No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Next generation of protein microarray support materials: evaluation for protein and antibody microarray applications
Abstract
The performance of protein and antibody microarrays is dependent on various factors, one of which is the use of an appropriate microarray surface for the immobilisation of either protein or antibody samples. We have investigated the properties of seven new surfaces in the context of both protein and antibody microarray technology. We have demonstrated the functionality of all new slide coatings and investigated the mean signal to spotted concentration ratio, determined detection limits and calculated coefficients of variation. Moreover, new concepts for slide coatings such as dendrimer and poly(ethylene glycol)-epoxy slides were evaluated and improved qualities of novel slide surfaces were observed. Optimal slide coatings for antibody and protein chips were proposed and the requirements for both technologies were discussed.
... In fact, we have recently generated data questioning the printing performances due to significant and inconsistent antibody binding properties displayed by a common and frequently-used source plate. In contrast, the solid microarray surfaces should display high antibody binding capacity and biocompatibility, while any non-specific (background) binding should be minimized [5,[7][8][9][10][11]. In our efforts, a hydrophilic polymer slide, to which the antibodies are randomly adsorbed, have so far served as the best-performing solid support [5]. ...
... In this context, we have developed a recombinant antibody microarray technology platform for protein profiling of crude, directly-labelled proteomes [4][5][6]. The design and performance of antibody (protein) microarrays and how they are processed are dependent on several factors, of which the interplay between the antibodies (proteins) and the solid surfaces plays a pivotal role [5,[7][8][9][10][11][12][13]. In more detail, the solid surfaces, including both the source plate to which the antibodies are loaded prior to printing and the surface onto which the antibodies are printed, will have a direct impact on the printing performances, array format (slide and well based), assay performances, assay processing (degree of automation) and sensing (slide-and plate-based scanners) [8,14,15]. ...
... In more detail, the solid surfaces, including both the source plate to which the antibodies are loaded prior to printing and the surface onto which the antibodies are printed, will have a direct impact on the printing performances, array format (slide and well based), assay performances, assay processing (degree of automation) and sensing (slide-and plate-based scanners) [8,14,15]. Early development work by us and others has, however, focused almost entirely on the design and evaluation of slide-based planar solid supports for antibody microarrays [5,[7][8][9][10][11][12]. Albeit successful, resulting in high-performing antibody microarray set-ups, e.g., [5,16], the potential of (novel) solid surfaces has not been (re-)evaluated in recent years. ...
Antibody microarrays have emerged as an important tool within proteomics, enabling multiplexed protein expression profiling in both health and disease. The design and performance of antibody microarrays and how they are processed are dependent on several factors, of which the interplay between the antibodies and the solid surfaces plays a central role. In this study, we have taken on the first comprehensive view and evaluated the overall impact of solid surfaces on the recombinant antibody microarray design. The results clearly demonstrated the importance of the surface-antibody interaction and showed the effect of the solid supports on the printing process, the array format of planar arrays (slide- and well-based), the assay performance (spot features, reproducibility, specificity and sensitivity) and assay processing (degree of automation). In the end, two high-end recombinant antibody microarray technology platforms were designed, based on slide-based (black polymer) and well-based (clear polymer) arrays, paving the way for future large-scale protein expression profiling efforts.
... Various other slides like dendrimer slides (dendrimer layer with reactive epoxy groups), poly(ethylene glycol) (PEG)-epoxy slides (PEG layer with reactive epoxy groups), commercially available amine slides (amine groups (extended chain length silane)), epoxy slides (epoxy group), silanated slides (amine group), FAST slides (nitrocellulose based matrix) and polystyrene cell culture slide (polystyrene) have been investigated for efficient immobilization of proteins [73].For a uniform orientation of proteins, affinity tag surfaces can be used e.g. nickel coated slide for use with HisX6 [74]. ...
... Protein binds to surface by physical adsorption as shown in Figure 12.13(b). Although these surfaces are most favourable with regard to protein conformation, however they suffer from large variations in signal intensity [73]. iii. ...
... 14 Diagram for detection limits of different surface materials for antibody microarrays. (Reprinted from[73] with kind permission from Elsevier). ...
A microarray is an orderly arrangement (grid pattern) of sample molecules
(i.e. probes with known sequences) immobilized on a particular substrate (e.g. glass,
silicon, etc.) at microscopic length scale. The probe can be a DNA, protein, antibody/
antigen or enzyme. Therefore, an artificially synthesized grid pattern of probes can be
used to recognize unknown complementary gene sequences, amino acid sequences
within proteins, other biological entities by using antigen-antibody binding kinetics
or selective enzymatic activity on substrates in order to recognize levels of these substrates
in biofluids. Microarrays find wide usage in clinical diagnostics of diseases,
genome sequencing for sensing and therapeutics work, drug discovery, environmental
and toxicological research and so on. In summary, the microarrays are comprised
of two main elements i.e., (a) a probe molecule and (b) an analyte which needs to be
detected. Further, any microarray should have a signal transduction step enabling
the user to read out chemical kinetics happening at the surface. Thus patterning of
the probe molecule becomes a fundamental issue and patterning further necessitates
an organizing step which should be able to send and bind such probe molecules into
different regions. Thus, the interaction of the binding surface to the probe molecule
is a very important step in fabricating a microarray. In this chapter, we have focused
on different types of microarrays such as protein microarrays, DNA microarray and
antibody microarrays with respect to of their fabrication techniques, substrates (viz
glass, silicon, polymer, gold etc.) used adhesion issues and binding kinetics.
... Proteins can be immobilized onto substrates in a non-covalent interaction by adsorption on glass slides coated with poly-llysine [10], agarose [11], polyacrylamide, hydrogels [12], with membranes made with polystyrene, poly-vinylidene fluoride (PVDF), thin film nitrocellulose, hydrophobic or hydrophilic surfaces [13], or in a covalent manner with functionalized glass surface, such as epoxy group-functionalized slides [14]. A drawback of random covalent immobilization is a partial block of the active sites of the proteins, which become less accessible to the interacting molecules. ...
... Initially, the first studies using protein chips were focused on antibodies for antigen screens [15][16][17]. Glycerol is added to enhance the binding and stability of proteins: this effect has been related to a prolonged hydration of proteins [13]. ...
In the past few years, protein chips have become a powerful tool for diagnostics and analysis of protein functions and protein-protein interactions. This technology allows fast, easy and parallel detection of multiple addressable elements from a minimal amount of sample in a single experiment. It has been applied to analyse protein and protein-protein interaction as well as antibody-antigen interaction, enzyme-substrate and enzyme-inhibitor binding.
... The PEG can be capped with a succinimidyl ester to covalently bind the protein, while the polymer layer can be designed to maintain the hydration of the proteins, thus minimizing the chance for denaturation ( Figure 3C). Evaluations of the different surfaces available for constructing protein arrays have been performed and these results point out important advantages and disadvantages associated with these surfaces (Angenendt et al., 2002(Angenendt et al., , 2003; Kusnezow et al., 2003). ...
