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3D Protein Microarrays: Performing Multiplex Immunoassays on a Single Chip
Abstract
The enzyme-linked immunosorbent assay (ELISA) is typically applied in the format of microtiter plates. To increase throughput and reduce consumption of precious samples, efforts have been made to transfer ELISA to the microchip format using conventional microarrays, microfluidic systems, and chips bearing microwells. However, all three formats lack the possibility to screen several analytes on several immobilized binders at a time or require complicated liquid handling, surface modifications, and additional equipment. Here, we describe an immunoassay performed on a standard microscope slide without the requirement for wells or tubes to separate the samples using standard surfaces and machinery already available for microarray technology. The new multiple spotting technique (MIST) comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on top of the immobilized binder. We show that the analytes bind their ligands immediately within the confined space of separate droplets on the chip surface, thereby eliminating the need for extra incubation time. We illustrate the feasibility of the new technique by spotting dilution rows of proteins or monoclonal and polyclonal antibodies on top of their immobilized binders. Moreover, we demonstrate specificity by applying a mixture of antibodies in a multiplex format and demonstrate that the technique is compatible with conventional microarray protocols, such as total incubation. Finally, we indicate that the technique is capable of quantifying as little as 400 zmol (240,000 molecules) of analyte.
... Especially for the simultaneous multicolour detection in analytical protein microarrays, such as antibody microarrays, bright and pH stable dyes with narrow emission and excitation spectra are optimal candidates (Angenendt, 2005). Commonly used scanners (nonconfocal or confocal) allow the application of up to four fluorophores simultaneously and permit direct comparison and relative quantification of up to four different samples (Angenendt et al., 2003a;Wingren and Borrebaeck, 2004). Alternative fluorescent protein labelling strategies involve semiconductor quantum dots, which are brighter and more photostable than organic dyes (Wu et al., 2003) and fluorophores linked to puromycin analogs, incorporated into the protein during in vitro translation (Doi et al., 2002). ...
... attomole) range in order to perform true proteome analysis (Kusnezow and Hoheisel, 2002). To date, most setups using fluorescent detection without signal amplification report limits of detection (LODs) in the ng/ml, i.e. nM to pM range, corresponding to total measures in the low femtoto attomole range (Miller et al., 2003;Pavlickova et al., 2003;Haab et al., 2001;Sreekumar et al., 2001;Angenendt et al., 2002Angenendt et al., , 2003a. Using custom made solid supports based on covalent coupling chemistry were able to further reduce assay sensitivities to the low pg/ml range. ...
... Adapted from traditional protein analysis, nitrocellulose is well-known as an excellent substrate for protein adsorption, providing both a preserving environment and binding capacities comparable to covalent coupling (Angenendt et al., 2003a). In our hands, nitrocellulose supports were compatible with mass spectrometric (Borrebaeck et al., 2001), colorimetric (paper I) as well as fluorescence detection (paper II, III, V) and showed excellent biocompatibility with the probe. ...
... The first issue, cross-reactivity can be minimized by the use of only one detecting antibody at a time but this limits multiplexing abilities. Another way to reduce cross-reactivity was introduced by Angenendt et al using the so-called 3D MA technology that uses a spotter device to treat the preprinted spots individually with the different affinity reagents (Angenendt et al 2003). Regarding the second issue, rigorous characterization and validation of candidate antibodies is needed to avoid false results. ...
... A fluorescently labeled secondary antibody was applied to detect molecular interactions. This method minimizes the required sample amount and eliminates the need of extra incubation times (Angenendt et al 2003). Another, novel concept was proposed by Pla-Roca and colleagues called antibody colocalizing microarray, where capture antibodies are spotted, and the chip is incubated with the sample then placed back onto the arrayer and then the detecting antibodies are spotted individually to their appropriate spots instead of treating the slide with a mixture of antibodies. ...
Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.
... Solution-based affinity binding is commonly characterized using methods such as UV-absorption [8], differential and titration calorimetry [9], matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) [10] and electrophoretic separation [11]. Surface-based affinity binding, which is widely used in affinity biosensors [12], can be investigated with methods such as protein arrays [13], immunoassays [14], and thermal-shift assays [15], which use fluorescent or radioactive labeling groups to signal binding events. Such labeling of target or receptor molecules is in general time-consuming and labor intensive, and is not capable of distinguishing signals from analytes in inactive and active forms [16]. ...
This paper presents label-free characterization of temperature-dependent biomolecular affinity binding on solid surfaces using a microcantilever-based device. The device consists of a Parylene cantilever one side of which is coated with a gold film and functionalized with molecules as an affinity receptor to a target analyte. The cantilever is located in a poly(dimethylsiloxane) (PDMS) microfluidic chamber that is integrated with a transparent indium tin oxide (ITO) resistive temperature sensor on the underlying substrate. The ITO sensor allows for real-time measurements of the chamber temperature, as well as unobstructed optical access for reflection-based optical detection of the cantilever deflection. To test the temperature-dependent binding between the target and receptor, the temperature of the chamber is maintained at a constant setpoint, while a solution of unlabeled analyte molecules is continuously infused through the chamber. The measured cantilever deflection is used to determine the target–receptor binding characteristics. We demonstrate label-free characterization of temperature-dependent binding kinetics of the platelet-derived growth factor (PDGF) protein with an aptamer receptor. Affinity binding properties including the association and dissociation rate constants as well as equilibrium dissociation constant are obtained, and shown to exhibit significant dependencies on temperature.
... Generally, protein immobilization has been achieved through one of the several methods including noncovalent adsorption (physical adsorption), covalent attachments onto chemically modified surfaces with glutaric dialdehydes or activated esters, and photoactivatable crosslinkers [13][14][15]. Among these methods, photo-activated crosslinkers, such as aryl azides [16][17][18] and aryldiazirine moieties [19][20][21], have been generally used to covalently immobilize proteins onto a solid substrate. ...
a b s t r a c t A simple and selective technique which immobilizes protein onto a solid substrate by using UV illu-mination has been developed. In protein immobilization, a Bovine serum albumin (BSA) performed bifunctional role as a cross-linker between substrate and proteins and as a blocker inhibiting a nonspecific protein adsorption. A new photo-induced protein immobilization process has been investigated at each step by fluorescence microscopy, ellipsometry, and Fourier transform infrared (FT-IR) spectroscopy. A UV photomask has been used to induce selective protein immobilization on target regions of the surface of the SiO 2 substrates under UV illumination with negligible nonspecific binding. The UV illumination also showed improved photostability than the conventional methods which employed bifunctional photo-crosslinker molecules of photo-reactive diazirine. This new UV illumination-based photo-addressable protein immobilization provides a new approach for developing novel protein microarrays for multi-plexed sensing as well as other types of bio-immobilization in biomedical devices and biotechnologies.
