Researchers at Kansas State University have developed a novel injection system to solve the problem of biased introduction of analytes for electrophoresis systems by electrokinetic injection. Sample introduction is a critical, yet often overlooked, step in electrophoretic separations. There are two main injection modes in electrophoresis – hydrodynamic and electrokinetic – and they can have a significant effect on the analytical performance of the method. In hydrodynamic injection, the sample is introduced by means of a pressure difference between the inlet and outlet valves in the system. Electrokinetic injections use voltage differences to introduce the sample; in this case, analytes migrate into the capillary based on their electrophoretic mobility and the electro-osmotic flow and, therefore, it shows a bias for the introduction of the more mobile, faster moving analytes.
Chris Culbertson (Kansas State University, USA) works on developing novel sample handling and separation components for lab-on-a-chip devices. In a recent paper published in Analytical Chemistry, Chris describes a novel microfluidic injection system based on polydimethyl siloxane (PDMS) with integrated dielectric elastomer actuators (IDEAs) to solve the problem of biased injection. The IDEAs produce hydrodynamic fluid pulses and Chris was able to demonstrate that, when samples were introduced using the IDEA device, the results of repeat injections of fluorescein thiocarbamyl-labeled amino acids were more consistent. Importantly, there was no significant difference in terms of resolution or efficiency when compared with electrokinetic injections.
Chris told Bioanalysis about the possibilities for future implementation: “we feel that IDEAs can be used for multiple forms of fluid manipulation on microfluidic devices, including mixing, pumping and cell lysis, in addition to sample introduction. One field in which IDEAs may have a profound impact is segmented flow systems, where IDEAs may be able to provide fine control over plug volume, plug frequency and the addition of reagents to pre-formed plugs.”
However, the biggest challenges for IDEA technology lie ahead, explained Chris: “We didn’t have many problems demonstrating the viability of the concept using devices that have all-PDMS channels. PDMS is an exceptional material for the dielectric elastomer layer and a widely-used material for microfluidic devices, but it isn’t always the optimal material to use for chemical analysis due to its hydrophobicity and predisposition for absorbing and adsorbing small molecules and proteins. To overcome these potential problems, we are at present integrating previous technology that we have developed for coating channels into the devices so that they are amenable to a wide variety of chemical analysis modes.”
In the future, widespread adaptation of this technology could be advantageous for many types of microfluidic systems.
Sources: Price AK, Culbertson CT. Generation of nonbiased hydrodynamic injections on microfluidic devices using integrated dielectric elastomer actuators, Anal. Chem. DOI: 10.1021/ac9015837 (2009) (Epub ahead of print); Culbertson CT. Research page www.k-state.edu/chem/people/faculty/culbertson.html
A group at the University of Tokyo have successfully developed a noncompetitive open-sandwich immunoassay (OS–IA) to quantify 11-deoxycortisol, a corticosteroid used as a diagnostic biomarker for pituitary-adrenal function.
Associate Professor Hiroshi Ueda, who led the research group, explained the advantages of their assay: “the existing immunoassay methodology for small molecules is dominated by competitive assays, whose sensitivity is theoretically limited by the affinity of the antibody. Since our open-sandwich immunoassay is free of such a competitive step, it is by its nature unlimited by affinity, which means we have enormous possibility in sensitivity and working range.”
The main obstacle in developing this assay was in obtaining a good source of antibodies, therefore, the group collaborated with another group with good antibody-producing hybridoma. However, Professor Ueda told Bioanalysis that this will no longer be a problem since they are “now able to clone good antibody genes from immunized animals directly using a propriety phage display system. We are now trying hard to work on this; we dream of an age when our method will revolutionize immunodiagnostics, at least for small molecules,” he enthused.
The next step in this research is to apply the OS–IA to other related analytes, including cortisol and cortisone. The group have already succeeded in measuring estradiol with impressive sensitivity. In the future, applications will include fabrication of ‘Bio-Chips’ or microfluidics, enabling faster detection than plate-based assays.
Sources: Ihara M, Suzuki T, Kobayashi N, Goto J, Ueda H. Open-sandwich enzyme immunoassay for one-step noncompetitive detection of corticosteroid 11-ceoxycortisol. Anal. Chem. 81 (20), 8298–8304 (2009); Dong J, Ihara M, Ueda H. Antibody Fab display system that can perform open-sandwich ELISA. Anal. Biochem. 386, 36–44, (2009); Sakata T, Ihara M, Makino I, Miyahara Y, Ueda H. Open sandwich-based immuno-transistor for label-free and noncompetitive detection of low molecular weight antigen. Anal. Chem. 81(18), 7532–7537 (2009).