Introduction The traditional approach of studying expression of one gene at a time is very time-consuming and expensive. Consequently in 1995, researchers at Stanford University developed DNA microarrays, which is a tool to study expression of thousands of genes at a time. Initial microarrays were intended to study expression of human expressed sequenced tags. Since then, DNA microarrays have witnessed a tremendous amount of interest and investment because of their potential impact on the discovery process and human health. The microarrays have been compared to the development of silicon microchips mainly due to their rapid technology development and applications in many diverse areas. This emerging technology promises to unravel the functions of genes in this post-genomics period. The microarray industry consists of companies that manufacture coated substrates, oligonucleotide manufacturers, printers for robotic deposition of probes, kits for isolation and labeling of RNA targets, scanners for acquiring images, software for data analysis, and pre-fabricated/spotted arrays. Although pre-fabricated/spotted arrays are available in the market, researchers from academia prefer to make their own arrays mainly due to financial considerations. On the contrary, the industrial researchers prefer to use prefabricated arrays for genome-wide analysis. The technology consists of several sub-component processes, which are typically performed manually, thereby introducing variability. In addition, the technology suffers due to lack of standardized procedures across platforms and laboratories. The ultimate utility of the microarrays is yet to be determined as the technology is still under development and needs at least another decade to mature into a fully robust and automated technology suited for major human health applications, including clinical diagnostics. The following description provides an overview of microarray technologies and a glimpse of what to expect in the future. The Past Genetic information is carried in the form of deoxyribonucleic acid (DNA) in the genomes of all organisms. The size and composition of DNA sequence in the genome determines the form, function, and complexity of an organism. Thousands of genes and their products in living organisms provide a well-orchestrated functional response to the environment. Figure 1 pictorially represents the important role played by genes in human physiology. Some external or internal stimulus results in transcription of genes, which involves binding of the transcription factors to the promoter region(s) of the genes and synthesis of the messenger RNA molecules using the DNA strand as a template via RNA polymerase. The RNA is then processed, particularly in the eukaryotic cells and translated to form proteins in the cytoplasm using the ribosomal apparatus. Most of these translated proteins undergo modification(s) to become functionally active and carry out their physiological role. Until the mid 90's, gene expression analysis was typically conducted using conventional techniques such as Northern blotting. Northern blotting is a technique developed in the 70's (Sambrook et al., 1989), which enables researchers to characterize the expression of genes at the mRNA level. Northern blotting involves separating the RNA population in denaturing agarose gels, followed by transfer and immobilization of separated RNA onto a nylon or nitrocellulose membrane. The membrane is then hybridized with a radioactively labeled probe of a known genetic sequence that can interact with a single target of interest within the complex immobilized RNA mixture. After hybridization, the membrane is exposed to X-ray film and the positive signal from a band is utilized to determine i) whether the target was present in the complex mixture and ii) the relative abundance of that target. Thus, this method provided researchers a tool for characterizing gene activity, albeit at a very slow pace, since only one gene is typically characterized per experiment. Technological advances in the field of genomics have resulted in sequencing genomes from several organisms, including the human genome. The results of the human genome project were published in 2001 (Venter et al., 2001; International Human Genome Sequencing Consortium, 2001), with the major goals of identifying all of the genes in human DNA, determining the sequences of those genes and storing the information in public databases. The human genome project created a library of knowledge that allows researchers to take an active, discovery-focused approach to understand human physiology at the molecular level. This new, more scientific approach should ideally replace and/or provide synergies to the trial and error methods that are presently utilized for drug discovery in high throughput laboratories (Paterson, 2003). The ~30,000 genes which comprise the human
... 2,4,5 For example, studies have shown that variability between replicate spots on the same chip can be as high as 43%. 6 This variability comes, in part, from manufacturing imperfections and cross-reactivity; 2,7 however, even under ideal conditions the level of detection can still vary upwards of 1000× from one Ab to the next. 8 Moreover, some Abs lose activity altogether when placed on a surface. ...
Antibody microarrays have the potential to revolutionize molecular detection in scientific, medical, and other biosensor applications, but their current use is limited because of poor reliability. It is hypothesized that one reason for their poor performance results from strong antibody-surface interactions that destabilize the antibody structure and create steric interference for antigen recognition. Using a recently developed coarse-grain protein-surface model that has been parameterized against experimental data, antibody-surface interactions for two antibody orientations on two types of surfaces have been investigated. The results show that regardless of attachment geometry, antibodies tend to collapse onto hydrophobic
surfaces and exhibit lower overall stability compared to antibodies on hydrophilic
surfaces or in bulk solution. The results provide an unprecedented view into the dynamics of antibodies on surfaces and offer new insights into the poor performance exhibited by current antibody microarrays.
This article may be downloaded for personal use only. Any other use requires prior permission of the author and AIP Publishing.
The article appeared in The Journal of Chemical Physics 143, 061101 (2015) and may be found at http://dx.doi.org/10.1063/1.4928455.
... A variety of surface treatments for ASR attachment, using physical adsorption or covalent immobilization, have been developed. 23,24 If the droplet microfluidic technique is preferred to carry out an immunoassay, the washing step becomes arduous to remove the residual substances since various macromolecules are encapsulated in a droplet. In addition, the immobilization of ASRs also becomes difficult and must rely on special material, such as magnetic beads. ...
This paper presents a droplet-based immunoassay chip allowing each droplet to be positioned in a passive droplet-positioning cavern under continuous flow. In addition, the chip surface can immobilize any kind of histidine-tagged capture agents for performing simultaneous multiplex immunoassays. Distinct families of monodispersed droplets were generated since a diaphragm, which is a thin elastomeric flap film suspended from the top of the main channel, forms a double T junction for shearing the aqueous liquids by the carrier flow. These two types of monodispersed droplets traverse the main channel to the downstream detection area and enter the passive positioning caverns for further immunoassay. A layer of Ni–Co film was coated on the substrate by electrodeposition in order to immobilize the multiplex histidine-tagged capture molecules. In this study, the tumor suppressor protein p53 and the extracellular signal-related kinase 1 (ERK1) were used as the capture agents. Then, both histidine-tagged proteins p53 and ERK1 were immobilized by the Ni–Co layer in a microarray format for subsequent immunoassay and fluorescence detection. The experimental results show that the detected fluorescence intensity is proportioned to the concentration of the encapsulated content in a small droplet. This proposed droplet-based immunoassay chip can immobilize multiplex histidine-tagged proteins, irrelevant to the species of proteins, to carry out simultaneous immunoassays and allow the operation sequence to be conducted automatically through the manipulation of droplets.
... goldbeschichteten Glas-Objektträgern [13] oder Organosilan-SAMs auf Siliziumoxid-Oberflächen [14]. Beschichtungen aus Acrylamid [15], Nitrocellulose [9, 10, 16], Agarose [17] oder funktionalisierten Dextranen [18] bieten eine dreidimensionale Matrix, an die Proteine physikalisch adsorbieren. Für die Immobilisierung von Fängermolekülen über einen molekularen Linker können Linkermoleküle entweder chemisch oder bei der rekombinanten Herstellung direkt über die Gensequenz mit dem Protein konjugiert werden: Protein-Biotin-, Protein-Streptag ® -oder Protein-His-Tag-Konjugate können auf [19], StrepTactin ® -oder Ni-NTA-modifizierten ...
Nanopartikel mit einer Schale aus Proteinen stellen ein Material mit extrem großer, biofunktioneller Oberfläche dar. Dieser vielseitige Werkstoff wurde in der vorliegenden Arbeit einem breiten Anwendungsspektrum verfügbar gemacht. Kern-Schale-Nanopartikel aus einem anorganischen SiO2-Kern und einer organischen Schale aus funktionellen Silanen wurden verwendet, um daran als Fängerelemente Proteine mit spezifischen Bindeeigenschaften zu immobilisieren. Die Funktionalisierung der Partikeloberflächen konnte mit großer Flexibilität an verschiedene Anforderungen angepasst werden: Fänger-Moleküle konnten in zufälliger Orientierung kovalent oder auch gerichtet auf den Partikeloberflächen immobilisiert werden. Die biofunktionalisierten Nanopartikel wurden anschließend mittels lithografischer Techniken oder Kontakt-Druck-Verfahren in Form von mikrostrukturierten Schichten stabil auf aktivierten Glasoberflächen abgeschieden. Die partikelgebundenen Fänger-Proteine wurden stabilisiert, sodass ihre biologische Funktionalität innerhalb der trockenen Partikelschichten erhalten blieb. Auf diese Weise wurden dreidimensionale Affinitätsoberflächen im Microarrayformat erzeugt, die dauerhaft lagerfähig waren. Die hohe Bindekapazität der dreidimensionalen Microspots resultierte beim Nachweis von Proteinen in einem dynamischen Bereich, der fünf Größenordnungen überspannte. Nanopartikel-Microarrays waren sowohl mit Fluoreszenzdetektion kompatibel als auch mit MALDI-Massenspektrometrie (MALDI-MS), den Standard-Ausleseverfahren der modernen Molekularbiologie und der Proteomforschung. Die hohe Bindekapazität und große Fängerdichte machen Biochips aus Nanopartikel-Microspots besonders relevant für Anwendungen, die für den empfindlichen Nachweis eines Analyten möglichst viele der in der Probe vorhandenen Analytmoleküle auf die Sensor-Oberfläche aufkonzentrieren wollen. Der modulare Aufbau der Chips ermöglicht die Trennung von Kopplungschemie und Microstrukturierung, sodass für die Entwicklung von Hybrid-Tests unterschiedliche Fängerelemente maßgeschneidert auf einer Sensoroberfläche immobilisiert werden können.