... The amount of serum required to develop an array containing over 10,000 features can be the same as that required to develop a single feature in a well of a 96-well plate. Genetix microarrayers have been used for a range of such applications [4][5][6][7][8] . There are some cases, however, in which microwells have an advantage in that they can be easier to handle with existing instrumentation. ...
Screening tens to hundreds of proteins for enzymatic activity or protein interactions can now be performed in a quantitative and economical manner using protein microarrays. Recent validated examples include assays for human protein kinases and protein interactions with p53. Here we describe the QArray series of instruments for the printing and assaying of such arrays. Of particular note is printing of arrays into microwell plates, a methodology that combines the benefits of arrays and the convenience of a conventional assay format.
... Multiplex immunoassays on a single chip can be performed on different surface modifications including 3-D immobilization matrices. (196) Generally, these are useful tools for the determination of chemical and biological warfare agents, and in food safety. Multitoxin sensor arrays exhibit an opportunity to develop miniaturized analytical devices that can be integrated into microfluidic systems. ...
Biosensors, as defined by Pure and Applied Chemistry, are ‘chemical sensors in which the recognition system utilizes a biochemical mechanism. The biological recognition system translates information from the biochemical domain, usually an analyte concentration, into a chemical or physical output signal with a defined sensitivity’.⁽¹⁾ It is also appointed that chemical or biological sensors contain two basic components connected in series: a chemical or biomolecular recognition system (receptor) and a physicochemical transducer. According to this prerequisite, this overlook is confined to sensor devices that combine a biomolecular recognition element with an optical signal transducer. Homogeneous or intracellular assays using fluorescent molecular probes or nanoparticles are not considered, although they are frequently termed as molecular sensors or nanosensors in the literature. Fluorescence-based biosensors are generalized as those devices that derive an analytical signal from a photoluminescent (either fluorescence or phosphorescence) emission process. Chemi- or bioluminescent detection systems are only briefly discussed in this review.
... The advantage of the microarray technology is that experiments can be carried out under essentially identical reaction conditions, while only using minute amounts of material for parallel interrogation. Conventional microarrays do not involve cells [302][303][304]. Cell-based microarrays have just begun to make inroads in the high-throughput screening field in the last few years [304], with potential applications such as global gene function analysis, selection of biomaterials, and drug cytotoxicity studies. ...
Biomedical manufacturing, which has seen rapid growth over the past decade, is an emerging research area for the manufacturing community. This growth trajectory is exemplified and coupled with a broadening scope of applications with biomedical manufacturing technology, including advancements in the safety, quality, cost, efficiency, and speed of healthcare service and research. The goal of this topical review is to offer a comprehensive survey of the current state-of-the-art in biomedical manufacturing and to summarize existing opportunities and challenges as a basis to guide future research activities in this emerging area. This paper categorizes the key manufacturing process types that are currently being leveraged for the biomedical field of use, including machining, joining, additive manufacturing, and micro/multi-scale manufacturing. For each of these manufacturing processes, notable applications are cited and discussed to provide insights and perspectives into how manufacturing processes can play an integral role in creating new and more sophisticated health care services and products.
... Nevertheless, this first approach was a success and they could show that the generated protein amount in the cell-free mix is high enough to get satisfying results in the microarray experiments [14]. The detriments of PISA, in particular the high amount of DNA and lysate needed for each experiment could be diminished with the multiple spotting technique [15] applied in 2006 for cell-free expression of proteins ( Fig. 1B) [16]. Basically the method is identical to PISA, but has one major difference-each spot on the array is treated twice. ...
Today, whole genomes are analysed by Next Generation Sequencing systems, or displayed by DNA microarray in situ synthesis. However, genomic data are mainly static and do not exactly reveal the complexity of protein-interaction networks of any organism or cell. To investigate protein interactions, the generation of protein microarrays, displaying whole proteomes is a prerequisite. But traditional protein microarray generation is time-consuming, costly and is restricted by a wide range of technical difficulties concerning cell culturing, protein purification and transfer onto microarray. Some of these obstacles can be bypassed by application of cell-free expression systems, enabling fast in situ synthesis of protein microarrays without need for cell culturing. This review provides a historical timeline of the different methods to generate protein microarrays by cell-free expression, highlights differences and similarities and reports the current state of the different approaches.This article is protected by copyright. All rights reserved
... The pool of polyclonal antibodies might be less sensitive to changes in the environment because if one of the epitopes are destroyed there are others that could be detected by other antibody fractions of the pool (38). The potential that lies in the polyclonal antibodies to recognize different types of epitope, both linear and structural epitopes (39) makes them useful in many kinds of application (40)(41)(42)(43)(44)(45)(46). Another advantage that makes the polyclonal antibodies so useful is that they are relatively easy to produce compared to other types of binders. ...
... These large populations of antibody types are, as a pool, also less sensitive to changes in the assay conditions; an increase in pH, temperature, salt concentration or denaturant may affect the antigen so that a 3-D epitope used by a certain antibody is disrupted or that binding to a linear sequence becomes impossible. The use of a population of different antibody types increases the chance that at least one type of antibody can still bind the target and therefore, they can be used in a variety of platforms (Angenendt et al., 2003;Engvall et al., 1971;Uhlén et al., 2005;Wide et al., 1967;Yalow et al., 1959). However, one major disadvantage is that polyclonal antibody production is not entirely reproducible; even if the same type of animal is immunized with an identical antigen, this could result in a different polyclonal serum. ...
... These large populations of antibodies are therefore, as a pool, less sensitive to changes in assay conditions and can often be used in a variety of different platforms (Wide et al., 1967;Engvall and Perlmann, 1971;Angenendt et al., 2003;. One disadvantage is the lack of reproducibility of polyclonal antibody production. ...
... These features have already found – and continue to lead to – a host of ap- plications [8], chiefly in creating tools for research in biology [9,10], and for chemical processing – e.g. high-throughput screening [11], immunoassay [12,13] , or development of organic reactions [14]. ...
The lecture will review the recent advances in the techniques for formation of bubbles of gas and droplets of liquid in two-phase
microfluidic systems. Systems comprising ducts that have widths of the order of 100 μm produce suspensions of bubbles and
droplets characterized by very narrow size distributions. These systems provide control over all the important parameters
of the foams or emulsions, from the volumes of the individual bubbles and droplets, through the volume fraction that they
occupy, the frequency of their formation, and the distribution of sizes, including monodisperse, multimodal and non-Gaussian
distributions. The lecture will review the fundamental forces at play, and the mechanism of formation of bubbles and droplets
that is responsible for the observed monodispersity.
