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Angenendt, P., Glokler, J., Murphy, D., Lehrach, H. & Cahill, D. J. Toward optimized antibody microarrays: a comparison of current microarray support materials. Anal. Biochem. 309, 253-260
Abstract
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.
... 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.
... Le choix de la méthode d'immobilisation covalente et de l'activation de surface doit de plus prendre en compte les conditions du test (risque d'immobilisation non spécifique, stabilité de la liaison covalente…), mais aussi la nature de la biomolécule. En comparant différentes activations de surface pour l'immobilisation d'anticorps, Angenendt et al. ont en effet mis en évidence l'influence de la nature de l'anticorps immobilisé sur la qualité de l'immobilisation (Angenendt et al. 2002). ...
The work reported in this thesis focuses on biomolecules immobilization for the development of multiparametric analysis tools on a biochip coupled with a colorimetric detection, applied to the characterization of biological samples and to diagnosis.The first concern was the development of high-throughput automated hybridization tests and immunotests on a filtration plate. This method led to the elaboration of an automated platform for extended blood group genotyping in collaboration with the Etablissement Français du Sang Rhône-Alpes (EFS-RA). It enables to analyze 96 samples in four hours and to characterize six genotypes per sample. Its analytical performances were validated on a panel of 293 blood donors.The second part of this work aimed to elaborate a new strategy for oligonucleotide immobilization on an innovative polymer (PolyshrinkTM) for the development of miniaturized analysis systems. Several approaches were evaluated and led to an in-situ immobilization of oligonucleotides in hydrogel dots technique. This method leads to 6 µm hydrogel dots with a diameter of 60 µm. Moreover it was demonstrated that such immobilized oligonucleotides were able to detect targets specifically and quantitatively using either a chemiluminescent or a colorimetric detection.
... An ideal surface should be able to retain polypeptide functionality with relatively high signal-to-noise ratios and guarantee both high protein-binding capacity and long half-life. Different slide surfaces are in use, including aldehyde-and epoxyderivatized glass surfaces, so-called Fullmoon slides, or Schott NHS-derivatized slides for random linkage of the proteins through amine- (Jones et al. 1998;Kusnezow et al. 2003), nitrocellulose- (Kramer et al. 2004;Stillman and Tonkinson 2000), or gel-coated slides for coupling through diffusion and absorption (Angenendt et al. 2002;Charles et al. 2004) and nickel-coated slides for non-covalent binding of His6-tagged proteins (Feilner et al. 2005). In any case, the immobilization step has to operate in efficient, reliable, and quantitative fashion. ...
Personalized medicine, i.e., the use of information about a person’s genes, proteins, metabolites, and environment to prevent, diagnose, and treat disease, has been much talked about in recent years. So some observers are wondering what the excitement is all about cumulating in the following statement: “Personalized health care is nothing new. Doctors have always tried to fit the therapy to the patient’s need if possible.” But what has happened more recently is that one has now begun to go a level deeper, i.e., to explore the biology of the disease and its treatment at the molecular level. However, molecular medicine does not per se define personalized medicine, but the molecular tools are important as they should enable greater relevance in the information provided by corresponding diagnostic tests (see below) (Edwards et al. 2008; Weedon et al. 2006; Romeo et al. 2007; Hegel et al. 1999; Wildin et al. 2001; Grant et al. 2006; Rothman and Greenland 2005; Raeder et al. 2006; Hegele et al. 2000; Capell and Collins 2006; Delepine et al. 2000; Janssens et al. 2006; Xiayan and Legido-Quigley 2008; Figeys and Pinto 2001; Müller 2002, 2010; Pearson et al. 2007; Janssens et al. 2008; Risch and Merikangas 1996; Janssens and van Duijn 2008; McCarthy 2003; McCarthy et al. 2003; Stumvoll et al. 2005; Lyssenko et al. 2005; Florez et al. 2003).
... In the simplest sense, protein microarrays are immobilized protein spots [312,313]. Thus, proteins can be arrayed on solid surfaces, capillary systems or immobilized on beads [314,315]. The spots may be homogeneous or heterogeneous and may consist of a bait molecule, such as an antibody, a cell or phage lysate, a nucleic acid, drug or a recombinant protein or peptide [316]. ...
Since the introduction of chemotherapy for cancer treatment in the early 20th century considerable efforts have been made to maximize drug efficiency and at the same time minimize side effects. As there is a great interpatient variability in response to chemotherapy, the development of predictive biomarkers is an ambitious aim for the rapidly growing research area of personalized molecular medicine. The individual prediction of response will improve treatment and thus increase survival and life quality of patients. In the past, cell cultures were used as in vitro models to predict in vivo response to chemotherapy. Several in vitro chemosensitivity assays served as tools to measure miscellaneous endpoints such as DNA damage, apoptosis and cytotoxicity or growth inhibition. Twenty years ago, the development of high-throughput technologies, e.g. cDNA microarrays enabled a more detailed analysis of drug responses. Thousands of genes were screened and expression levels were correlated to drug responses. In addition, mutation analysis became more and more important for the prediction of therapeutic success. Today, as research enters the area of -omics technologies, identification of signaling pathways is a tool to understand molecular mechanism underlying drug resistance. Combining new tissue models, e.g. 3D organoid cultures with modern technologies for biomarker discovery will offer new opportunities to identify new drug targets and in parallel predict individual responses to anticancer therapy. In this review, we present different currently used chemosensitivity assays including 2D and 3D cell culture models and several -omics approaches for the discovery of predictive biomarkers. Furthermore, we discuss the potential of these assays and biomarkers to predict the clinical outcome of individual patients and future perspectives.
... 8 Moreover, some Abs lose activity altogether when placed on a surface. 9 In short, better understanding of the factors that affect the behavior of Abs on different types of surfaces is needed to help engineer better Ab microarrays. ...
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.
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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.