... Thus, it is very important to choose appropriate array surfaces for the success of antibody microarrays. A number of immobilization strategies have been evaluated extensively in several studies for preserving the on-chip activity, specificity, and stability of antibodies (Angenendt et al. 2003). They can be broadly classified into three major categories, including absorption, covalent conjugation, and affinity binding. ...
Despite the success of DNA microarrays in uncovering gene mutations and gene expression patterns linked to specific diseases, genomic profiling provides little insight into the rapid dynamics of cellular signaling networks with respect to the actual levels, subcellular locations, and functional activities of proteins and their interactions. Such information is essential for a fuller understanding of the molecular events underlying malignant transformation. While much more technologically challenging than oligonucleotide microarrays, in recent years, protein microarrays have also emerged as full-fledged discovery tools for disease biomarkers discovery, as evidenced by an increasing number of studies in which protein microarrays have been employed. In this review, we will highlight some of the recent technological improvements in relation to the two most commonly used types of protein microarrays, i.e., antibody microarrays and reverse-phase protein lysate microarrays. We assess the potential of the future integration of protein microarrays in clinical practices through review of their applications in studies of a wide range of diseases as well as in therapeutic drug discovery efforts. The challenges and outlook of protein microarrays in the era of personalized medicine are also considered.
... In general, the immobilization of proteins is accomplished by a range of methods including, (a) simple physisorption (Voros 2004), or (b) by electrostatic interaction (Zhi and Haynie 2004), or (c) by covalently linking through amines (Melles, Anderson et al. 2005), or (d) using thiol-containing compounds (Zhu, Bilgin et al. 2001), or (e) attachment by affinity interactions. The recent immobilization methods use materials such as a silanized layer (Babacan, Pivarnik et al. 2000), sol-gel matrices (Unen, Engbersen et al. 2001), polymer membrane (Su and Li 2004), Langmuir-Blodgett film (Walter, Bussow et al. 2000), or self-assembled monolayer (SAM) (Angenendt, Glokler et al. 2003). However, simple nonspecific immobilization techniques on flat surfaces utilize soft membranes such as PVDF (Walter, Bussow et al. 2000) and nitrocellulose membrane (Joos, Schrenk et al. 2000) or poly(L-lysine). ...
... Hence, an appropriate chip surface for the immobilization of protein samples is the key to protein chips. 10 Since immobilized metal affinity chromatography (IMAC) is one of the most popular methods for purification of recombinant proteins, [11][12][13][14] it has been applied to the development of protein chips. [15][16][17][18][19][20][21] In IMAC, a "tag" consisting of a series of histidine residues (His-tag) is usually attached to one terminus of the protein; moreover, an intermediate metal ion, such as nickel, is usually used to capture the Histagged protein. ...
Based on the principle of immobilized metal affinity chromatography (IMAC), it has been found that a Ni-Co alloy-coated protein chip is able to immobilize functional proteins with a His-tag attached. In this study, an intelligent computational approach was developed to promote the performance and repeatability of a Ni-Co alloy-coated protein chip. This approach was launched out of L18 experiments. Based on the experimental data, the fabrication process model of a Ni-Co protein chip was established by using an artificial neural network, and then an optimal fabrication condition was obtained using the Taguchi genetic algorithm. The result was validated experimentally and compared with a nitrocellulose chip. Consequentially, experimental outcomes revealed that the Ni-Co alloy-coated chip, fabricated using the proposed approach, had the best performance and repeatability compared with the Ni-Co chips of an L18 orthogonal array design and the nitrocellulose chip. Moreover, the low fluorescent background of the chip surface gives a more precise fluorescent detection. Based on a small quantity of experiments, this proposed intelligent computation approach can significantly reduce the experimental cost and improve the product's quality.
© 2015 Society for Laboratory Automation and Screening.
... It is also extremely important to effectively block the remaining surface prior to the assay, since it minimizes the unspecific binding of analytes or detection molecules. As a consequence, high signal-to-noise ratios can be obtained, which leads to an improved sensitivity 35,36 . Arraying techniques were also successfully adopted from DNA microarray production methods 37 . ...
... This has lead some authors to refer to these devices as miniarrays. We classify them among a longer tradition of 3D microarrays and expect them to meet the promise of greater sensitivity harbored along the diverse variants of this technique (74,59,(75)(76)(77)(78)(79)(80)(81). ...
On a past volume of this monograph we have reviewed general aspects of the varied technologies available to generate peptide arrays. Hallmarks in the development of the technology and a main sketch of preparative steps and applications in binding assays were used to walk the reader through details of peptide arrays. In this occasion, we resume from that work and bring in some considerations on quantitative evaluation of measurements as well as on selected reports applying the technology.
... [20] Some antibodies have been shown to be active in standard assaying techniques such as ELISA, while the activity can not be measured on surfaces. [21] Also, signal intensities can vary as much as 43% on the same chip [22]. ...
... These microarray surfaces fall into two major categories : non-gel-coated two-dimensional surfaces and gel-coated three-dimensional surfaces. Non-gel coated surfaces are surfaces modified with different chemicals, such as poly-L-lysine Snapyan et al., 2003), epoxy (Angenendt et al., 2003), or aldehyde ; while gel-coated surfaces are coated with polyacrylamide (Arenkov et al., 2000), agarose , nitrocellulose, etc. The production of substrates for protein microarrays has been commercialized , and various products have been compared in terms of the spot characteristics, limit of detection (LOD), and signal-to-noise (S/N) ratio (Angenendt et al., 2002;. ...
... There are a number of approaches to this problem, which differ fundamentally according to whether the proteins are immobilised through non-speci fi c, poorly de fi ned interactions or through a speci fi c set of known interactions. The former approach is attractive in its simplicity and is compatible with puri fi ed proteins derived from native or recombinant sources (MacBeath and Schreiber 2000 ; Angenendt et al. 2003 ) but suffers from a number of risks. Most notable among these is that the uncontrolled nature of the interactions between each protein and the surface might at best give rise to a heterogeneous population of proteins or at worst destroy activity altogether due to partial or complete surface-mediated unfolding of the immobilised protein. ...
Protein microarrays have many potential applications in the systematic, quantitative analysis of protein function, including in biomarker discovery applica-tions. In this chapter, we review available methodologies relevant to this fi eld and describe a simple approach to the design and fabrication of cancer-antigen arrays suitable for cancer biomarker discovery through serological analysis of cancer patients. We consider general issues that arise in antigen content generation, microar-ray fabrication and microarray-based assays and provide practical examples of experimental approaches that address these. We then focus on general issues that arise in raw data extraction, raw data preprocessing and analysis of the resultant preprocessed data to determine its biological signi fi cance, and we describe compu-tational approaches to address these that enable quantitative assessment of serologi-cal protein microarray data. We exemplify this overall approach by reference to the creation of a multiplexed cancer-antigen microarray that contains 100 unique, puri fi ed, immobilised antigens in a spatially de fi ned array, and we describe speci fi c methods for serological assay and data analysis on such microarrays, including test cases with data originated from a malignant melanoma cohort.
... Despite the capability of the MS-based methods in discovering novel phosphosites, phosphokinase arrays have emerged as preferred tools to measure well-characterized phosphosites of functional significance using the phosphosite-specific antibodies. These arrays can be categorized into antibody arrays [5,6] and reverse-phase protein lysate arrays [7][8][9]. Several phosphokinase antibody arrays have been developed (Table S1), including Phospho Explorer Array (Full Moon BioSystems), Signal Trans-duction AntibodyArray (Hypromatrix), Kinex Antibody Microarray (Kinexus Bioinformatics), Human Phospho-Kinase Antibody Array (R&D system), and Panorama Antibody Array XP725 (Sigma-Aldrich). ...