... This is also important for additives used in the precursors for adjusting their viscosity to the needs of photolithography. Suitable additives are poly (vinyl alcohol) (PVA), polymeric sugars like Ficoll TM or dextran and poly (ethylene glycol) (PEG) and pHEMA (Angenendt et al. 2003;Barsky et al. 2003). In summary, it was shown that low-water-content hydrogels exhibit excellent properties for spatial controlled enzyme immobilization in microsystems. ...
Hydrogels are valuable materials for use in biosensors. They can be used for immobilization as well as for creating protecting
layers controlling diffusion and enhancing biocompatibility. Highly stable biosensors use hydrogels for entrapment of enzymes
on microelectrodes. The stability of enzymes in hydrogel membranes for biosensors can be enhanced by choosing the right microenvironment
using micro-hydrogels. The thermodynamic stability of the entrapped enzymes in micro-gels can be characterized via differential
scanning calorimetry (nano-DSC). Hydrogel-based biosensors were characterized by nano-DSC showing that hydrogel membranes
are excellent for creating long-term stable enzyme biosensors. Additionally, smart hydrogels can be used as stimuli responsive
materials enabling sensing as well as actuating performance.
KeywordsMulti-analyte biosensor–Microelectrode-array–Enzyme immobilization–Enzyme stability–Differential scanning calorimetry (DSC)–Chip-calorimeter
... Therefore, the analysis and quantification of E. coli in water and food by ELISA method has received widespread attention [9,10]. However, the shortcomings of ELISA method cannot be ignored, e.g., it is difficult, antibody preparation takes a long time and it has a high detection limit in protein analysis [11,12]. Moreover, some samples with small molecular weight have no immunogenic activity, and need be coupled to macromolecular proteins for ELISA detection. ...
Pathogenic Escherichia coli (E. coli) widely exist in Nature and have always been a serious threat to the human health. Conventional colony forming units counting-based methods are quite time consuming and not fit for rapid detection for E. coli. Therefore, novel strategies for improving detection efficiency and sensitivity are in great demand. Aptamers have been widely used in various sensors due to their extremely high affinity and specificity. Successful applications of aptamers have been found in the rapid detection of pathogenic E. coli. Herein, we present the latest advances in screening of aptamers for E. coli, and review the preparation and application of aptamer-based biosensors in rapid detection of E. coli. Furthermore, the problems and new trends in these aptamer-based biosensors for rapid detection of pathogenic microorganism are also discussed.
... Since the early 1980's, microfluidic systems have advanced significantly to satisfy the growing demand for the miniaturization of bioassay devices, with applications ranging from disease diagnostics [1][2][3][4] to cell behavior studies [5][6][7][8] . Here, the main goal has been to provide cheaper, simpler and more reliable means for simultaneous detection of multiple biomarkers, such as DNA and protein fragments, in a single system [9][10][11][12] . Out of the available platforms, microarray-based microfluidic bioassay devices are ris-the number of available recognition biomolecules in the devices, throughout the course of the assay. ...
Microfluidic systems integrated with protein and DNA micro- and nanoarrays have been the most sought-after technologies to satisfy the growing demand for high-throughput disease diagnostics. As the sensitivity of these systems relies on the bio-functionalities of the patterned recognition biomolecules, the primary concern has been to develop simple technologies that enable biomolecule immobilization within microfluidic devices whilst preserving bio-functionalities. To address this concern, we introduce a two-step patterning approach to create micro- and nanoarrays of biomolecules within microfluidic devices. First, we introduce a simple aqueous based microcontact printing (μCP) method to pattern arrays of (3-aminopropyl)triethoxysilane (APTES) on glass substrates, with feature sizes ranging from a few hundred microns down to 200 nm (for the first time). Next, these substrates are integrated with microfluidic channels, to then covalently couple DNA aptamers and antibodies with the micro- and nanopatterned APTES. As these biomolecules are covalently tethered to the device substrates, the resulting bonds enable them to withstand the high shear stresses originated from the flow in these devices. We further demonstrated the flexibility of this technique, by immobilizing multiple proteins onto these APTES-patterned substrates using liquid-dispensing robots to create multiple microarrays. Next, to validate the functionalities of these microfluidic biomolecule microarrays, we perform (i) aptamer-based sandwich immunoassays to detect human interleukin 6 (IL6); and (ii) antibody-based sandwich immunoassays to detect human c-reactive protein (hCRP) with the limit of detection at 4 nM, a level below the range required for clinical screening. Lastly, the shelf-life potential of these ready-to-use microfluidic microarray devices is validated by effectively functionalizing the patterns with biomolecules up to 3 months post-printing. In summary, with a single printing step, this aminosilane patterning technique enables the creation of functional microfluidic micro- and nano biomolecule arrays, laying the foundation for high-throughput multiplexed bioassays.
... Owing to the immune activity between antigen and antibody, ELISA method has superior specificity, high sensitivity and short detection time, and has been used in detection of different subtypes of E. coli [11][12][13]. However, due to the costly and cumbersome preparation process of antibodies, the detection cost of ELISA has increased greatly [14,15]. Another disadvantage is their limited shelf lives, which limits the viability of antibody-based biosensors in ELISA technology [16]. ...
An aptamer-based electrochemical biosensor was successfully developed and applied in the rapid detection of pathogenic Escherichia coli (E. coli) in licorice extract. The thiolated capture probes were firstly immobilized on a gold electrode, and then the biotinylated aptamer probes for E. coli were introduced by hybridization with the capture probes. Due to the stronger interaction between the aptamer and the E. coli, a part of the biotinylated aptamers will dissociate from the capture probes in the presence of E. coli. The residual biotinylated aptamer probes can quantitatively bind with streptavidin-alkaline phosphatase. Subsequently, α-naphthyl phosphate substrate was catalytically hydrolyzed to generate electrochemical response, which could be recorded by a differential pulse voltammetry. The dependence of the peak current on the logarithm of E. coli concentration in the range from 5.0 × 102 colony forming units (CFU)/mL to 5.0 × 107 CFU/mL exhibited a linear trend with a detection limit of 80 CFU/mL. The relative standard deviation of 5 successive scans was 5.3%, 4.5% and 1.1% for 5.0 × 102, 5.0 × 105 and 5.0 × 107 CFU/mL E. coli, respectively. In the detection of the licorice extract samples, the results obtained from the proposed strategy and traditional culture counting method were close to each other, but the time consumption was only ~1/30 compared with the traditional method. These results demonstrate that the designed biosensor can be potentially utilized for rapid microbial examination in traditional Chinese medicine and relevant fields.