... After demonstrating that the assays show clear dose-response with varying concentrations of proteins, we further tested the variations of the assays by measuring pre-aliquoted and frozen quality control (QC) samples with different reader stations on different days ( Figure S2). The calculated coefficients of variation (CV) of the assays were less than 25% in all the cases that we tested, which is comparable to or better than the performance of conventional microarray techniques [28][29][30]. ...
Cancer proteomics is the manifestation of relevant biological processes in cancer development. Thus, it reflects the activities of tumor cells, host-tumor interactions, and systemic responses to cancer therapy. To understand the causal effects of tumorigenesis or therapeutic intervention, longitudinal studies are greatly needed. However, most of the conventional mouse experiments are unlikely to accommodate frequent collection of serum samples with a large enough volume for multiple protein assays towards single-object analysis. Here, we present a technique based on magneto-nanosensors to longitudinally monitor the protein profiles in individual mice of lymphoma models using a small volume of a sample for multiplex assays.
Methods: Drug-sensitive and -resistant cancer cell lines were used to develop the mouse models that render different outcomes upon the drug treatment. Two groups of mice were inoculated with each cell line, and treated with either cyclophosphamide or vehicle solution. Serum samples taken longitudinally from each mouse in the groups were measured with 6-plex magneto-nanosensor cytokine assays. To find the origin of IL-6, experiments were performed using IL-6 knock-out mice.
Results: The differences in serum IL-6 and GCSF levels between the drug-treated and untreated groups were revealed by the magneto-nanosensor measurement on individual mice. Using the multiplex assays and mouse models, we found that IL-6 is secreted by the host in the presence of tumor cells upon the drug treatment.
Conclusion: The multiplex magneto-nanosensor assays enable longitudinal proteomic studies on mouse tumor models to understand tumor development and therapy mechanisms more precisely within a single biological object.
... [1] The wide variation in the properties of proteins (polarity, hydrophobicity etc) has given rise to a large number of different surfaces being used as the array's solid support. Different surfaces give rise to different binding methods such as adsorption (nitrocellulose, [2] poly-Llysine [3]), absorption (hydrogel [4]), covalent bonding (various silanes [5,6]) and affinity attachment (histidine tags [7]) ( Figure 1). One of the primary drawbacks when attempting to fully utilise protein microarrays is the physical size of the protein spots generated. ...
Protein microarrays are one of the leading tools for analysing the proteome due to a range of factors including; their relatively simplistic creation and use in an assay, low cost and long shelf life. One of the primary limiting factors in conventional microarray assay systems is that they create a spot of protein ~100-300 μm in diameter. The miniaturisation of these spots will allow for the creation of much denser arrays and hence will increase the number of simultaneous protein detections possible whilst decreasing the overall physical size of the array. One of the methods for creating protein nanoarrays currently showing promise is Dip-Pen Nanolithography (DPN), a scanning probe microscopy technique. This paper outlines the creation of two types of ultra small functional protein microarrays using DPN on a nitrocellulose surface and their use to detect protein:protein interaction.
... ii. Three-dimensional gel or membrane coated surfaces: These include gels like polyacrylamide, agarose and nitrocellulose [105,107,108]. Protein binds to surface by physical adsorption as shown in Figure 12.13(b). ...
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.
... In this study, we exploit the epoxide ring opening reaction of amine-tethered carbohydrates on epoxide-terminated SAMs [42] to print carbohydrate microarrays on silicon and glass substrates. Epoxide-terminated SAMs are particularly versatile for the fabrication of biological arrays [43,44] and we have recently demonstrated that epoxide-terminated substrates are easily modified using µCP [45]. The incubation of the syn-thetic lectin FITC-HisHis as well as two natural lectins on the immobilized carbohydrates provides insight into the affinity and selectivity of lectin-carbohydrate interaction. ...
The molecular recognition of carbohydrates and proteins mediates a wide range of physiological processes and the development of synthetic carbohydrate receptors ("synthetic lectins") constitutes a key advance in biomedical technology. In this article we report a synthetic lectin that selectively binds to carbohydrates immobilized in a molecular monolayer. Inspired by our previous work, we prepared a fluorescently labeled synthetic lectin consisting of a cyclic dimer of the tripeptide Cys-His-Cys, which forms spontaneously by air oxidation of the monomer. Amine-tethered derivatives of N-acetylneuraminic acid (NANA), β-D-galactose, β-D-glucose and α-D-mannose were microcontact printed on epoxide-terminated self-assembled monolayers. Successive prints resulted in simple microarrays of two carbohydrates. The selectivity of the synthetic lectin was investigated by incubation on the immobilized carbohydrates. Selective binding of the synthetic lectin to immobilized NANA and β-D-galactose was observed by fluorescence microscopy. The selectivity and affinity of the synthetic lectin was screened in competition experiments. In addition, the carbohydrate binding of the synthetic lectin was compared with the carbohydrate binding of the lectins concanavalin A and peanut agglutinin. It was found that the printed carbohydrates retain their characteristic selectivity towards the synthetic and natural lectins and that the recognition of synthetic and natural lectins is strictly orthogonal.
Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0.05) decreased relative to the amount of PA remained in the solution after passing through unmodified as well as protein A modified poly(AAm-AGE) cryogel columns, indicates efficient PA removal from spiked PBS over 60 min of circulation. The high adsorption capacity towards anthrax toxin PA of the cryogel adsorbents indicated potential application of these materials for treatment of Bacillus anthracis infection.
Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
In August 1999, at the Joint Statistical Meetings in Baltimore, two of us [DA & JC] were invited to present a paper on "The Analysis of DNA Microchip Data." This was the first presentation in any Joint Statistical Meetings on the topic of DNA microarrays, as they are now called. In just a few years, the field has exploded and, in each of the recent Joint Statistical Meetings, there have been over a hundred presentations related to DNA microarrays! The Baltimore paper outlined many of the issues that are still being discussed today, including intensity-dependent normalization, the use of methods that are robust to outliers, improving the sensitivity of the analysis by borrowing strength across genes, and interpretation of results in a way that is germane to the overall objectives of a study. However, there have been many developments since then, and this book represents our effort at organizing this material into a semicoherent whole.