Monitoring protein phosphorylation at the cellular level is important to understand the intracellular signaling. Among the phosphoproteomics methods, phosphokinase antibody arrays have emerged as preferred tools to measure well-characterized phosphorylation in the intracellular signaling. Here, we present a dendron-coated phosphokinase antibody array (DPA) in which the antibodies are immobilized on a dendron-coated glass slide. Self-assembly of conically shaped dendrons well-controlled in size and structure resulted in precisely controlled lateral spacing between the immobilized phosphosite-specific antibodies, leading to minimized steric hindrance and improved antigen-antibody binding kinetics. These features increased sensitivity, selectivity, and reproducibility in measured amounts of protein phosphorylation. To demonstrate the utility of the DPA, we generated the phosphorylation profiles of brain tissue samples obtained from Alzheimer's disease (AD) model mice. The analysis of the profiles revealed signaling pathways deregulated during the course of AD progression.
... In-house coating produces inconsistent surfaces that lead to poor spot reproducibility 13 and, in some cases, may lead to quantification problems. 18 Of equal importance, temperature and humidity affect the "printability" of the materials. While no detailed studies on the effects related to temperature were carried out, printing was always carried out at room temperature (23±3 °C). ...
Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays.
... The diversity of protein structures in allergen extracts poses an additional challenge for identifying a universal assay surface and the ideal solution conditions (e.g. probe concentration, buffer composition, pH, incubation times, etc.) that best maintain capture protein functionality equally for all the probe molecules in a microarray (Angenendt et al. 2003;Seurynck-Servoss et al. 2007). PBS spotting buffer was specified by the array printing commercial service (ArrayIt) through their prior experience printing similar reagents. ...
We demonstrate the detection of low concentrations of allergen-specific Immunoglobulin E (IgE) in human sera using a Photonic Crystal Enhanced Fluorescence (PCEF) microarray platform. The Photonic Crystal (PC) surface, designed to provide optical resonances for the excitation wavelength and emission wavelength of Cy5, was used to amplify the fluorescence signal intensity measured from a multiplexed allergen microarray. Surface-based sandwich immunoassays were used to detect and quantify specific IgE antibodies against a highly purified cat allergen (Fel d1). A comparison of the lowest detectable concentration of IgE measured by the PC microarray system and a commercially available clinical analyzer demonstrated that the PCEF microarray system provides higher sensitivity. The PCEF system was able to detect low concentrations of specific IgE (∼0.02kU/L), which is 5-17-fold more sensitive than the commercially available FDA-approved analyzers. In preliminary experiments using multi-allergen arrays, we demonstrate selective simultaneous detection of IgE antibodies to multiple allergens.
... A systematic comparison of protein microarrays on epoxy surface and other tested slides such as those coated with PEG-epoxy, amine, and dendrimer showed similar detection limits. The authors of that report also demonstrated that all tested surfaces exert saturation of mean signal intensities of spotted proteins and antibodies in the range of 22.5 fmol/spot [129]. ...
Despite remarkable progress in understanding biology and disease at the level of nucleic acids, insights into the relevant biochemical processes frequently remain preliminary, since much regulation and activity occurs at the protein level through control of gene expression and variations of protein conformation. In particular, the effect of such variations on protein interactions is critical for a better description of biology and disease. Protein microarray technology provides a means to such ends and is a growing field of proteomics, with a high potential for analytical and functional applications in biology and medicine. On the basis of sequence information from individuals, it is possible to characterize disease-specific protein isoforms that result from mutations, polymorphisms, and splice variants with personalized protein microarrays. During my thesis, I developed such a technique.
As a first step, solid-phase PCR is applied to copy a particular tissue’s RNA/cDNA onto the microarray surface, using for each gene a specific primer pair that is attached to the chip surface. The generated DNA templates are firmly attached to and specifically oriented on the array surface. The solid-phase PCR successfully amplified DNA of up to 3 kb, also allowing multiplex amplification of DNA. The arrayed DNA copies then act as templates for an in situ cell-free expression, yielding a protein microarray that presents the protein content of a particular tissue of an individual person. Expression control was conducted by a multiple spotting technique (MIST).
C-terminus detection showed that translation was complete, yielding full-length proteins.
During the process of setting up the technique of producing individualized protein microarrays, the MIST technology was optimized concomitantly. The various steps involved were analyzed to determine optimal conditions for template preparation, protein expression and interaction detection. Protein microarrays of 3500 human proteins were produced with these procedures and their performance was tested in model studies of protein–protein interactions.
... 23,24 Common methods for attachment of antibodies to solid surfaces include physical adsorption, chemical cross linking, covalent bonding, or entrapment in a gel network. 25,26 Numerous studies of the use of chemical methods for sitespecific immobilization of whole antibody molecules onto inorganic substrates have been reported. One of the most common methods employs the prior immobilization of antibody-binding protein A or G, 27 or the use of polymers with carefully designed functional groups to control antibody orientation. ...
Stainless steel 316L material is commonly used for the production of coronary and peripheral vessel stents. Effective biofunctionalization is a key to improving the performance and safety of the stents after implantation. This paper reports the method for the immobilization of recombinant antibody fragments (scFv) on stainless steel 316L to facilitate human endothelial progenitor cell (EPC) growth and thus improve cell viability of the implanted stents for cardiovascular applications. The modification of stent surface was conducted in three steps. First the stent surface was coated with titania based coating to increase the density of hydroxyl groups for successful silanization. Then silanization with 3 aminopropyltriethoxysilane (APTS) was performed to provide the surface with amine groups which presence was verified using FTIR, XPS and fluorescence microscopy. The maximum density of amine groups (4.8*10-5 mol/cm(2) ) on the surface was reached after reaction taking place in ethanol, for 1 h at 60° C and 0.04M APTS. On such prepared surface the glycosylated scFv were subsequently successfully immobilised. The influence of oxidation of scFv glycan moieties and the temperature on scFv coating were investigated. The fluorescence and confocal microscopy study indicated that the densest and most uniformly coated surface with scFv was obtained at 37°C after oxidation of glycan chain. The results demonstrate that the scFv cannot be efficiently immobilized without prior aminosilanization of the surface. The effect of the chemical modification on the cell viability of EPC line 55.1 (HucPEC-55.1) was performed indicating that the modifications to the 316L stainless steel are non-toxic to EPCs. This article is protected by copyright. All rights reserved.
... The interplay between arrayed antibodies and the surface, modulated by the spotting buffer [34], is essential for antibody functionality and spot features [7,22,35,36]. Frequently, excess of antibodies have been dispensed in order to make sure that the spots are fully saturated. Our data implied that the spots grew from the center and outwards with increasing antibody concentration, and that the antibodies appeared to populate the spots in a clone dependent manner. ...
In the quest for deciphering disease-associated biomarkers, high-performing tools for multiplexed protein expression profiling of crude clinical samples will be crucial. Affinity proteomics, mainly represented by antibody-based microarrays, have during recent years been established as a proteomic tool providing unique opportunities for parallelized protein expression profiling. But despite the progress, several main technical features and assay procedures remains to be (fully) resolved. Among these issues, the handling of protein microarray data, i.e. the biostatistics parts, is one of the key features to solve. In this study, we have therefore further optimized, validated, and standardized our in-house designed recombinant antibody microarray technology platform. To this end, we addressed the main remaining technical issues (e.g. antibody quality, array production, sample labelling, and selected assay conditions) and most importantly key biostatistics subjects (e.g. array data pre-processing and biomarker panel condensation). This represents one of the first antibody array studies in which these key biostatistics subjects have been studied in detail. Here, we thus present the next generation of the recombinant antibody microarray technology platform designed for clinical immunoproteomics.
... Thus, it is very important to choose appropriate array surfaces for the success of antibody microarrays. A number of immobilization strategies have been evaluated extensively in several studies for preserving the on-chip activity, specificity, and stability of antibodies (Angenendt et al. 2003). They can be broadly classified into three major categories, including absorption, covalent conjugation, and affinity binding. ...