... MIST allows multiplex analysis on a single microarray thereby overcoming one major disadvantage of microarray technology. By spotting the second reactant in the same position, in which the first component was fixed to the surface, multiple separate tests are performed in parallel, using minimal volumes of analyte (Angenendt et al., 2003a). ...
Microarray technology plays an increasing role in proteomic research. We give an overview about recent
developments in this technology focusing on molecular interaction studies using protein and antibody microarrays. We
report about technical aspects in the development of protein microarrays and describe different surfaces and detection
modes. Furthermore, we review the applications of protein microarrays in different molecular interaction studies including
interactions of proteins with antibodies, proteins, DNA, small molecules and enzymes. Advantages and limitations of the
microarray-based methods with other in vitro methods have been compared. We present the increasing applications of
protein and antibody microarrays in basic research, diagnostics, drug discovery, and in vitro-risk assessment of nutrients.
... The open microarray architecture is one of the major advantages of microarray technology allowing several antibodies to be screened on several antigens at the same time which requires complicated liquid handling. This is highlighted by the multiple spotting techniques (MIST), which comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on the top of the immobilized binder [32,33]. A major advantage of microarray technology is the production of functional proteins with methods such as the protein in situ microarray (PISA). ...
Phage-display technology constitutes a powerful tool for the generation of specific antibodies against a predefined antigen. The main advantages of phage-display technology in comparison to conventional hybridoma-based techniques are: (1) rapid generation time and (2) antibody selection against an unlimited number of molecules (biological or not). However, the main bottleneck with phage-display technology is the validation strategies employed to confirm the greatest number of antibody fragments. The development of new high-throughput (HT) techniques has helped overcome this great limitation. Here, we describe a new method based on an array technology that allows the deposition of hundreds to thousands of phages by micro-contact on a unique nitrocellulose surface. This setup comes in combination with bioinformatic approaches that enables simultaneous affinity screening in a HT format of antibody-displaying phages.
... Furthermore, the standard protein arrays are more difficult to store and to keep functional for longer periods of time than DNA arrays.These IVTT systems allow the expression of proteins in situ directly in the reaction wells or on a glass slide. In the multiple spotting technology (MIST), for example,68 the DNA is spotted on the slide in a first round of spotting, followed by a second round where an Escherichia coli-based IVTT mixture is spot in the same place. With the DNA array to protein array (DAPA) approach, it becomes possible to repeatedly print at least twenty copies of a protein array from one DNA template array.69 ...
The analysis of protein interaction networks is one of the key challenges in the study of biology. It connects genotypes to phenotypes, and disruption often leads to diseases. Hence, many technologies have been developed to study protein‐protein interactions (PPIs) in a cellular context. The expansion of the PPI technology toolbox however complicates the selection of optimal approaches for diverse biological questions. This review gives an overview of the binary and co‐complex technologies, with the former evaluating the interaction of two co‐expressed genetically tagged proteins, and the latter only needing the expression of a single tagged protein or no tagged proteins at all. Mass spectrometry is crucial for some binary and all co‐complex technologies. After the detailed description of the different technologies, the review compares their unique specifications, advantages, disadvantages, and applicability, while highlighting opportunities for further advancements.
... Several alternative technologies have been developed to overcome these limitations, namely, direct synthesis of proteins in situ from the corresponding DNA arrays by means of cell-free expression systems, such as the protein in situ array, nucleic acid programmable protein array (NAPPA), or DNA array to protein array (DAPA; Angenendt, Glökler, Konthur, Lehrach, & Cahill, 2003;Angenendt, Kreutzberger, Glökler, & Hoheisel, 2006;He et al., 2008). In NAPPA, for instance, the DNA encoding the relevant proteins is printed onto the array from which proteins are simultaneously produced with an in vitro transcription-translation system. ...
Protein‐protein interactions (PPIs) represent an essential aspect of plant systems biology. Identification of key protein players and their interaction networks provide crucial insights into the regulation of plant developmental processes as well as into interactions of plants with their environment. Despite the great advance in the methods for the discovery and validation of PPIs, still several challenges remain. First, the PPI networks are usually highly dynamic and the in vivo interactions are often transient and difficult to detect. Therefore, the properties of the PPIs under study need to be considered to select the most suitable technique, because each has its own advantages and limitations. Second, besides knowledge on the interacting partners of a protein of interest, characteristics of the interaction, such as the spatial or temporal dynamics, are highly important. Hence, multiple approaches have to be combined to obtain a comprehensive view on the PPI network present in a cell. Here, we present the progress in commonly used methods to detect and validate PPIs in plants with a special emphasis on the PPI features assessed in each approach and how they were or can be used for the study of plant interactions with their environment.
... A couple of years after the development of the NAPPA method, a technique called MIST emerged ( Figure 4C). The authors applied a multiple spotting technique (Angenendt, Glökler et al. 2003) for the detection of the fluorescent signal and protein expression in nanoliter volume (Angenendt, Nyarsik et al. 2004). Later, they used this methodology to develop in situ synthesized protein arrays (Angenendt, Kreutzberger et al. 2006). ...
The incorporation of cell-free transcription and translation systems into high-throughput screening applications enables the in situ and on-demand expression of peptides and proteins. Coupled with modern microfluidic technology, the cell-free methods allow the screening, directed evolution, and selection of desired biomolecules in minimal volumes within a short timescale. Cell-free high-throughput screening applications are classified broadly into in vitro display and on-chip technologies. In this review, we outline the development of cell-free high-throughput screening methods. We further discuss operating principles and representative applications of each screening method. The cell-free high-throughput screening methods may be advanced by the future development of new cell-free systems, miniaturization approaches, and automation technologies.
Proteomic approaches play an important role in the study of complex biological systems. The application of
proteomic technologies in plant science has been strongly supported by the completion of genome sequence projects of
the model plants Arabidopsis thaliana and rice. This review focuses on the state of proteomic technologies with special
emphasis on their application in plant biology. An overview of recent developments in 2-dimensional gel electrophoresis
and liquid chromatography-based multidimensional protein identification technology, MudPIT is provided. These
techniques are commonly combined with mass spectrometric methods for identification of proteins. Furthermore, protein
expression profiling by antibody arrays and the selection of required recombinant antibodies by phage display are
described. Interaction studies, using functional protein microarrays or the yeast two-hybrid system are presented as
powerful techniques to gain insights into the function of proteins. Advantages and limitations of the described methods as
well as their current and potential future applications in plant research are discussed.