With the success of high-throughput DNA microarrays, protein biochips have been intensively investigated and broadly used in bioscience research, clinic diagnosis, drug discovery, and other applications. However, there is great need to significantly improve the sensitivity of protein chips, especially in early diagnosis. A major challenge of improving sensitivity is that protein detection does not have an effective amplification method, such as PCR for DNA microarrays. Construction of unique biofilms for efficient immobilization of protein probes and innovation of new amplification schemes could play a critical role in performance improvement of protein biochips. With dramatic developments in microfabrication, nanotechnologies, and biotechnologies, enormous progress has been made, particularly in improving biosensing sensitivity. This article reviews new advances in protein biochip technologies with emphasis on novel approaches for efficient probe immobilization and nanomaterials-assisted signal amplification for high performance protein chips. Prominent progress in integration of protein microarrays with microfluidic platforms is briefly discussed. The major challenges and perspectives on the future of protein biochips are also addressed.
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.
The key to constructing a protein microarray is the stable immobilization of proteins and the retention of their biological activities. In this study, immobilization of the carcinoembryonic antigen (CEA) antibody onto a silicon dioxide surface was investigated by physical adsorption, direct chemical covalent conjugation, spacer-added chemical covalent conjugation, and biological affinity interactions. Based on the specific antibody-antigen interactions, the sandwich reaction, enzyme-linked immunosorbent assay (ELISA) was chosen to evaluate various immobilization strategies. The most efficient immobilization strategy was with glutaraldehyde as a coupling reagent between the CEA antibody and the amino surface. The attachment of a spacer-armcomprising poly-L-lysine significantly improved the immobilization efficiency and simultaneously decreased nonspecific adsorption. High immobilization efficiency and stronger nonspecific adsorption were also observed when the CEA antibody was immobilized by bioaffinity interactions.
This paper investigated a method that proteins were immobilized on platinum surface based on silicon substrate according to the concept self-assembled monolayers. At first, we produced actived carboxyl groups through surface modification of platinum surface on silicon substrate in reaction solution, and then utilized condensation reaction between proteins and carboxyl groups to immobilize proteins (cattle IgG). Secondly, we showed SEM photographs of surface morphologies of immobilization cattle IgG on silicon wafer. EDS energy spectrum microanalysis of cattle IgG immobilization on silicon wafer was also obtained by means of SEM. By contrast,it was evident that proteins (cattle IgG) can be immobilized effectively on the platinum surface on silicon using the experimental methods. Finally, there were tests of I-V characteristic and I-T characteristic of immobilized cattle IgG which demonstrated its temperature coefficient is .
Functional protein microarray is an important tool for high-throughput and large-scale systems biology studies. Besides the progresses that have been made for protein microarray fabrication, significant advancements have also been achieved for applying protein microarrays on determining a variety of protein biochemical activities. Among these applications, detection of protein binding properties, such as protein-protein interactions (PPIs), protein-DNA interactions (PDIs), protein-RNA interactions, and antigen-antibody interactions, are straightforward and have substantial impacts on many research fields. In this review, we will focus on the recent progresses in protein-protein, protein-DNA, protein-RNA, protein-small molecule, protein-lipid, protein-glycan, and antigen-antibody interactions. We will also discuss the challenges and future directions of protein microarray technologies. We strongly believe that protein microarrays will soon become an indispensible tool for both basic research and clinical applications.
The review summarizes the results of long-term studies having a general idea of the modeling of solid-state processes based on the principle of biological recognition. In all studies considered in this review, new-generation stationary phases, so-called macroporous monolithic sorbents, were used as supporting media. The developed biological recognition systems established the basis of the methods for the fine isolation and highly sensitive analysis of biological substances having different structures and functionality.
In order to develop biosensing system with the waveguide-mode sensor, fabrication of biosensing interface on the silica surface of the sensing chip was carried out using triethoxysilane derivatives with anti-leptin-antibody. Triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties were synthesized to immobilize antibody and to suppress nonspecific adsorption of proteins, respectively. The chip modified with triethoxysilane derivatives bearing oligoethylene glycol moiety suppressed nonspecific adsorption of proteins derived from human serum effectively by rinse with PBS containing surfac-tant (0.05 % Tween 20). On the other hand, it was confirmed that antibody was immobilized on the chip by immersion into anti-body solution to show response of antigen-antibody reaction, where the chip was modified with triethoxysilane derivatives bear-ing succinimide ester moiety. When the interface was fabricated with antibody and a mixture of triethoxysilane derivatives bear-ing succinimide ester and oligoethylene glycol moieties, the response of antigen-antibody reaction depended on composition of the mixture and enhanced with the increase of ratio for triethoxysilane derivatives bearing succinimide ester moiety reflecting the antibody concentration immobilized on the chip. While introduction of excess triethoxysilane derivatives bearing succin-imide ester moiety induced nonspecific adsorption of proteins derived from human serum, the immobilized antibody on the chip kept its activity after one-month-storage in refrigerator. Taking into consideration those factors, the biosensing interface was fabricated using triethoxysilane derivatives with anti-leptin-antibody to examine performance of the waveguide-mode sensor. It was found that the detection limits for human leptin were 50 ng/mL in PBS and 100 ng/mL in human serum. The results demon-strate that the waveguide-mode sensor powered by the biosensing interface fabricated with those triethoxysilane derivatives and antibody has potential to detect several tens ng/mL of biomarkers in human serum with unlabeled detection method.
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.