Array-comparative genomic hybridisation (array-CGH) and single nucleotide polymorphism array hybridisation (SNP-array) enable genome-wide detection of copy number alterations (CNA). These techniques outperform conventional chromosomal karyotyping in relation to detection of CNAs. The discovery of previously cryptic alterations associated with constitutional or acquired cytogenetic changes with DNA copy number gains or losses quickly led to the identification of novel syndromes or previously unseen tumour-related changes. Array platforms are often targeted to pathognomonic regions to detect well-characterised unbalanced chromosomal rearrangements. Platforms with genome-wide coverage detect additional CNA representing normal genomic variation (copy number variation: CNV) or variation with yet unknown significance. The use of SNP-array facilitating the detection of segmental uniparental disomies might be advantageous compared to classical array-CGH approaches. However, none of the array platforms permit the detection of balanced genomic rearrangements such as translocation, inversion or insertions or some polyploidies. The use of array platforms in cytogenetic testing quickly became a routine diagnostic tool and will replace conventional cytogenetic testing in many instances. In addition, these techniques have the potential to identify genomic changes relevant for the establishment of prognostic stratification of different neoplastic conditions. © 2012 Springer-Verlag Berlin Heidelberg. All rights are reserved.
... 7,[12][13][14] On some microarrays, even the same antibodies arrayed at different locations on the same chip have been shown to vary in activity by up to 43%. 15 Since the microarray's performance is dependent on the activity of the antibody, building a reliable microarray would benefit from the knowledge of the behavior of the antibody on the surface and how the surface affects its structure. This knowledge is key to improving the design and manufacture of next generation devices. ...
Despite their potential benefits, antibody microarrays have fallen short of performing reliably and have not found widespread use outside of the research setting. Experimental techniques have been unable to determine what is occurring on the surface of an atomic level, so molecular simulation has emerged as the primary method of investigating protein/surfaceinteractions. Simulations of small proteins have indicated that the stability of the protein is a function of the residue on the protein where a tether is placed. The purpose of this research is to see whether these findings also apply to antibodies, with their greater size and complexity. To determine this, 24 tethering locations were selected on the antibody Protein Data Bank (PDB) ID: 1IGT. Replica exchange simulations were run on two different surfaces, one hydrophobic and one hydrophilic, to determine the degree to which these tethering sites stabilize or destabilize the antibody. Results showed that antibodies tethered to hydrophobicsurfaces were in general less stable than antibodies tethered to hydrophilicsurfaces. Moreover, the stability of the antibody was a function of the tether location on hydrophobicsurfaces but not hydrophilicsurfaces.
Next generation antibody microarray devices have the potential to outperform current molecular detection methods and realize new applications in medicine, scientific research, and national defense. However, antibody microarrays, or arrays of antibody fragments ("fabs"), continue to evade mainstream use in part due to persistent reliability problems despite improvements to substrate design and protein immobilization strategies. Other factors could be disrupting microarray performance, including effects resulting from antigen characteristics. Target molecules embody a wide range of sizes, shapes, number of epitopes, epitope accessibility, and other physical and chemical properties. As a result, it may not be ideal for microarray designs to utilize the same substrate or immobilization strategy for all of the capture molecules. This study investigates how three antigen properties, such as size, binding site valency, and molecular flexibility, affect fab binding. The work uses an advanced, experimentally validated, coarse-grain model and umbrella sampling to calculate the free energy of ligand binding and how this energy landscape is different on the surface compared to in the bulk. The results confirm that large antigens interact differently with immobilized fabs compared to smaller antigens. Analysis of the results shows that despite these differences, tethering fabs in an upright orientation on hydrophilic surfaces is the best configuration for antibody microarrays.
The interaction of proteins with surfaces is important in numerous applications in many fields-such as biotechnology, proteomics, sensors, and medicine-but fundamental understanding of how protein stability and structure are affected by surfaces remains incomplete. Over the last several years, molecular simulation using coarse grain models has yielded significant insights, but the formalisms used to represent the surface interactions have been rudimentary. We present a new model for protein surface interactions that incorporates the chemical specificity of both the surface and the residues comprising the protein in the context of a one-bead-per-residue, coarse grain approach that maintains computational efficiency. The model is parameterized against experimental adsorption energies for multiple model peptides on different types of surfaces. The validity of the model is established by its ability to quantitatively and qualitatively predict the free energy of adsorption and structural changes for multiple biologically-relevant proteins on different surfaces. The validation, done with proteins not used in parameterization, shows that the model produces remarkable agreement between simulation and experiment.
The formation of functional polymers by implementing small-molecule organic chemistry reactions to homopolymers has attracted great attention lately due to an increasing number of novel potential applications. This post-polymerization modification (PPM) approach circumvents a number of problems associated with direct polymer synthesis and enables the creation of polymeric systems that are difficult or impossible to produce otherwise. This holds especially true in the field of polymer brushes where the tethering of the polymer chains to a surface complicates direct polymerization of bulky monomers. The advantages of PPM reactions do not stop with polymer synthesis. This review highlights a variety of innovations that stem directly from this approach. A selection of these applications includes modified barrier properties, catalysis, surface patterning, separations, and biologically active substrates.
Plasma immersion ion implantation (PIII) modifies the surface properties of polymers, enabling them to covalently immobilize proteins without using linker chemistry. We describe the use of PIII treated polycarbonate (PC) slides as a novel platform for producing microarrays of cluster of differentiation (CD) antibodies. We compare their performance to identical antibody microarrays printed on nitrocellulose-coated glass slides that are currently the industry standard. Populations of leukocytes are applied to the CD microarrays and unbound cells are removed revealing patterns of differentially immobilized cells that are detected in a simple label-free approach by scanning the slides with visible light. Intra-slide and inter-slide reproducibility, densities of bound cells, and limits of detection were determined. Compared to the nitrocellulose-coated glass slides, PIII treated PC slides have a lower background noise, better sensitivity, and comparable or better reproducibility. They require three-fold lower antibody concentrations to yield equivalent signal strength, resulting in significant reductions in production cost. The improved transparency of PIII treated PC in the near-UV and visible wavelengths combined with superior immobilization of biomolecules makes them an attractive platform for a wide range of microarray applications.
In protein research, protein microarray facilitates high-throughput study of protein abundance and function. An appropriate microarray surface that can be used to immobilize protein samples is a prerequisite for the investigation of molecular interactions. Ni–Co alloy coated protein microarray chip has been found to adsorb histidine-tagged proteins effectively based on the method of immobilized metal affinity chromatography. Due to the ingredient of bi-metallic elements, different electroplating conditions resulted in distinct binding affinities. Therefore, the influence of Ni–Co material properties on the immobilization of histidine-tagged protein was systematically investigated in this study. In the experiments, the contact angle measurement suggested that no strong relationship can be established between the wettability of chip surface and its corresponding protein immobilization. ESCA test demonstrated that the major ingredients of the Ni–Co alloy coated protein microarray chip were Ni and Co. In addition, the XRD test concluded that a Ni–Co protein chip that consists mostly of hcp lattice has better binding capability. SEM micrographs provide direct image evidence. These material tests summarize that the Ni–Co alloy coated protein microarray chip adsorbs His-tagged proteins through its surface morphology. Therefore, it can provide specific binding due to the affinity adsorption between the intermediate metals and the protein.
In the last decade, many proteomic technologies have been applied, with varying success, to the study of tissue samples of breast carcinoma for protein expression profiling in order to discover protein biomarkers/signatures suitable for: characterization and subtyping of tumors; early diagnosis, and both prognosis and prediction of outcome of chemotherapy. The purpose of this review is to critically appraise what has been achieved to date using proteomic technologies and to bring forward novel strategies - based on the analysis of clinically relevant samples - that promise to accelerate the translation of basic discoveries into the daily breast cancer clinical practice. In particular, we address major issues in experimental design by reviewing the strengths and weaknesses of current proteomic strategies in the context of the analysis of human breast tissue specimens.