The present paper reports a fully automated microfluidic system for the DNA amplification process by integrating an electroosmotic pump, an active micromixer and an on-chip temperature control system. In this DNA amplification process, the cell lysis is initially performed in a micro cell lysis reactor. Extracted DNA samples, primers and reagents are then driven electroosmotically into a mixing region where they are mixed by the active micromixer. The homogeneous mixture is then thermally cycled in a micro-PCR (polymerase chain reaction) chamber to perform DNA amplification. Experimental results show that the proposed device can successfully automate the sample pretreatment operation for DNA amplification, thereby delivering significant time and effort savings. The new microfluidic system, which facilitates cell lysis, sample driving/mixing and DNA amplification, could provide a significant contribution to ongoing efforts to miniaturize bio-analysis systems by utilizing a simple fabrication process and cheap materials.
Surface modification using living radical polymerization (LRP) chemistry is a powerful technique for surface modification of polymeric substrates. This research demonstrates the ability to use LRP as a polymer substrate surface-modification platform for covalently grafting polymer chains in a spatially and temporally controlled fashion. Specifically, dithiocarbamate functionalities are introduced onto polymer surfaces using tetraethylthiuram disulfide. This technique enables integration of LRP-based grafting for the development of an integrated, covalent surface-modification method for microfluidic device construction. The unique photolithographic method enables construction of devices that are not substrate-limited. To demonstrate the utility of this approach, both controlled fluid flow and cell patterning applications were demonstrated upon modification with various chemical functionalities. Specifically, poly(ethylene glycol) (375) monoacrylate and trifluoroethyl acrylate were grafted to control fluidic flow on a microfluidic device. Before patterning, surface-functionalized samples were characterized with both goniometric and infrared spectroscopy to ensure that photografting was occurring through pendant dithiocarbamate functionalities. Near-infrared results demonstrated conversion of grafted monomers when dithiocarbamate-functionalized surfaces were used, as compared to dormant control surfaces. Furthermore, attenuated total reflectance/infrared spectroscopy results verified the presence of dithiocarbamate functionalities on the substrate surfaces, which were useful in grafting chains of various functionalities whose contact angles ranged from 7 to 86°. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1404–1413, 2006
In recent years, array-based technologies, including DNA microarrays and protein-detecting microarrays have been getting more attractive and promising as high-throughput research/diagnostic tools. DNA microarrays comprise oligo-DNA immobilized onto solid surfaces. Whereas protein-detecting microarrays are categorized in several formats depending on classes of capturing agents that selectively bind to proteins of interest. Protein-based capturing agents such as antibodies and fusion proteins are commonly immobilized onto solid surfaces. Alternatively, DNA/RNA aptamers, synthetic peptides, and small molecules including drugs and carbohydrates also act as capturing agents. In particular, peptides as capturing agents are very versatile, because they are chemically prepared according to the well-established standard synthetic procedures and enable site-specific and directed surface modifications. In this review, we describe the basic features of protein-detecting microarray technologies (capturing agents, surface chemistry, and signal generation/readout); thereafter we suggest that the use of synthetic peptides is a very promising and powerful tool to monitor not only protein–peptide interactions but also enzymatic activities.
Optical detection method based protein chips for detection of the various pathogens such as Escherichia coli O157:H7, Salmonella typhimurium, Yersinia enterocolitica, and Legionella pneumophila in contaminated environment were developed. In order to endow the orientation of antibody molecules on solid surface, protein
G was introduced. Gold (Au) surface was modifi ed with 11-mercaptoundecanoic acid (11-MUA) and the protein G was immobilized
on the Au surface. And the spots of different antibodies against pathogens (E. coli O157:H7, S. typhimurium, Y. enterocolitica, and L. pneumophila) on protein G of Au surface were arrayed using a microarrayer. The responses of the various pathogens such as E. coli O157:H7, S. typhimurium, Y. enterocolitica, and L. pneumophila to the protein chip was investigated by surface plasmon resonance (SPR), fl uorescence microscopy and imaging ellipsometry
(IE). The lowest detection limit of the fl uorescence based protein chip was 102 CFU/mL and the protein chip using IE could successfully detect the pathogens in concentrations varying from 103 to 107 CFU/mL.
Keywords: Fluorescence microscopy, imaging ellipsometry, protein chip, pathogen, protein G, surface plasmon resonance
Enzyme immunoassays (EIAs) based on electrochemical detection offer several potential advantages and have been applied in clinical, medical, biotechnological, food and environmental analysis. Among the enzyme labels employed, horseradish peroxidase (HRP), alkaline phosphatase (ALP) and glucose oxidase (GOx) are the most common. This brief review reflects recent advances, challenges, and trends of electrochemical EIAs focusing on HRP, ALP or GOx as labels over the past five years. We especially emphasize current development of EIAs combined with other developments, including nanotechnology and miniaturization.
This review is an account of our efforts to develop a versatile and flexible microfluidic technology for surface-processing applications and miniatur- izing biological assays. The review is presented in the context of current trends in microfluidic technology and addresses some of the major chal- lenges for confining chemical and biochemical processes on surfaces: the sealing of a microchannel with a surface, the world-to-chip interface, the displacement of liquids in small conduits, the sequential delivery of multiple solutions, the accurate patterning of surfaces, the coincident detection of various analytes, and the detection of analytes in a small and dilute sample. Our solutions to these problems include the use of reversible sealing, capillary phenomena for powering and controlling liquid transport, and non-contact microfluidics for spotting and drawing (on surfaces) with flow conditions. These solutions offer many advantages over conventional tech- niques for handling minute amounts of liquids and may find applications in lithography, biopat- terning (e.g., the patterning of biomolecules), diagnostics, drug discovery, and also cellular assays.
Microcantilevers have caught great attention as label-free and ultra-sensitive biological sensors. When molecular adsorption
occurs on only one surface of cantilever, the resulting differential surface stress leads to cantilever bending, thus providing
a method of detecting molecular adsorption (Figure 2.1). How does the transduction from molecular adsorption to surface stress
change occur? Though the underlying science of the transduction is yet to be completely understood, the thermodynamic argument
suggests that the reaction-induced free energy reduction on one cantilever surface is balanced by the strain energy increase
due to bending, such that at equilibrium the free energy of the whole system reaches the minimum [11]. In other words, the penalty of increasing the strain energy must be compensated by a larger reduction in free energy due
to reaction, reflecting the interplay between mechanics and chemistry. Hence, the cantilever bending can be construed as a
measure of free energy reduction due to the chemical reaction on one surface. What isworth noting is that because free energy
reduction is common for all reactions, the cantilever-based sensing is universal platform for studying reactions. The ability
of analyzing molecules without the use of optical or radioactive labels makes this approach rather attractive for biology
and medicine.