A method of electrically detecting of protein described is developed
using self-assembled multilayer gold nanoparticles (AuNPs) on a
SiO2/Si substrate between gold electrodes. Electrical
measurements are performed at room temperature using a probe station. A
monoclonal antibody is immobilized on the top surface of the first layer
of AuNPs (14 nm). The second layer of AuNPs is formed through specific
binding among a target antigen [hepatitis C virus, (HCV)], the
monoclonal antibody, and the conjugate of a AuNP-polyclonal antibody.
Once the specific binding among the monoclonal antibody, target antigen,
and polyclonal antibody occurs, a significant electric current is
detected through multilayer self-assembled gold nanoparticles between
nanogap electrodes. No significant current (<1 pA) can be measured
through a monolayer of AuNPs. A significant difference between the IV
curves of the monolayer and the multilayer of AuNPs is used to identify
whether the target antigen exists in the tested sample.
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.
Phospholipid, which is a building block of biological membranes, plays an important role in compartmentalization of cellular reaction environment and control of the physicochemical conditions inside the reaction environment. Phospholipid bilayer membrane has been proposed as a natural biocompatible platform for attaching biological molecules like proteins for biosensing related application. Due to the enormous potential applications of biomimetic model biomembranes, various techniques for depositions and patterning of these membranes onto solid supports and their possible biotechnological applications have been reported by different groups. In this work, patterning of phospholipid thin-films is accomplished by interferometric lithography as well as using lithographic masks in liquid phase. Surface Enhanced Raman Spectroscopy and Atomic Force microscopy are used to characterize the model phospholipid membrane and the patterning technique. We describe an easy and reproducible technique for direct patterning of azo-dye (NBD)-labeled phospholipid (phosphatidylcholine) in aqueous medium using a low-intensity 488 nm Ar+ laser and various kinds of lithographic masks.
A recent acoustic MEMS transducer, a mass sensor based on FBAR (film bulk acoustic resonator), is shown to detect addition of a single DNA base. As the FBAR sensor can detect minute amount of DNA (and/or RNA) markers without the need for optical imaging, it will enable us to develop an “easy to use” and reliable system to detect the presence of pathogen's nucleic acids in non-invasive fluids, such as urine and saliva, when arrayed on a small chip and coupled with a simple sample preparation and an “on-chip” isothermal nucleic acid amplification techniques. Another recent acoustic MEMS, self-focusing acoustic transducers (SFATs) are ideally suited for on-chip DNA and protein synthesis, high frequency ultrasonic imaging, cell lysis or high-intensity ultrasonic treatment with excellent localization on particular cells over a micron-sized area. The focused acoustic beam generated by SFAT is strong and precise enough to eject micron-sized droplets at any desired direction, activate cell-detaching cavitation, and/or lyse cells.
Protein macroarray is a new, simple and multiple biochemistry detection system, in which the test spots are more than 1mm diameter and results directly visible and instrumentation is not necessary. This technology, however, possesses recognized problems with spot quality and uniformity, issues that can limit its application. Previous methods have been developed for spot quality control, but they are complicated or require specific instrumentation. Therefore, we have developed a spot quality control buffer supplement with Ponceau S (SQCB-PS) as a direct and visible means of monitoring spot quality on nitrocellulose membrane prior to hybridization. In this report, 1% (w/v) Ponceau S and 10% (v/v) glycerol in spotting buffer were found to be optimal for spot quality control and food-borne pathogens multiple detection. Furthermore, the sensitivity and specificity of the protein macroarray were not compromised by spotting buffer with Ponceau S. Under optimal conditions, this visible spot quality control method makes the detection more reliable, and should enhance the wider use of the technique.
Copyright © 2015 Elsevier B.V. All rights reserved.
In recent years, various label-free biosensing technologies have been developed for studying the real-time kinetics of diverse biomolecular interactions. These biosensors partially take the place of fluorescence-based methods by providing a comparable sensitivity as well as retaining the conformational and functional integrality of biomolecules to be investigated. However, to completely eliminate the need of fluorescence, throughput is the next big consideration. Microarrays provide a high-throughput platform for screening tens of thousands of biomolecular interactions simultaneously, and many compatible fluorescent scanners have been commercially available. The combination of microarrays and label-free biosensors will be of great interest to researchers in related fields. Microarrays are fabricated by spotting, imprinting, or directly synthesizing biomolecules on solid supports such as glasses, silicon wafers, and other functionalized substrates, and they have been applied to detect DNAs, proteins, toxins, and so on in surface plasmon resonance (SPR) imaging systems and oblique-incidence reflectivity difference (OI-RD) microscopes. Current challenges include increasing sensitivity, reducing sampling time, improving surface chemistry, identifying captured molecules, and minimizing reagent consumption. Future research directions are to improve the instruments themselves, modify the microarray surface for more efficient analyte capture, and combine the systems with mass spectrometry and microfluidics.
© 2015 Society for Laboratory Automation and Screening.
Nanofluidic devices promise high reaction efficiency and fast kinetic responses due to the spatial constriction of transported biomolecules with confined molecular diffusion. However, parallel detection of multiple biomolecules, particularly proteins, in highly confined space remains challenging. This study integrates extended nanofluidics with embedded protein
microarray to achieve multiplexed real-time biosensing and kinetics monitoring. Implementation of embedded standard-sized antibody microarray is attained by epoxy-silane surface modification and a room-temperature low-aspect-ratio bonding technique. An effective sample transport is achieved by electrokinetic pumping via electroosmotic flow. Through the nanoslit-based spatial confinement, the antigen-antibody binding reaction is enhanced with ∼100% efficiency and may be directly observed with fluorescence microscopy without the requirement of intermediate washing steps. The image-based data provide numerous spatially distributed reaction kinetic curves and are collectively modeled using a simple one-dimensional convection-reaction model. This study represents an integrated nanofluidic solution for real-time multiplexed immunosensing and kinetics monitoring, starting from device fabrication, protein immobilization, device bonding, sample transport, to data analysis at Péclet number less than 1.