A fast, noninvasive metabolomics-based test has the potential to improve the outcome of infertility treatment for hundreds of thousands of couples, according to researchers at the Department of Obstetrics, Gynecology and Reproductive Sciences at the Yale School of Medicine. The findings were presented at the American Society for Reproductive Medicine meeting (Atlanta, Georgia, USA) by lead researcher Emre Seli, MD.
Typically, when undergoing in vitro fertilization (IVF), the woman is hormonally stimulated to overproduce eggs, which are then fertilized in the lab. Normally, several viable embryos would be produced and it can be tricky to choose the right ones for implantation: the embryologist makes the decision based on the number of cells, evenness of growth and degree of fragmentation. Seli explained: “it’s a guessing game that can end in IVF failure or multiple pregnancies.”
The metabolomics approach developed by Seli and his team has the potential to identify the embryos most likely to survive before implantation, meaning that fewer would need to be implanted and thus reducing the risk of multiple pregnancies. Seli’s group studied the metabolomic profile of spent embryo cultures and then used this data to generate a viability score for embryos prior to implantation. In the study, carried out in collaboration with Molecular Biometrics, Inc., they found that the application of the viability score improved pregnancy outcomes in women undergoing IVF at four centers in Europe and Australia.
“These findings have important implications for the more than 125,000 IVF cycles performed yearly in the USA” said Seli. “The high multiple pregnancy rates associated with IVF have significant public health consequences, such as decreased survival and increased risk of lifelong disability associated with severe prematurity.”
The team previously found that metabolomic profiling could give an instant snapshot of the physiology of a cell. This noninvasive approach may provide a useful adjunct to the current embryo grading systems based on the structure of the embryo and the rate at which the embryo divides.
Source: Identifying the metabolism of a healthy embryo could improve infertility treatment http://opa.yale.edu/news/article.aspx?id=6993
A newly-developed magnetic immunoprobe has the potential to be used as a rapid and accurate means of detecting biomarkers in serum. In a recent Analytical Chemistry paper, researchers at the National Tsing-Hua University and Academia Sinica (Taiwan), describe a new method for immobilizing antibodies on magnetic nanoparticles.
Traditionally, antibodies were immobilized on magnetic nanoparticles in a random manner, but this new method allows them to be covalently linked at specific locations and orientations. The new method involves the formation of a boronate on the surface of the nanoparticles; once the boronic acid is available on the surface, the immobilization of antibodies can be easily achieved by incubation of the antibody with the boronic acid-containing material. Although the formation of boronate is a reversible reaction, the immobilized antibody is relatively stable, due to the multivalent interaction between the sugar moiety of the antibody and the surface-bound boronic acid.
When applied to profiling amyloid P, amyloid A and C-reactive protein levels in serum, the probe was able to distinguish between normal patients and those with cancer or cardiovascular disease. The probe also showed excellent performance in terms of stability and sensitivity and the group suggested that this was due to an enhancement in antigen-binding activity.
Lead author on the paper, Chun-Cheng Lin (National Tsing-Hua University) spoke to Bioanalysis about the challenges involved in developing this probe: “significantly reduced activity of antibody in serum samples (compared to standard antigen solution) presented the most challenging issue. Through the introduction of a precleaning step, the problem was resolved.” He went to explain that the remaining free boronic acid on the surface may cause undesired interactions, resulting in false-positive detection results and necessitating a further capping step when complex samples, such as human serum, are used. The group is currently applying boronic acid-functionalized magnetic nanoparticles to the study of glycoproteomics.
Chun-Cheng Lin and Yu-Ju Chen (another group leader) are very enthusiastic about the possibilities opened up by their work: “the synthetic immobilization strategy may also enhance the detection sensitivity of many other analytical techniques that require antibody immobilization on the surface of a solid support, such as ELISA, protein chip (SELDI) and immunoaffinity column.”
Source: Lin PC, Chen SH, Wang KY et al. Fabrication of oriented antibody-conjugated magnetic nanoprobes and their immunoaffinity Application. Anal. Chem. DOI: 10.1021/ac9012122 (2009) (Epub ahead of print).