With the thrust of scientific endeavormoving from genomics to proteomics, the protein array provides a powerful means by which
to examine hundreds to thousands of proteins in parallel. A result of the many genome projects has been the advance of automation
and robotic procedures to manipulate biomolecules using a high-throughput, systematic approach. The promise of the protein
microarray is the ability to interrogate a large number of proteins simultaneously in a high-density format for disease diagnosis,
prognosis or efficacy of therapeutic regime as well as for biochemical analysis. Similar to aDNAmicroarray, each spot on a
protein array can be identified based on its addressability on the planar surface.
IntroductionGeneral Considerations for the Automation of Antibody GenerationDevelopment of an Antibody Generation PipelineConclusion
Antibody microarrays are routinely employed in the lab and in the clinic for studying protein expression, protein-protein, and protein-drug interactions. The microarray format reduces the size scale at which biological and biochemical interactions occur, leading to large reductions in reagent consumption and handling times while increasing overall experimental throughput. Specifically, antibody microarrays, as a platform, offer a number of different advantages over traditional techniques in the areas of drug discovery and diagnostics. While a number of different techniques and approaches have been developed for creating micro and nanoscale antibody arrays, issues relating to sensitivity, cost, and reproducibility persist. The aim of this review is to highlight current state-of the-art techniques and approaches for creating antibody arrays by providing latest accounts of the field while discussing potential future directions.
Protein is one of major bio-functional performers. As one of several crucial proteomic research approaches, protein microarray has these following advantages: high-throughput, high sensitivity, quick detection and so on. Meanwhile, there are some critical factors that are important to the further development of protein microarray technology, for example, how to express and purify proteins for the research of protein microarray, how to immobilize proteins onto the substrate and keep the bio-function of proteins immobilized. Nano-biotechnology and cell-free expression system have been used to fabricate protein microarray by the way of immobilizing target genes onto the substrate and directly expressing corresponding proteins, which provides a new strategy to fabricate more complicated microarray. The stragegy and its progress were summarized-fabrication of protein microarray based on DNA, including immobilization of target genes, cell-free expression to proteins, immobilization of renascence proteins, advantages and drawbacks of the methods of protein chip fabrication etc.
A selective laser foaming process is developed to fabricate localized three-dimensional (3D) tissue scaffolds with biodegradable polymer, polylactic acid (PLA). Such scaffolds could be used in an array for high throughput, tissue-based biomedical assays. In this study, the effects of major process parameters and the morphology of resulting porous structure were investigated. The results show that laser foaming of gas impregnated polylactic acid generates inverse cone shaped wells with interconnected porous structure on the wall. The size of the well can be controlled with laser power and exposure time, while the pore size of the scaffold can be manipulated with the saturation pressure. Copyright © (2014) by the Society of Manufacturing Engineers All rights reserved.
Protein adsorption onto polymer surfaces is a very complex and ubiquitous phenomenon whose integrated process impacts essential applications in our daily lives such as food packaging materials, health devices, diagnostic tools, and medical products. Increasingly, novel polymer materials with greater chemical intricacy and reduced dimensionality are used for various applications involving adsorbed proteins on their surfaces. Hence, the nature of protein-surface interactions to consider is becoming much more complicated than before. A large body of literature exists for protein adsorption. However, most of these investigations have focused on collectively measured, ensemble-averaged protein behaviors that occur on macroscale and chemically unvarying polymer surfaces instead of direct measurements at the single protein or sub-protein level. In addition, interrogations of protein-polymer adsorption boundaries in these studies were typically carried out by indirect methods, whose insights may not be suitably applied for explaining individual protein adsorption processes occurring onto nanostructured, chemically varying polymer surfaces. Therefore, an important gap in our knowledge still exists that needs to be systematically addressed via direct measurement means at the single protein and sub-protein level. Such efforts will require multifaceted experimental and theoretical approaches that can probe multilength scales of protein adsorption, while encompassing both single proteins and their collective ensemble behaviors at the length scale spanning from the nanoscopic all the way to the macroscopic scale. In this review, key research achievements in nanoscale protein adsorption to date will be summarized. Specifically, protein adsorption studies involving polymer surfaces with their defining feature dimensions and associated chemical partitions comparable to the size of individual proteins will be discussed in detail. In this regard, recent works bridging the crucial knowledge gap in protein adsorption will be highlighted. New findings of intriguing protein surface assembly behaviors and adsorption kinetics unique to nanoscale polymer templates will be covered. Single protein and sub-protein level approaches to reveal unique nanoscale protein-polymer surface interactions and protein surface assembly characteristics will be also emphasized. Potential advantages of these research endeavors in laying out fundamentally guided design principles for practical product development will then be discussed. Lastly, important research areas still needed to further narrow the knowledge gap in nanoscale protein adsorption will be identified.
Biosensors, as defined by Pure and Applied Chemistry, are ‘chemical sensors in which the recognition system utilizes a biochemical mechanism. The biological recognition system translates information from the biochemical domain, usually an analyte concentration, into a chemical or physical output signal with a defined sensitivity’.⁽¹⁾ It is also appointed that chemical or biological sensors contain two basic components connected in series: a chemical or biomolecular recognition system (receptor) and a physicochemical transducer. According to this prerequisite, this overlook is confined to sensor devices that combine a biomolecular recognition element with an optical signal transducer. Homogeneous or intracellular assays using fluorescent molecular probes or nanoparticles are not considered, although they are frequently termed as molecular sensors or nanosensors in the literature.Fluorescence-based biosensors are generalized as those devices that derive an analytical signal from a photoluminescent (either fluorescence or phosphorescence) emission process. Chemi- or bioluminescent detection systems are only briefly discussed in this article.
Poly(thionine)-Au, a novel multifunctional substrate with excellent redox signal, enzyme-like activity, and easy antibody immobilisation, was synthesised using HAuCl4 as the oxidising agent and thionine as the monomer. The prepared poly(thionine)-Au composite exhibited an admirable electrochemical redox signal at -0.15 V and excellent H2O2 catalytic ability. In addition, gold nanoparticles in this composite were found to directly immobilise antibodies and further improve conductivity. In addition, a label-free electrochemical immunosensor was developed using poly(thionine)-Au as the sensing substrate for ultrasensitive detection of cytokeratin antigen 21-1 (CYFRA 21-1), an immunoassay found in human serum. The prepared immunosensor showed a wide liner range from 100 ng mL⁻¹ to 10 fg mL⁻¹ and an ultralow detection limit of 4.6 fg mL⁻¹ (the ratio of signal to noise (S/N) = 3). Additionally, this method was used to analyse human serum samples and yielded results consistency with those of ELISA, implying its potential application in clinical research. The poly(thionine)-Au composite can be easily extended to other polymer-based nanocomposites, which is significant for other electrochemical immunoassays.