The antibody bioarray is a promising tool for the detection of proteins, which can be used as disease biomarkers. Therefore, we have developed a sandwich immunoassay-type bioarray using a microfluidic method to detect human interleukins that have diagnostic values. In the method development, we studied the effect of different factors that affect capture antibody immobilization, antibody–antigen interactions, and detection methods on the bioarray surface. The fluorescence signal obtained from different detection strategies was compared based on the signal fold-increase. This comparison showed that the use of covalent immobilization of capture antibodies, as opposed to their physical adsorption on the bioarray surface, increases the signal by 1.5 fold. Moreover, the use of protein G to achieve a better oriented immobilized capture antibody has resulted in a more than 3-fold signal enhancement.
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.
Infectious bronchitis virus (IBV), an avian coronavirus, significantly affects the performance of both the egg-laying and meat-type birds causing the foremost of economic loss in poultry industry. This study aims to develop a rapid, low-cost and sensitive biosensor for IBV detection by using molybdenum disulfide (MoS2). MoS2 is a two-dimensional nano-sheet which has strong high fluorescence-quenching ability when applied to a dye-labeled antibody. In this work, we developed an antibody-functionalized MoS2 based fluorescent immunosensor, which utilized the fluorescence resonance energy transfer (FRET) between the MoS2 and fluorescence dye during the antibody-antigen interaction. The assay was performed on a low-cost cotton thread-based microfluidic platform due to the good wicking property and flexibility. Upon the optimization of assay conditions, the immunosensor demonstrated remarkable sensitivity of 4.6×102 EID50 per mL and specificity with a dynamic linear response range of 102 EID50 per mL to 106 EID50 per mL for IBV standard solutions. The developed immunoassay successfully detected the IBV spiked chicken serum with satisfactory results. The foregoing presents its potential application for on-farm detection.
Billions of people suffer from allergies, though in many cases, the source allergen is unknown. If one knows which allergens to avoid, this would result in an improved quality of life. Since a rapid, high-throughput, automatic allergen detection method is of great need, an integrated system combining microfluidic techniques and microarray chips was developed herein to automate the allergen detection process. The developed microfluidic system could automatically carry out reagent incubation, hybridization, transport, and washing. The microarray chip could be easily detached from the microfluidic chip afterwards, enabling it to be read under a fluorescent scanner. The experimental results indicated that the developed microfluidic system can automatically perform all the incubation processes, including hybridization, reagent transportation, and washing. It is worth noting that the active mixing was applied for the present study which is different from our previous study using micro-channels for passive incubation. Comparable, or even slightly superior, results to a conventional benchtop approach were obtained in ~30% less time with ~25% less samples/reagents. It was also demonstrated that the signal intensities were superior to those using the bench method while detecting immunoglobulin E samples. The developed system could therefore provide a rapid, reliable, and automated approach for detecting allergen-specific antibodies in human serum.
The specific recognition and binding of promoter and RNA polymerase is the first step of transcription initiation in bacteria, and largely determines transcription activity. Therefore, direct analysis of the interaction between promoter and RNA polymerase in vitro may be a new strategy for promoter characterization, to avoid interference due to the cell's biophysical condition and other regulatory elements. In this study, the specific interaction between T7 promoter and T7 RNA polymerase was studied as a model system using force spectroscopy based on atomic force microscopy (AFM). The specific interaction between T7 promoter and T7 RNA polymerase was verified by control experiments, and the rupture force in this system was measured as 307.2±6.7 pN. The binding between T7 promoter mutants with various promoter activities and T7 RNA polymerase was analyzed. Interaction information including rupture force, rupture distance and binding percentage were obtained in vitro, and reporter gene expression regulated by these promoters was also measured according to a traditional promoter activity characterization method in vivo. Using correlation analysis, it was found that the promoter strength characterized by reporter gene expression was closely correlated with rupture force and the binding percentage by force spectroscopy. These results indicated that analysis of the interaction between promoter and RNA polymerase using AFM based force spectroscopy was an effective and valid approach for the quantitative characterization of promoters.
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.
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.
This review highlights optical imaging technologies for the fluorescent read out of sensor arrays. Chemosensor arrays for the determination of pH, oxygen partial pressure or metal ions found particular applications in biomedical and environmental analysis. On the other hand, the monitoring of biomolecular interactions, e.g. of DNA sequences or proteins, is an important tool in pharmaceutical research and medical diagnosis. Microwell plate-based assays provided the possibility to analyze a large number of samples in parallel in a very short time. The development of microarray technologies was a step forward in miniaturization of high-throughput (or multiplexed) assay formats. The analysis of both microwell plate and microarray-based assays are subject of this survey, focussing on fluorescence lifetime imaging methods.
This chapter presents a range of statistical methods for antibody microarray normalization and data analysis. Commonly used techniques for cluster generation, differential analysis, and classification are covered. The focus is on the implementation of each technique to the technology and its suitability in relation to sample types and experiment design.
Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures.
In the present work we describe a biochip that allows the detection of 26 cluster differentiation (CD) antigens, HLA-DR, and IgM antigens on lymphocyte surface. The biochip consists of antibodies (IgG) specific for the selected antigens, immobilized on transparent plastic surfaces in 1.5-mm spots. We have used these biochips to investigate normal and tumor lymphocytes. We demonstrate that these biochips can be used to determine the percentage of the cells expressing different surface antigens in the lymphocyte suspension. The data obtained are in a good agreement with the results of the flow cytometry. The transparent biochip support permits to apply standard staining methods to the cells bound to the biochip and to study their morphology.