The development of strategies to covalently couple biomolecules to surfaces is necessary for constructing sensing arrays for biological and biomedical applications. One attractive conjugation reaction is hydrazone formation - the reaction of a hydrazine with an aldehyde or ketone - as both hydrazines and aldehydes/ketones are largely bioorthogonal, which makes this particular reaction suitable for conjugating biomolecules to a variety of substrates. We show that the mild reaction conditions afforded by hydrazone conjugation enable the conjugation of DNA and proteins to the substrate surface in significantly higher yields than can be achieved with traditional bioconjugation techniques, such as maleimide chemistry. Next, we designed and synthesized a photocaged aryl ketone that can be conjugated to a surface and photochemically activated to provide a suitable partner for subsequent hydrazone formation between the surface-anchored ketone and DNA- or protein-hydrazines. Finally, we exploit the latent functionality of the photocaged ketone and pattern multiple biomolecules on the same substrate, effectively demonstrating a strategy for designing substrates with well-defined domains of different biomolecules. We expect that this approach can be extended to the production of multiplexed assays by using an appropriate mask with sequential photo-exposure and biomolecule conjugation steps.
Multi-analyte immunoassays on microarrays and on multiplex DNA microarrays have been described for quantitative analysis of small organic molecules (e.g., antibiotics, drugs of abuse, small molecule toxins), proteins (e.g., antibodies or protein toxins), and microorganisms, viruses, and eukaryotic cells. In analytical chemistry, multi-analyte detection by use of analytical microarrays has become an innovative research topic because of the possibility of generating several sets of quantitative data for different analyte classes in a short time. Chemiluminescence (CL) microarrays are powerful tools for rapid multiplex analysis of complex matrices. A wide range of applications for CL microarrays is described in the literature dealing with analytical microarrays. The motivation for this review is to summarize the current state of CL-based analytical microarrays. Combining analysis of different compound classes on CL microarrays reduces analysis time, cost of reagents, and use of laboratory space. Applications are discussed, with examples from food safety, water safety, environmental monitoring, diagnostics, forensics, toxicology, and biosecurity. The potential and limitations of research on multiplex analysis by use of CL microarrays are discussed in this review.
Figure
Achievements in the development of CL microarray analysis platforms
Protein microarray or protein chip is an important tool in proteomics. However, duplicating the success of the DNA chip for the protein chip has been difficult. This account discusses a key issue in protein microarray development, i.e., surface chemistry. Ideally, the surface chemistry for protein microarray fabrication should satisfy the following criteria: the surface resists nonspecific adsorption; functional groups for the facile immobilization of protein molecules of interest are readily available; bonding between a protein molecule and a solid surface is balanced to provide sufficient stability but minimal disturbance on the delicate three-dimensional structure of the protein; linking chemistry allows the control of protein orientation; the local chemical environment favors the immobilized protein molecules to retain their native conformation; and finally, the specificity of linking chemistry is so high that no pre-purification of proteins is required. Strategies to achieve such an ideal situation are discussed, with successful examples from our laboratories illustrated. Finally, the need of surface technology for membrane protein microarray fabrication is addressed.
Antibody microarrays have the potential to revolutionize molecular detection for many applications, but their current use is limited by poor reliability, and efforts to change this have not yielded fruitful results. One difficulty which limits the rational engineering of next-generation devices is that little is known, at the molecular level, about the antibody-antigen binding process near solid surfaces. Atomic-level structural information is scant because typical experimental techniques (X-ray crystallography and NMR) cannot be used to image proteins bound to surfaces. To overcome this limitation, this study uses molecular simulation and an advanced, experimentally validated, coarse-grain, protein-surface model to compare fab-lysozyme binding in bulk solution and when the fab is tethered to hydrophobic and hydrophilicsurfaces. The results show that the tether site in the fab, as well as the surfacehydrophobicity, significantly impacts the binding process and suggests that the optimal design involves tethering fabs upright on a hydrophilicsurface. The results offer an unprecedented, molecular-level picture of the binding process and give hope that the rational design of protein-microarrays is possible.
In this study, antibody microarrays on a nano-scale controlled surface were prepared. Aspects of performance such as signal intensity, dependence of signal intensity on target antigen concentration, etc. were evaluated and compared with those of micro-arrays on amine, aldehyde, and epoxy surfaces. The signal intensities of the anti-TNF-α antibody micro-array fabricated on the nano-scale controlled surface were found to be 2-8 times higher than those prepared on the other surfaces. Additionally, the anti-TNF-α and anti-IL-1β antibody microarrays evidenced linear correlations between their signal intensities and concentrations of target antigen applied to the microarrays in a range between 3.0 nM-1.0n.M. Furthermore, the antibody microarrays detected two different antigens simultaneously with similar signal intensity to those achieved in single antigen detection experiments.
CD4-gp120 interaction is the first step for HIV-1 entry into host cells. A highly conserved pocket in gp120 protein is an attractive target for developing gp120 inhibitors or novel HIV detection tools. Here we incorporate seven phenylalanine derivatives having different sizes and steric conformations into position 43 of domain 1 of CD4 (mD1.2) to explore the architecture of the ‘Phe43 cavity’ of HIV-1 gp120. The results show that the conserved hydrophobic pocket in gp120 tolerates a hydrophobic side chain of residue 43 of CD protein, which is 12.2 Å in length and 8.0 Å in width. This result provides useful information for developing novel gp120 inhibitors or new HIV detection tools.
A new flow-through method for rapid capture and detection of microorganisms is developed using optically-flat microengineered membranes. Selective and efficient capture of Salmonella is demonstrated with antibodies coated on membranes (microsieves) having a pore size much larger than the microorganism itself. The silicon-nitride membranes are first photochemically coated with 1,2-epoxy-9-decene yielding stable Si–C and N–C linkages. The resultant epoxide-terminated microsieves are subsequently biofunctionalized with anti-Salmonella antibodies. The capture efficiency of antibody-coated microsieves with different pore sizes (2.0–5.0 μm) is studied with Salmonella enterica enterica serotype Typhimurium suspensions (107 cfu.mL–1). The antibody-coated microsieves capture 52% (2 μm microsieves), 30% (3.5 μm microsieves), and 12% (5 μm microsieves) of Salmonella from the suspension. The influence of flow rate (0.8–16 μL.min–1.mm–2) on the capture efficiency of antibody-coated 3.5 μm microsieves is investigated. The capture efficiency increases from ~30% to ~70% when the flow-rate decreases from 16 to 0.8 μL.min–1.mm–2. Antibody-coated 3.5 μm microsieves can capture Salmonella rapidly and directly from fresh milk suspension (capture 35% at concentration of 80 cfu.mL–1). The use of antibody-coated microsieves as microbial selective capture devices is thus shown to be highly promising for the direct capture of microorganisms.
The protein chip is a powerful emerging technology for biomedical applications. In contrast to the DNA chip, however, great challenges face the development of optimal surface materials for protein chips that are able to maintain the activity of embedded proteins with increased sensitivity, while being produced at sufficiently low cost to replace the materials used in current technology. We previously developed a sol-gel protein chip technology with significantly improved physical properties and sensitivity. Here, in addition to modifying the protein chip materials, we found that the addition of more salt with proteins in the preparation of 3-dimensional protein chips can increase the sensitivity of the protein chip. The present study results will support the technological advancement for protein chip developers and biochip research.
Autoimmunity is highly coincident with immunodeficiency. In a small but growing number of primary immunodeficiencies, autoantibodies are diagnostic of a given disease and implicated in disease pathogenesis. In order to improve our understanding of the role of autoantibodies in immunodeficiencies and to discover novel autoantibodies, new proteomic tools are needed. Protein microarrays have the ability to screen for reactivity to hundreds to many thousands of unique autoantigens simultaneously on a single chip using minimal serum input. Here, we review different types of protein microarrays and how they can be useful in framing the study of primary and secondary immunodeficiencies.