Here we describe the design and evaluation of a fluidic device for the automatic processing of microarrays, called microarray processing station or MPS. The microarray processing station once installed on a commercial microarrayer allows automating the washing, and drying steps, which are often performed manually. The substrate where the assay occurs remains on place during the microarray printing, incubation and processing steps, therefore the addressing of nL volumes of the distinct immunoassay reagents such as capture and detection antibodies and samples can be performed on the same coordinate of the substrate with a perfect alignment without requiring any additional mechanical or optical re-alignment methods. This allows the performance of independent immunoassays in a single microarray spot.
In answer to the ever-increasing need in biomolecular research and clinical diagnostics to carry out many assays simultaneously in on tube, several microcarrier-based multiplex technologies (suspension arrays) have arisen in the past few years. Simultaneous detection of different target molecules that are present in one sample is possible by incubating the sample with a mixture of differently encoded microcarriers, each carrying another probe which can specifically interact with one of the targets. This means that each target will bind to a differently encoded microcarrier. When the targets are caught, several methods exist to label those 'positive' microcarriers. By means of this label, and by means of the code, it becomes possible to verify whether a target was caught at its surface, and which target was caught, respectively. Those multiplex measurements work quantitatively, because the more a target is present in the sample, the more targets will bind to their corresponding microcarrier. Five years ago, our research group proposed the use of spatial selective photobleaching, as an alternative method for the development of digitally encoded microcarriers, which were called 'memobeads'. It was suggested that this method could overcome the multiplexing limitations of existing technologies. The present study aimed to optimize the surface characteristics of t hose memobeads, and to verify whether they could then be applied to multiplex protein tests and nucleic acid tests. Furthermore, it was investigated in which way these memobead assays (and in general the assays performed with every kind of suspension arrays) could be improved to make them more efficient and sensitive.
The immobilization of proteins and their molecular interactions on various polymer-modified glass substrates [i.e. 3-aminopropyltriethoxysilane (APTS), 3-glycidoxypropyltrimethoxysilane (GPTS), poly (ethylene glycol) diacrylate (PEG-DA), chitosan (CHI), glutaraldehyde (GA), 3-(trichlorosilyl)propyl methacrylate (TPM), 3'-mercaptopropyltrimethoxysilane (MPTMS), glycidyl methacrylate (GMA) and poly-l-lysine (PL).] for potential applications in a nanoarray protein chip at the single-molecule level was evaluated using prism-type dual-color total internal reflection fluorescence microscopy (dual-color TIRFM). A dual-color TIRF microscope, which contained two individual laser beams and a single high-sensitivity camera, was used for the rapid and simultaneous dual-color detection of the interactions and colocalization of different proteins labeled with different fluorescent dyes such as Alexa Fluor® 488, Qdot® 525 and Alexa Fluor® 633. Most of the polymer-modified glass substrates showed good stability and a relative high signal-to-noise (S/N) ratio over a 40-day period after making the substrates. The GPTS/CHI/GA-modified glass substrate showed a 13.5-56.3% higher relative S/N ratio than the other substrates. 1% Top-Block in 10 mM phosphate buffered saline (pH 7.4) showed a 99.2% increase in the blocking effect of non-specific adsorption. These results show that dual-color TIRFM is a powerful methodology for detecting proteins at the single-molecule level with potential applications in nanoarray chips or nano-biosensors.
Antibody-based biosensors provide sensitive and rapid analytical tools for the detection of a range of pathogens and associated
toxins. In this paper, the recent progress in antibody-based biosensors for environmental monitoring is reviewed with particular
emphasis on generation and immobilization methods of antibody capture probes. We also describe the current available data
on antibody-based detection of pathogens and toxins. Optimal assay design and the strengths and limitations of current sensor
technologies for detection of biological agents in environment on site are also discussed.
Recent progress in molecular biology has made available several biotechnological tools that take advantage of the high detectability and rapidity of bioluminescence and chemiluminescence spectroscopy. These developments provide inroads to in vitro and in vivo continuous monitoring of biological processes (e.g. gene expression, protein–protein interaction and disease progression), with clinical, diagnostic and drug discovery applications. Furthermore, combining luminescent enzymes or photoproteins with biospecific recognition elements at the genetic level has led to the development of ultrasensitive and selective bioanalytical tools, such as recombinant whole-cell biosensors, immunoassays and nucleic acid hybridization assays. The high detectability of the luminescence analytical signal makes it appropriate for miniaturized bioanalytical devices (e.g. microarrays, microfluidic devices and high-density-well microtiter plates) for the high-throughput screening of genes and proteins in small sample volumes.
In this work, Cd3[Co(CN)6]2 and Cu3[Co(CN)6]2 (CdNCs and CuNCs) nanocubes were synthesized simply by a one-step process at room temperature in the presence of chitosan (CS). It was found that CdNCs and CuNCs produced obviously distinctive anodic peak currents at -0.7V and -0.1V (vs. Ag/AgCl), whose separation enabled differentiation between two analytes. They were used as novel electrochemical probes in multiplex electrochemical detection for carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) in a single run. The good performance of the new electrochemical probes was obtained. The linearity range was from 0.025 to 250ngmL(-1) for both CEA and AFP. The detection limit of CEA was 0.0175ngmL(-1) and that of AFP was 0.0109ngmL(-1) at a signal-to-noise of 3. Analysis of clinical serum samples using this immunosensor was well consistent with the data determined by the enzyme-linked immunosorbent assay (ELISA). The novel electrochemical probes could be generally used in multiplex protein detections.
A microarray containing three-dimensional (3D) tissue models is a promising substitute for the two-dimensional (2D) cell-based microarrays currently available for high throughput, tissue-based biomedical assays. A cell culture microenvironment similar to in vivo conditions could be achieved with biodegradable porous scaffolds. In this study, a laser foaming technique is developed to create an array of micro-scale 3D porous scaffolds. The effects of major process parameters and the morphology of the resulting porous structure were investigated. For comparison, cell culture studies were conducted with both foamed and unfoamed samples using T98G cells. The results show that by laser foaming gas-impregnated polylactic acid it is possible to generate an array of inverse cone shaped wells with porous walls. The size of the foamed region can be controlled with laser power and exposure time, while the pore size of the scaffold can be manipulated with the saturation pressure. T98G cells grow well in the foamed scaffolds, forming clusters that have not been observed in 2D cell cultures. Cells are more viable in the 3D scaffolds than in the 2D cell culture cases. The 3D porous microarray could be used for parallel studies of drug toxicity, guided stem cell differentiation, and DNA binding profiles.