Intelligent properties represent discipline-independent definitions for complex behaviors of natural (biomacromolecules, cells, organisms), artificial, (algorithms, devices) and hybrid (biosensors) systems. Intelligent properties can be thought of as subproperties of complex adaptive systems that possess: (i) self-maintenance, (ii) adaptivity, (iii) information preservation, and (iv) spontaneous increase in complexity. The intelligent subproperties include self-assembly, self-organization, self-repair, self-replication, self-maintenance, redundancy, self-diagnosis, recognition, learning, adaptivity, information storage, signaling, and evolution, to name a few. The properties of biomacromolecules, their complexes, cells, signaling networks, and organism subsystems are discussed within the context of their intelligent properties. Specific biomacromolecules' intelligent properties (e.g. DNA, bacteriorhodopsin) offer a range of application and hybrid device opportunities in creating nanostructures, nanocomputing, microelectronics, optical memory designs, and biosensors. Important biosensor nucleic acid and protein class examples are presented that have basic research and clinical applications. Sophisticated levels of intelligent properties exist within evolved control networks in cells. Therefore, cells have added value integrated into smart biosensors. Cellular networks evolved to control important cellular functions and specific examples of gene regulatory control elements are described. Some naturally occurring smart biomaterials have evolved intelligent properties that play critical roles in maintaining the homeostasis and integrity of an organism. An example is the fibrin gel, critical for blood clotting and wound repair. Our aim has been to better integrate molecular and cellular biochemistry with other scientific disciplines within the context of their applications, interfaces, and shared properties by providing a more universal property language —the intelligent properties—to enrich our understanding of biomolecular systems.
Keywords:
intelligent properties;
DNA;
proteins;
cells;
biosensors;
nanotechnology;
bacteriorhodopsin;
fibrin
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.
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.
Analysis of variations in DNA structure using a low-density microarray technology for routine diagnostic in evidence-based medicine is still relevant. In this work the applicability of 3-D macroporous monolithic methacrylate-based platforms for detection of different pathogenic genomic substitutions was studied. The detection of nucleotide replacements in F5 (Leiden G/A, rs6025), MTHFR (C/T, rs1801133) and ITGB3 (T/C, rs5918), involved in coagulation, and COMT (C/G, rs4818), TPH2 (T/A, rs11178997), PON1 (T/A rs854560), AGTR2 (C/A, rs11091046) and SERPINE1 (5G/4G, rs1799889), associated with pregnancy complications, was performed. The effect of such parameters as amount and type of oligonucleotide probe, amount of PCR product on signal-to-noise ratio, as well as mismatch discrimination was analyzed. Sensitivity and specificity of mutation detections were coincided and equal to 98.6%. The analysis of SERPINE1 and MTHFR genotypes by both NGS and developed microarray was performed and compared.
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.
Arrayed Imaging Reflectometry (AIR) is a highly sensitive label-free biosensor which can be used to detect hundreds of antigens on a single substrate. The signal monitored with AIR is the light intensity of an angled beam reflected off of a flat substrate which is composed of a protein-reactive film on a thermally grown silicon oxide layer. If the angle, wavelength, and polarization of the incident light beam is fixed, a near-zero reflectance condition can be obtained by adjusting the thickness of the thermally grown oxide. In a typical AIR biosensing experiment, antibodies are printed (using a piezoelectric microarrayer) on top of the oxide layer to create a minimum reflectance condition. If the substrate is exposed to a complex solution (such as serum), the patterned antibodies bind to their specific targets increasing the effective spot thickness, which perturbs the anti-reflective condition and causes a measurable signal increase. One of the main considerations with AIR is evaluating and controlling the bioactivity and efficiency of antibody immobilization after printing, since these factors significantly affect the dynamic range and limit of detection. Here, we present preliminary experiments towards using microgel nanoparticles as a simple and customizable construct to deposit antibodies on biosensor surfaces. This method can be generalized to work with other microarray technology formats, including those that are not label-free.
Systems biology intends to understand the biological systems as a whole, it may hold the key for eventually cure of some of the most challenging complex diseases, such as cancer, diabetes, obesity, mental disorders and etc. The major driving forces of systems biology are high-Throughput -omics technologies, i.e., genomics, transcriptomics, proteomics and metabolomics and etc. Featured as high-Throughput, miniaturized and capable of parallel analyses, protein microarray has already become a powerful tool for systems biology. In this chapter, we will focus on the application of protein microarrays for global analysis of biological systems and clinical samples, especially cancer related studies. We will discuss the four major types of protein microarray, i.e., proteome microarray, antibody microarray, reverse phase protein array (RPA) and lectin microarrays. We will also discuss the challenges that we are facing and the future trends of protein microarray technology and its applications for systems biology. We strongly believe that protein microarrays will become a standard technology in both basic research and clinical study. © 2012 Springer Science+Business Media Dordrecht. All rights reserved.
Aflatoxin is a major fungal toxin with enormous effect on animal and human health, agriculture, and trade, particularly in African countries with poor storage facilities and improper postharvest handling methods. The serious health effects associated with eating food contaminated with aflatoxins include liver cancer, impaired immune system and cognitive development in children, stunted growth, and injuries to the intestinal wall among others. Most farmers do not know the dangers of aflatoxins, let alone their existence, and therefore are not concerned about their mitigation. Quantitative analysis of aflatoxins is optional for locally consumed foods, but mandatory for crops designated for export in order to meet international regulatory standards. Community members who might otherwise be interested in analyzing their food for aflatoxin contamination before consumption may fail to do so to the high cost of analysis (more than $30 per test per sample), and laborious analytical techniques. These techniques include high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA), and fluorescence spectrophotometer, which are only available in the major food laboratories in the main cities, which are located hundred kilometers away from most farm lands. The aim of this thesis was to develop interventions to monitor aflatoxin concentration at the point of need, and to reduce levels and bioavailability in foods and the gut, respectively, in order to lower the public health risks of aflatoxins and promote aflatoxin-free trade. To achieve this aim, we developed an electrochemical cysteine based immunosensor, and analyzed its feasibility for on-site detection of aflatoxins. We also studied fermentation as a means to decontaminate aflatoxins. Last but not least we assessed the aflatoxin binding ability of Lactobacillus species isolated from the gut of Ugandan children.