The DNA microarray technique was supposed to help identifying and analyzing the expression level of tens of thousands of genes in the whole genome. But there is a serious problem concerning fabrication of the microarrays by chemical synthesis, such as specific and efficient linking of probes to a solid support. Therefore, we reckon that applying “click” chemistry to covalently anchor oligonucleotides on chemically modified supports may help construct microarrays in applications such as gene identification. Silanization of the glass support with organofunctional silane makes it possible to link azide groups on glass surface and the nucleic acid probe that is equipped with a pentynyl group. This is followed by direct spotting of the nucleic acid on the azide-modified glass support in the presence of copper ions, and this is a frequently applied method of “click” chemistry.
Intracellular proteins comprise numerous peptide motifs that interact with protein-binding domains. However, using sequence information alone, the identification of functionally relevant interaction motifs remains a challenge. Here, we present a microarray-based approach for the evaluation of peptides as protein-binding motifs. To this end, peptides corresponding to protein interaction motifs were spotted as a microarray. First, peptides were titrated with a pan-specific binder and the apparent K
d value of this binder for each peptide was determined. For phosphotyrosine-containing peptides, an anti-phosphotyrosine antibody was employed. Then, in the presence of the pan-specific binder, arrays were competitively titrated with cell lysate and competition constants were determined. Using the Cheng-Prusoff equation, binding constants for the pan-specific binder and inhibition constants for the lysates were converted into affinity constants for the lysate. We experimentally validate this method using a phosphotyrosine-binding SH2 domain as a further reference. Furthermore, strong binders correlated with binding motifs engaging in numerous interactions as predicted by Scansite. This method provides a highly parallel and robust approach to identify peptides corresponding to interaction motifs with strong binding capacity for proteins in the cell lysate.
Graphical Abstract
Using an antibody as a pan-specific binder the capacity of interaction motifs to bind to proteins from cell lysates can be probed. Competition of the antibody is observed for only those peptides to which a lysate protein binds
Microarrays are grids of biomolecules in which each element of the grid performs a specific assay such as identification of a specific binding molecule or measuring a particular enzymatic activity. The potential of microarrays to facilitate global analysis of genes and gene products has led to its adoption in nearly every area of biological science—from basic research to clinical diagnostics. This article provides a technical overview of microarrays including information on array formats, production, and use of microarrays for interrogating various molecular species such as DNA, RNA, and proteins.
Keywords:
microarrays;
enzymatic activity;
biological science;
biomolecules
Our research in immunotechnology focuses on the cells and molecules of the immune system for various biomedical applications. Applied research as well as projects addressing fundamental issues is being pursued within four main project areas: 1) antibody technology, 2) proteomics, 3) cancer, and 4) allergy. Within the proteomics project, we have taken advantage of the strong background in antibody engineering to design antibody-based micro-and nanoarrays for applications ranging from focused assays to proteome-scale analysis. Antibody-based microarray is a novel technology that holds great promise in proteomics. The microarray can be printed with thousands of recombinant antibodies carrying the desired specificities, the biological sample added (e.g., an entire proteome), and virtually any specifically bound analytes detected. The microarray patterns generated can then be converted into proteomic maps, or molecular fingerprints, revealing the composition of the proteome. Global proteome analysis and protein expression profiling, by using this tool, will provide new opportunities for biomarker discovery, drug target identification, and disease diagnostics and will give insight into disease biology. Ultimately, we apply this novel technology platform within our cancer and allergy projects to perform high-throughput disease proteomics.
Dendritic nanomaterials have attracted a great deal of scientific interest for use in various biomedical applications primarily due to their well-defined structure and capacity for multifunctionalization. In this chapter, we present a comprehensive overview of the recent advances in various dendrimers and their modified dendritic materials with a particular focus on their applications such as drug delivery, gene delivery, diagnostics and prognostics, and detoxification/prophylaxis. We also discuss the fundamentals of the biological interactions of dendritic nanomaterials in regard to non-specific cell interactions, multivalent binding, stimuli-responsive processes, and biodistribution. In addition, this chapter highlights the current drawbacks of dendrimers that have hindered their rapid clinical translation and introduce recent approaches to overcoming these challenges.
Zwitterionic polymers have anion and cation groups simultaneously, which make them highly hydrated and render them unique biocompatibility. Because of their unique zwitterionic pendant-side chain structures, sulfobetaine methacrylate (SBMA) polymers present the excellent anti-biofouling properties, i. e. the resistance to protein adsorption, the inhibition of bacterial and blood coagulation, which make them increasingly applied in a wide range of biological and medical related fields recently. The hydration theory, one of the widely accepted antifouling mechanisms, is briefly introduced. In this article, recent progress in the properties, synthesis methods and surface construction methods is reviewed, the applications of SBMA polymers in the materials for artificial organs, tissue engineering, biological separation, bio-monitoring system and temperature-sensitive materials are summarized. In addition, SBMA polymers holding great potential for use in biomaterials and issues existed in these applications are also discussed.
Protein dot spot technology is presented using unique proteins such as hydrophobin HFB (from Trichoderma reesei). HFB is a protein of low molecular weight (7.8 kD) and is an outstanding hydrophobic molecule. The authors employ HFB as a carrier for protein dot spots. In this study, fluorescent-labeled HFB was spotted on a hydrophobic glass substrate using a solution drop contact spotter. HFB adsorbed on the glass surface uniformly. The results show that HFB provides prominent adsorption characteristics as a carrier protein. The small HFB molecule can be tagged onto other protein molecules through a conventional genetic procedure. By using HFB as a carrier protein, practical protein chip technology can be realized.
Protein microarrays meet the need for large-scale, low cost and accurate assays in the post-genomic era. Efforts in revealing the internal relationship between surface chemistry and microarray quality may greatly help the realization of widespread protein microarray use. In this paper, we prepared iPDMS with various surface properties, which were studied in combination with other parameters such as [protein], spot size and environment conditions to identify the causes of array defects (exosmosis, coffee ring and overflow) and corresponding strategies to avoid them. Through this research, we determined that the optimal parameters and conditions of protein microarray fabrication for an iPDMS sheet were 50° < contact angle < 90°: array volume ≤10 nL, [capture antibody] at 50 μg mL−1, fixed at 25 °C, and 60% humidity. We also identified some characteristics of protein microarray fabrication that may benefit other solid supporting materials (SSM) and serve as a good model of proteins interacting with various bio-interfaces.
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.
To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.
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.
The DNA microarray-based analysis of single nucleotide polymorphisms (SNPs) is important for the correlation of genetic variations
and individual phenotypes, and for locating disease-causing genes. To facilitate the development of surfaces suitable for
immobilization of oligonucleotides, we report here a novel method for the surface immobilization of DNA using pre-fabricated
polyamidoamine (PAMAM) starburst dendrimers as mediator moieties. Dendrimers containing 64 primary amino groups in their outer
sphere are covalently attached to silylated glass supports and, subsequently, the dendritic macromolecules are modified with
glutaric anhydride and activated with N-hydroxysuccinimide. As a result of the dendritic PAMAM linker system the surfaces reveal both a very high immobilization
efficiency for amino-modified DNA-oligomers, and also a remarkable high stability during repeated regeneration and re-using
cycles. The performance of dendrimer-based DNA microarrays in the discrimination of SNPs is demonstrated.
We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.
Critical changes in protein expression that enable tumors to initiate and progress originate in the local tissue microenvironment, and there are increasing indications that these microenvironmental alterations in protein expression play critical roles in shaping and directing this process. As a model to better understand how patterns of protein expression shape the tissue microenvironment, we analyzed protein expression in tissue derived from squamous cell carcinoma of the oral cavity through an antibody microarray approach for high-throughput proteomic analysis. Utilizing laser capture microdissection to procure total protein from specific microscopic cellular populations, we demonstrate that quantitative, and potentially qualitative, differences in expression patterns of multiple proteins within epithelial cells reproducibly correlate with oral cavity tumor progression. Furthermore, differential expression of multiple proteins was also found in stromal cells surrounding and adjacent to regions of diseased epithelium that directly correlated with tumor progression of the epithelium. Most of the proteins identified in both cell types are involved in signal transduction pathways, thus we hypothesize that extensive molecular communication involving complex cellular signaling between epithelium and stroma play a key role in driving oral cavity cancer progression.