A novel device comprising polydimethyl-siloxane (PDMS) microlenses bonded to a microfluidic compact disk (CD) is proposed for enzyme-linked immunosorbent assay (ELISA) applications. The PDMS microlenses were fabricated using a simple soft replica molding method and were bonded to the microfluidic CD using oxygen plasma treatment. A commercial software tool (ZEMAX) has been used to analyze the focal length of the microlens. A laser-induced fluorescence bio-detection system, consisting of the integrated microfluidic CD/PDMS microlenses and an optical detection module, was constructed and used to examine the enzymatic reaction of 3-(4-hydroxy) phenly propionic acid. The experimental results show that the PDMS microlens focusing effect yields a significant improvement in the intensity of the detected fluorescence signals. As a result, the proposed device represents an ideal solution for ELISAs and other high-sensitivity bio-detection applications.
We present a method to fabricate, assemble dope-coded biosensing particles, and demonstrate a scalable high throughput protein detection application. The coded biosensing particles (8 mum in diameter and 280 nm thick) are composed of biosensing/coding/magnetic/adhesive layers and coded via patterned boron doping. Coding via doping is powerful in that it can be easily decoded, permits scalability of bit sizes down to tens of nanometers, generates a large number of codes, and retains uniform particle size and shape independent of particle code for consistent protein analysis. Following suspension phase protein binding, the dope-coded biosensing particles are extracted with an external magnet and analyzed with an atomic force microscope.
We describe a novel label-free method to analyse protein interactions on microarrays as well as in solution. By this technique the time resolved native protein fluorescence in the UV is probed. The method is based on alterations of the protein upon ligand binding, and, as a consequence, of alterations of the environment of the proteins' aromatic amino acids. These amino acids act as internal probes, and as a result, the fluorescence lifetime of the proteins change due to binding to a ligand partner such as another protein. We were able to demonstrate the feasibility of the method with many compounds, including protein-protein, protein-antibody, protein-nucleic acid and protein-small ligand pairs. Unlike to many other label-free techniques, the sensitivity of the method does not depend on the size of the counterbinding ligand and therefore is particularly suitable for drug monitoring, when small molecules are involved.
Recent developments in proteomic technologies have enabled the high-throughput, multiplex measurement of large panels of antibodies in biological fluids of patients with immune-driven diseases. Antigen microarrays are increasingly being used to delineate the natural history of autoantibody formation and epitope spread, and thus gain insight into the pathogenesis of autoimmune diseases, as well as into host immunity and its shortcomings. Characterization of autoimmunity that precedes the onset of clinically apparent disease has the potential to guide disease prevention using either conventional immunosupression or novel, antigen-specific tolerizing therapies. In addition, autoantibody profiling has the potential to identify molecular subtypes of a disease, which could allow for prediction of disease outcomes such as severity, tissue damage, and response to therapy.
We have developed a novel protein chip technology that allows the high-throughput analysis of biochemical activities, and used this approach to analyse nearly all of the protein kinases from Saccharomyces cerevisiae. Protein chips are disposable arrays of microwells in silicone elastomer sheets placed on top of microscope slides. The high density and small size of the wells allows for high-throughput batch processing and simultaneous analysis of many individual samples. Only small amounts of protein are required. Of 122 known and predicted yeast protein kinases, 119 were overexpressed and analysed using 17 different substrates and protein chips. We found many novel activities and that a large number of protein kinases are capable of phosphorylating tyrosine. The tyrosine phosphorylating enzymes often share common amino acid residues that lie near the catalytic region. Thus, our study identified a number of novel features of protein kinases and demonstrates that protein chip technology is useful for high-throughput screening of protein biochemical activity.
Many new gene products are being discovered by large-scale genomics and proteomics strategies, the challenge is now to develop high throughput approaches to systematically analyse these proteins and to assign a biological function to them. Having access to these gene products as recombinantly expressed proteins, would allow them to be robotically arrayed to generate protein chips. Other applications include using these proteins for the generation of specific antibodies, which can also be arrayed to produce antibody chips. The availability of such protein and antibody arrays would facilitate the simultaneous analysis of thousands of interactions within a single experiment. This chapter will focus on current strategies used to generate protein and antibody arrays and their current applications in biological research, medicine and diagnostics. The shortcomings of these approaches, the developments required, as well as the potential applications of protein and antibody arrays will be discussed.
A transfer tool adjustment procedure for the generation of micro- and macroarrays is described. It is based on control spotting of solutions containing radioactive or fluorescent labels and the quantification of each obtained spot by standard image-analyzing software. This method provides a simple, rapid, and efficient way to control the quality and liquid delivery properties of spotting transfer tools.
Microarray technology has become a crucial tool for large-scale and high-throughput biology. It allows fast, easy and parallel detection of thousands of addressable elements in a single experiment. In the past few years, protein microarray technology has shown its great potential in basic research, diagnostics and drug discovery. It has been applied to analyse antibody-antigen, protein-protein, protein-nucleic-acid, protein-lipid and protein-small-molecule interactions, as well as enzyme-substrate interactions. Recent progress in the field of protein chips includes surface chemistry, capture molecule attachment, protein labeling and detection methods, high-throughput protein/antibody production, and applications to analyse entire proteomes.
Immunoassays are widely used for medical diagnostics and constitute the principal method of detecting pathogenic agents and thus of diagnosing many diseases. These assays, which are most often performed in well plates, would be greatly improved by a practical method to pattern a series of antigens on a flat surface and to localize their binding to many analytes. But no obvious method exists to expose a planar surface successively to a series of antigens and analytes. Here, we present miniaturized mosaic immunoassays based on patterning lines of antigens onto a surface by means of a microfluidic network (muFN). Solutions to be analyzed are delivered by the channels of a second muFN across the pattern of antigens. Specific binding of the target antibodies with their immobilized antigens on the surface results in a mosaic of binding events that can readily be visualized in one screening using fluorescence. It is thus possible to screen solutions for antibodies in a combinatorial fashion with great economy of reagents and at a high degree of miniaturization. Such mosaic-format immunoassays are compatible with the sensitivity and reliability required for immunodiagnostic methods.
This paper describes microfluidic devices that contain connections that can be opened by the user after fabrication. The devices are fabricated in poly(dimethylsiloxane) (PDMS) and comprise disconnected fluidic channels that are separated by 20 microm of PDMS. Applying voltages above the breakdown voltage of PDMS (21 V/microm) opened pathways between disconnected channels. Fluids could then be pumped through the openings. The voltage used and the ionic strength of the buffer in the channels determined the size of the opening. Opening connections in a specific order provides the means to control complex reactions on the device. A device for ELISA was fabricated to demonstrate the ability to store and deliver fluids on demand.
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