In this thesis, a novel on-site detection device was developed through an electroless deposition of silver (plating) onto a glass slide, immobilization of cysteine amino acid on the deposited silver, followed by conjugation of aflatoxin B1 to cysteine groups, and blocking of free cysteine groups with horseradish peroxidase. The performance of the immunosensor was monitored using the indirect competitive immunoassay format, whereby free and immobilized aflatoxin B1 on the sensor competed for the binding site of free anti-aflatoxin B1 antibody. The sensor generated a differential staircase voltammogram peak that was inversely proportional to the concentration of aflatoxin B1. The immunosensor was validated with a range of aflatoxin concentrations from 0 to 15 μg kg-1, and produced a lower limit of detection of 0.7 μg kg-1 maize flour. The results obtained by the novel immunosensor positively correlated with the established laboratory-based HPLC
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and ELISA-detection of aflatoxin B1, with correlation coefficients of 0.94 and 0.98, respectively. Unlike HPLC, ELISA, and many other laboratory-based quantitative methods for detection of aflatoxins, the developed novel immunosensor is portable, weighing approximately 0.5 kg. Therefore, a trader or regulatory agent can move with it to places where people buy and/or sell produce. On-site detection of aflatoxin may enhance awareness, and therefore lead to a healthier population, low incidence of rejected exports due to aflatoxin contamination, and higher earnings in foreign exchange.
In this thesis we also tested the performance of the novel on-site method in real time by determining the concentration of aflatoxins in maize flour samples on the market and households in Kampala, Uganda. The novel immunosensor revealed that aflatoxin contamination in maize flour at household levels is 2.5-fold the concentration of aflatoxin found in maize flour from the markets. This indicates that the risk of acute and chronic aflatoxicosis increases down the crop distribution chain. Therefore, practices such as proper drying of grains, frequent testing for aflatoxin contamination, and proper storage conditions should become everyday practices in order to minimize exposure to aflatoxin. The immunosensor also revealed that hulled maize samples are more frequently contaminated, with concentration levels of approximately 4.3 times greater than dehulled maize.
The probiotic kwete was successfully produced using starter culture with the probiotic Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106. Probiotic kwete was acceptable to the consumers, with a score of ≥ 6 on a 9-point hedonic scale. The products were stable over a month’s study period, with a mean pH of 3.9, titratable acidity of 0.6% (w/v), and L. rhamnosus counts >108 cfu g−1. The yoba bacteria in the probiotic kwete decontaminated aflatoxins B1, B2, G1, and G2 to over a 1000-fold reduction. However, the toxicity on the final product still needs to be elucidated. In vitro fluorescence spectroscopy confirmed binding of aflatoxin B1 to L. rhamnosus yoba 2012 with an efficiency of more than 80% for a one microgram per ml of aflatoxin concentration. Daily consumption of probiotic kwete should lower the risk of aflatoxicosis and aflatoxin-induced ill health such as liver cancer. Moreover, this innovative fermentation system is affordable and transferable to many contexts, and to other nations. The fermentation can improve nutrition security, particularly for children, thus improving their growth and development. It can easily be taken up as a business model by the youth and women, thereby reducing poverty and enhancing economic growth.
The exposure of 10 children aged 55–60 months from a cohort of 512 to aflatoxin B1 was confirmed, with mean urine aflatoxin M1 levels ranging from 15–
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170 pg mg-1 creatinine. The children were frequently fed on hulled and dehulled maize flour and peanut, which contained 9 ± 9 μg kg-1, 5 ± 8 μg kg-1, and 13 ± 8 μg kg-1 levels of aflatoxin B1, respectively. The strains of Lactobacillus species isolated from the children’s fecal samples removed aflatoxin B1 at varying levels. The abundance of Lactobacillus in the gut microbiota of 140 children from the same cohort at 24 and 36 months positively correlated with stunting. The correlation could be attributed to change in the diet of the children from breastfeeding to plant-based solid foods, which could be heavily contaminated with aflatoxins. These solid foods also promote intake of Lactobacillus species.
The current thesis demonstrates the feasibility of using the newly developed on-site immunosensor device for the analysis of aflatoxins, and reveals the prevalence and abundance of aflatoxin contamination in Uganda. The thesis further demonstrates that Lactobacillus species isolated from fecal samples of Ugandan children bind aflatoxins. Results in this thesis indicate that bacterial fermentation is not only a way to enrich and preserve foods, but can also serve to reduce widely occurring aflatoxin contamination. As a follow-up, a double-blind, placebo controlled trial is recommended with fermented maize porridge to substantiate reduction of the bio-availability of aflatoxin in consumed foods.
Glass slides were modified with (3-aminopropyl)-trimethoxysilane, glutaraldehyde, poly-L-lysine to fabricate protein microarrays. Evaluate and choose more optimal surface modification methods for the preparation of protein microarray. The proteins probe labeled by Cy3 was immobilized onto the activated glass slides, attachment rate was evaluated. Subsequently, the mouse IgG were printed on the modified glass slides, the target proteins were labeled by Cy3, responsive activity of protein on microarrays were evaluated. The attachment rate and the effects of immobilized proteins on the glass were compared. The result showed that the aldehyde modification is more suitable surface modification for the fabrication of protein microarrays.
We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation
or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible
expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded
onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6 sequence of expressed proteins (RGS·His antibody, Qiagen) detected 20% of the library as putative expression clones. Two
example genes, GAPDH and HSP90α, were identified on high-density filters using DNA probes and antibodies against their proteins.
A thin layered agarose film on microscope slides provides a versatile support for the preparation of arrayed molecular libraries.