Protein–protein interactions have been widely used to study gene expression pathways and may be considered as a new approach
to drug discovery. Here I report the development of a universal protein array (UPA) system that provides a sensitive, quantitative,
multi-purpose, effective and easy technology to determine not only specific protein–protein interactions, but also specific
interactions of proteins with DNA, RNA, ligands and other small chemicals. (i) Since purified proteins are used, the results
can be easily interpreted. (ii) UPA can be used multiple times for different targets, making it economically affordable for
most laboratories, hospitals and biotechnology companies. (iii) Unlike DNA chips or DNA microarrays, no additional instrumentation
is required. (iv) Since the UPA uses active proteins (without denaturation and renaturation), it is more sensitive compared
with most existing methods. (v) Because the UPA can analyze hundreds (even thousands on a protein microarray) of proteins
in a single experiment, it is a very effective method to screen proteins as drug targets in cancer and other human diseases.
Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen-antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor-ligand interactions.
We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses.
We have developed a novel technique for high-throughput screening of recombinant antibodies, based on the creation of antibody arrays. Our method uses robotic picking and high-density gridding of bacteria containing antibody genes followed by filter-based enzyme-linked immunosorbent assay (ELISA) screening to identify clones that express binding antibody fragments. By eliminating the need for liquid handling, we can thereby screen up to 18,342 different antibody clones at a time and, because the clones are arrayed from master stocks, the same antibodies can be double spotted and screened simultaneously against 15 different antigens. We have used our technique in several different applications, including isolating antibodies against impure proteins and complex antigens, where several rounds of phage display often fail. Our results indicate that antibody arrays can be used to identify differentially expressed proteins.
Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.
The generation of chemically activated glass surfaces is of increasing interest for the production of microarrays containing DNA, proteins, and low-molecular-weight components. We here report on a novel surface chemistry for highly efficient activation of glass slides. Our method is based on the initial modification of glass with primary amino groups using a protocol, specifically optimized for high aminosilylation yields, and in particular, for homogeneous surface coverages. In a following step the surface amino groups are activated with a homobifunctional linker, such as disuccinimidylglutarate (DSG) or 1,4-phenylenediisothiocyanate (PDITC), and then allowed to react with a starburst dendrimer that contains 64 primary amino groups in its outer sphere. Subsequently, the dendritic monomers are activated and crosslinked with a homobifunctional spacer, either DSG or PDITC. This leads to the formation of a thin, chemically reactive polymer film, covalently affixed to the glass substrate, which can directly be used for the covalent attachment of amino-modified components, such as oligonucleotides. The resulting DNA microarrays were studied by means of nucleic acid hybridization experiments using fluorophor-labeled complementary oligonucleotide targets. The results indicate that the novel dendrimer-activated surfaces display a surface coverage with capture oligomers about twofold greater than that with conventional microarrays containing linear chemical linkers. In addition, the experiments suggest that the hybridization occurs with decreased steric hindrance, likely a consequence of the long, flexible linker chain between the surface and the DNA oligomer. The surfaces were found to be resistant against repeated alkaline regeneration procedures, which is likely a consequence of the crosslinked polymeric structure of the dendrimer film. The high stability allows multiple hybridization experiments without significant loss of signal intensity. The versatility of the dendrimer surfaces is also demonstrated by the covalent immobilization of streptavidin as a model protein.
High-throughput protein arrays allow the miniaturized and parallel analysis of large numbers of diagnostic markers in complex samples. Using automated colony picking and gridding, cDNA or antibody libraries can be expressed and screened as clone arrays. Protein microarrays are constructed from recombinantly expressed, purified, and yet functional proteins, entailing a range of optimized expression systems. Antibody microarrays are becoming a robust format for expression profiling of whole genomes. Alternative systems, such as aptamer, PROfusion, nano- and microfluidic arrays are all at proof-of-concept stage. Differential protein profiles have been used as molecular diagnostics for cancer and autoimmune diseases and might ultimately be applied to screening of high-risk and general populations.
With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.
Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.
drimer slides show an excellent signal to concen-Wohrle
- R Benters
- C M Niemeyer
- D Drutschmann
- D Blohm
R. Benters, C.M. Niemeyer, D. Drutschmann, D. Blohm, D. drimer slides show an excellent signal to concen-Wohrle, Nucleic Acids Res. 30 (2002) E10. tration ratio. For the quantitative measurement of
Petri-In conclusion, we have tested eight different coin 3rd
- J S Prabhu
- L A Gutkind
- P J Liotta
- E F Munson
Prabhu, J.S. Gutkind, L.A. Liotta, P.J. Munson, E.F. Petri-In conclusion, we have tested eight different coin 3rd, D.B. Krizman, Proteomics 1 (2001) 1271. microarray coatings for the routine use of protein and
Genome Biol. 2 antibodies, PEG-epoxy slides are the surface coating
- B B Haab
- M J Dunham
- P O Brown
B.B. Haab, M.J. Dunham, P.O. Brown, Genome Biol. 2 antibodies, PEG-epoxy slides are the surface coating (2001) RESEARCH0004. ¨ of choice. For experiments that necessitate the
Trends Mol. for slide coatings such as dendrimer and PEG-epoxy Med
- G Walter
- K Bussow
- A Lueking
- J Glokler
G. Walter, K. Bussow, A. Lueking, J. Glokler, Trends Mol. for slide coatings such as dendrimer and PEG-epoxy Med. 8 (2002) 250. slides were introduced and improved qualities of
croarray technology using one protein and one Mitchell
- H Zhu
- M Bilgin
- R Bangham
- D Hall
- A Casamayor
- P Microarray
- N Bertone
- R Lan
- S Jansen
- T Bidlingmaier
- T Houfek
H. Zhu, M. Bilgin, R. Bangham, D. Hall, A. Casamayor, P. microarray coatings for protein and antibody mi-Bertone, N. Lan, R. Jansen, S. Bidlingmaier, T. Houfek, T. croarray technology using one protein and one Mitchell, P. Miller, R.A. Dean, M. Gerstein, M. Snyder, Science 293 (2001) 2101. antibody, the results presented here point towards
Walter, bodies, both, dendrimer slides as well as amine slides
- K Bussow
- E Nordhoff
- C Lubbert
- H Lehrach
K. Bussow, E. Nordhoff, C. Lubbert, H. Lehrach, G. Walter, bodies, both, dendrimer slides as well as amine slides Genomics 65 (2000) 1. are very well suited. Nevertheless, FAST slides also
Eic-khoff) 848. ly published ones were shown. Optimal slide coat-ings for antibody and protein chips were proposed
- A V Soldatov
- E N Nabirochkina
- S G Georgieva
A. V. Soldatov, E.N. Nabirochkina, S.G. Georgieva, H. Eic-khoff, Biotechniques 31 (2001) 848. ly published ones were shown. Optimal slide coat-ings for antibody and protein chips were proposed
Lehrach, suggests that most surfaces presented in this study G
- A Lueking
- M Horn
- H Eickhoff
- K Bussow
A. Lueking, M. Horn, H. Eickhoff, K. Bussow, H. Lehrach, suggests that most surfaces presented in this study G. Walter, Anal. Biochem. 270 (1999) 103. are superior in comparison to [9].
) 45. the coefficients of variation. Moreover, new concepts
- H Zhu
- M Snyder
H. Zhu, M. Snyder, Curr. Opin. Chem. Biol. 5 (2001) 45. the coefficients of variation. Moreover, new concepts
coatings that offer outstanding qualities in their D respective area of application. As expected Anal. surface for both antibody and protein microarray Biochem
- P Peluso
- D S Wilson
- D Do
- H Tran
- M Venkatasubbaiah Quincy
- B Heidecker
- K Poindexter
- N Tolani
P. Peluso, D.S. Wilson, D. Do, H. Tran, M. Venkatasubbaiah, coatings that offer outstanding qualities in their D. Quincy, B. Heidecker, K. Poindexter, N. Tolani, M. respective area of application. As expected, one Phelan, K. Witte, L.S. Jung, P. Wagner, S. Nock, Anal. surface for both antibody and protein microarray Biochem. 312 (2003) 113. applications could not be found. For quantitative