electrical signal generated from the result of the immunoreaction was measured and monitored by a PC-based system. The overall assay time was reduced from 3-8 h to about 55 min when compared to conventional immunoassays. The working range of the proposed microchip was from 10(-3) to 10(-1) microg/mL of the target antigen.     

An innovative strategy for direct electrochemical detection of microRNA biomarkers

We report an electrochemical method for direct, reagentless, and label-free detection of microRNA, based on a conjugated copolymer, poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-2-carboxyethyl-1,4-naphthoquinone), acting as hybridization transducer. Hybridization between the oligonucleotide capture probe and a microRNA target of 22 base pairs generates an increase in the redox current (“signal-on”), which is evidenced by square wave voltammetry. Selectivity is good, with little hybridization for non-complementary targets, and the limit of detection reaches 650 fM. It is also evidenced that this sensitivity benefits from the high affinity of DNA for RNA.

FRET-based polymer materials for detection of cellular microenvironments

Effective detection of cellular microenvironments and understanding of physiological activities in living cells remain a considerable challenge.In recent years,fluore scence(or Forster) resonance energy trans fe r(FRET) technology has emerged as a valuable method for real-time imaging of intracellular environment with high sensitivity,specificity and spatial resolution.Particularly,polymer-based imaging systems show enhanced stability,improved biodistribution,increased dye payloads,and amplified signal/noise ratio compared with small molecular sensors.This review summarizes the recent progress in FRET-based polymeric systems for probing the physiological environments in cells.     

Microfabricated ratchet structure integrated concentrator arrays for synthetic bacterial cell-to-cell communication assays

We describe a microfluidic concentrator array device that is integrated with microfabricated ratchet structures to concentrate motile bacterial cells in desired destinations with required cell densities. The device consists of many pairs of concentrators with a wide range of spacing distances on a chip, and allows cells in one concentrator to be physically separated from but chemically connected to cells in the other concentrator. Therefore, the device facilitates quantification of the effect of spacing distance on the cell-to-cell communication of synthetically engineered bacterial cells. In addition, the device enables us to control the cell number density in each concentrator unit by adjusting the concentration time and the density of cell suspensions, and the basic concentrator unit of the device can be repeatedly duplicated on a chip. Hence, the device not only facilitates an investigation of the effect of cell densities on cell-to-cell communication, but it can also be further applied to an investigation of cellular communication among multiple types of cells. Lastly, the device can be easily fabricated using a single-layered soft-lithography technology so that we believe it would provide a simple but robust means for many synthetic and systems biologists to simplify and speed up their investigations of the synthetic genetic circuits in bacterial cells.     

The potential of electrophoretic mobility shift assays for clinical mutation detection

As the understanding of the links between genetic mutations and diseases continues to grow, there is an increasing need for techniques that can rapidly, inexpensively, and sensitively detect DNA sequence alterations. Typically, such analyses are performed on PCR-amplified gene regions. Automated DNA sequencing by capillary array electrophoresis can be used, but is expensive to apply to large numbers of patient samples and/or large genes, and may not always reveal low-abundance mutations in heterozygous samples. Many different types of genetic differences need to be detected, including single-base substitutions and larger sequence alterations such as insertions, deletions, and inversions. Electrophoretic mobility shift assays seem well suited to this purpose and could be used for the efficient screening of patient samples for sequence alterations, effectively reducing the number of samples that must be subjected to full and careful sequencing. While there is much promise, many of the mobility shift assays presently under development have yet to be demonstrated to have the high sensitivity and specificity of mutation detection required for routine clinical application. Hence, further studies and optimization are required, in particular the application of these methods not only to particular genes but also to large numbers of patient samples in blinded studies aimed at the rigorous determination of sensitivity and specificity. This review examines the state-of-the-art of the most commonly used mobility shift assays for mutation detection, including denaturing gradient gel electrophoresis, TGGE, SSCP, heteroduplex analysis, and denaturing HPLC.     

New technologies in affinity assays to explore biological communication

The language that cells use to communicate consists of the small molecules, peptides, and proteins that are released into the extracellular environment. To decipher this language, analytical assays are needed that have high selectivity, high sensitivity, and fast temporal resolution. Affinity assays are a group of analytical methodologies that are adept at studying this communication. In this overview, we highlight several examples from the literature on various types of affinity assays used in different platforms to monitor biological communication of peptides and proteins.     

Competitive fluorescence polarization assays for the detection of phosphoinositide kinase and phosphatase activity

We describe the development and implementation of competitive fluorescence polarization (FP) based assays for determining activity of phosphoinositide 3-kinase (PI 3-K) and the type-II SH2-domain-containing inositol 5-phosphatase (SHIP2). These assays are based on the interaction of specific phosphoinositide binding proteins with fluorophore-labeled phosphoinositide and inositol phosphate tracers. Enzyme reaction products are detected by their ability to compete with the fluorescent tracers for protein binding, leading to an increase in the amount of free tracer and a decrease in polarization (mP) values. A variety of fluorophore-labeled tracers were evaluated, and assay sensitivity and specificity for products of PI 3-K and SHIP2 activity was determined. Assay performance was evaluated using recombinant PI 3-Kalpha and SHIP2 with diC(8)-PI(4,5)P(2) and diC(8)-PI(3,4,5)P(3) as respective substrates. IC(50) values for previously characterized PI 3-K inhibitors were within expected ranges. These assays are homogeneous, sensitive, and rapid, and suitable for HTS applications, and will facilitate screening for novel inhibitors of phosphoinositide kinases and phosphatases in drug development.