An activation step leading to the formation of aldehyde groups in the agarose creates reactive sites that allow covalent immobilization
of molecules containing amino groups. Arrays of oligonucleotides and PCR products were prepared by tip printing. After hybridization
with complementary fluorescence labeled nucleic acid probes strong fluorescence signals of sequence-specific binding to the
immobilized probes were detected. The intensity of the fluorescence signals was proportional to the relative amount of immobilized
oligonucleotides and to the concentration of the fluorescence labeled probe. We also used the agarose film-coated slides for
the preparation of protein arrays. In combination with specific fluorescence labeled antibodies these protein arrays can be
used for fluorescence linked immune assays. With this approach different protein tests can be performed in parallel in a single
reaction with minimal amounts of the binding reagents.
We have developed and tested a method for printing protein microarrays and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, the other representing an experimental sample in which the protein quantities were to be measured, were labeled by covalent attachment of spectrally resolvable fluorescent dyes.
Specific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signal representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To test the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs. 50% of the arrayed antigens and 20% of the arrayed antibodies provided specific and accurate measurements of their cognate ligands at or below concentrations of 0.34 microg/ml and 1.6 microg/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of 1 part in 106, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples.
These results suggest that protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications.
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.
An mRNA-protein fusion consists of a polypeptide covalently linked to its corresponding mRNA. These species, prepared individually or en masse by in vitro translation with a modified mRNA conjugate (the PROfusion™ process), link phenotype to genotype and enable powerful directed evolution schemes. We have exploited the informational content of the nucleic acid component of the mRNA-protein fusion to create an addressable protein microarray that self-assembles via hybridization to surface-bound DNA capture probes. The nucleic acid component not only directs the mRNA-protein fusion to the proper coordinate of the microarray, but also positions the protein in a uniform orientation. We demonstrate the feasibility of this protein chip concept with several mRNA-protein fusions, each possessing a unique peptide epitope sequence. These addressable proteins could be visualized on the microarray both by autoradiography and highly specific monoclonal antibody binding. The anchoring of the protein to the chip surface is surprisingly robust, and the system is sensitive enough to detect sub-attomole quantities of displayed protein without signal amplification. Such protein arrays should be useful for functional screening in massively parallel formats, as well as other applications involving immobilized peptides and proteins.
Background We describe a method for printing protein microarrays, and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, and the other representing an experimental sample in which the concentrations of specific proteins were to be measured, were labeled by covalent attachment of spectrally-resolvable fluorescent dyes. Specific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signals representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To characterize the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs. Results 50% of the arrayed antigens, and 20% of the arrayed antibodies, provided specific and accurate measurements of their cognate ligands at or below concentrations of 1.6 mu g/ml and 0.34 mu g/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of less than 1 part in 106, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples. Conclusion Protein microarrays can provide a simple and practical means to characterize patterns of variation in hundreds or thousands of different proteins, in clinical or research applications.
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.
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.
Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 x 100 x 20-microm gel pads of protein microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the microchip reaction chamber speeded up antigen-antibody interactions within the gel. Protein microchips were used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein microchip can be used several times in different immunoassays and enzymatic kinetic measurements.
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.
An mRNA-protein fusion consists of a polypeptide covalently linked to its corresponding mRNA. These species, prepared individually or en masse by in vitro translation with a modified mRNA conjugate (the PROfusion process), link phenotype to genotype and enable powerful directed evolution schemes. We have exploited the informational content of the nucleic acid component of the mRNA-protein fusion to create an addressable protein microarray that self-assembles via hybridization to surface-bound DNA capture probes. The nucleic acid component not only directs the mRNA-protein fusion to the proper coordinate of the microarray, but also positions the protein in a uniform orientation. We demonstrate the feasibility of this protein chip concept with several mRNA-protein fusions, each possessing a unique peptide epitope sequence. These addressable proteins could be visualized on the microarray both by autoradiography and highly specific monoclonal antibody binding. The anchoring of the protein to the chip surface is surprisingly robust, and the system is sensitive enough to detect sub-attomole quantities of displayed protein without signal amplification. Such protein arrays should be useful for functional screening in massively parallel formats, as well as other applications involving immobilized peptides and proteins.
The adaptive immune system induces T cells to change from a naive phenotype to a Th1/Th2 phenotype each of which produce characteristic types of cytokines. Knowledge of whether a specific immune response is Th1 or Th2 is a useful indicator for diseases with basis in immune function disorder. An assay that can rapidly analyze multiple cytokines indicative of these two cell types from small sample quantities can be an extremely useful research and diagnostic tool. Silanized glass slides were printed with multiple arrays of capture antibodies to detect eight different cytokines involved in the Th1/Th2 response along with control proteins for assessing assay performance. Arrays were developed by sequential addition of known antigen amounts, detector antibodies and a fluorescent detection system followed by imaging and quantification. These arrays were used to determine the specificity, sensitivity and reproducibility of the assay and the performance compared with conventional ELISA. This multiplexed assay is able to measure human Th1/Th2 cytokines in sample volumes lower than 20 microl. The assay sensitivity for the eight cytokines range from 0.3 microg/l for IL-4 to 6.4 microg/l for IL-5 which are either comparable to or higher than those reported for conventional ELISA or bead-based multiplex ELISA methods. This assay can be automated to measure expression levels of multiple Th1/Th2 cytokines simultaneously from tens to hundreds of biological samples. This assay platform is more sensitive and has a larger dynamic range as compared to a conventional ELISA in addition to significantly reducing the time and cost of assay. This platform provides a versatile system to rapidly quantify a wide variety of proteins in a multiplex format.
- P Arenkov
- A Kukhtin
- A Gemmell
- S Voloshchuk
- V Chupeeva
- A Mirzabekov
P. Arenkov, A. Kukhtin, A. Gemmell, S. Voloshchuk, V.
Chupeeva, A. Mirzabekov, Anal. Biochem. 278, (2000) 123–131.