Three new analytical platforms have been developed for studying the pharmacokinetics of protein drugs. The platforms, described in a recently-published paper, have been developed by a group at Northeastern University (MA, USA) in order to facilitate the analysis of different types of protein drugs.
The first method, based on albumin depletion, is very generic meaning it is applicable to any protein drugs; the group was able to detect 1 ng of drug in 30 µl of serum. The second method is based on protein A enrichment (with SDS–PAGE), allowing detection down to 0.02 ng. The third method is based on protein A enrichment without SDS–PAGE (detection to 10 ng), or with antidrug antibody enrichment (detection to 0.1 ng). There was shown to be good agreement when compared with an ELISA in a mimic study of the drug in monkey serum.
The group believes that these three platforms can be selectively applied in pharmacokinetic studies, depending on the required sensitivity, available time and resources, essentially enabling the analyst to balance throughput and sensitivity requirements. According to Professor Shiaw-Lin Wu, a member of the group that carried out the work, the wider applications of these methods, outside of pharmacokinetic studies, will come from the ability to use methods without the need for affinity enrichment.
Source: Lu Q, Zheng X, McIntosh T et al. Development of different analysis platforms with LC–MS for pharmacokinetic studies of protein drugs. Anal. Chem. DOI: 10.1021/ac901991x (2009) (Epub ahead of print).
A team at Hollings Marine Laboratory (Charleston, SC, USA) are using NMR-based metabolomics to better understand coral bleaching caused by the disruption of the symbiosis between coral animals and their zooxanthellae, which can occur as part of a natural cycle or as the result of unusual events, such as infection by pathogens, pollution or increased water temperature.
The zooxanthellae are tiny photosynthesizing algae that live within coral tissue and provide up to 90% of its energy. The algae produce an array of chemical products that give coral its characteristic colours. When the symbiosis is upset, the coral can turn white, hence the term ‘bleaching’.
One cause is infection by Vibrio coralliilyticus, which has been linked to bleaching in the Mediterranean, Red Sea, Indian Ocean and Great Barrier Reef. The group at Hollings Marine Laboratory used NMR to investigate the effects of temperature on the metabolome of V. coralliilyticus. They used water-methanol to extract the intracellular metabolites (or endometabolome) from cultures grown from 24–27°C, analyzed the extracts by NMR and then used principal component analysis to determine the differences between the avirulent form (24°C) and the virulent form (27°C). They observed that, as the temperature increased, betaine was downregulated, while succinate and glutamate were upregulated. The group hopes that these metabolic changes will help them to understand why small temperature changes can turn the innocuous V. coralliilyticus into a coral-bleaching menace and are planning to carry out additional metabolomics studies in the future to gain a deeper understanding of the delicate symbiotic relationship between coral and its zooxanthellae.
Sources: Marine lab team seeks to understand coral bleaching www.sciencedaily.com/releases/2009/10/091022114357.htm; Boroujerdi et al. NMR-based microbial metabolomics and the temperature-dependent coral pathogen Vibrio coralliilyticus. Environ. Sci. Technol. 43(20), 7658 (2009).
Chocolate or, more specifically, cocoa-phytochemical consumption is purported to have health benefits. A recent study carried out by Rafael Llorach and Cristina Andres-Lacueva at the University of Barcelona Nutrition and Food Science Department has assessed the effect of cocoa consumption on the urinary metabolome, in order to shed some light on the health benefits of cocoa phytochemicals.
In the recently published paper 10 subjects were randomly dosed with either cocoa powder with milk or water, or plain milk. Urine samples from before and after cocoa consumption were analyzed by HPLC-QTOF; the group then carried out multivariate data analysis on the samples to reveal an important effect on urinary metabolome during the 24 h after cocoa powder intake. However these changes were not influenced by matrix as no global differences were found between cocoa powder consumption with milk or with water. They found 27 cocoa-phytochemical-related metabolites in the post-cocoa consumption urine samples, as well as identifying diketopiperazines (cocoa processing-derived products) in urine samples for the first time. Metabolomic strategy has proved a powerful tool for identifying new markers of exposure and very useful for confirming the robustness of some expected cocoa metabolites such as polyphenols or alkaloid derivatives. Cristina Andres-Lacueva, lead author on the paper, described the benefits of this study: “This kind of work produces important information in order to building up the concept of the “food metabolome”. Moreover, these markers will be useful to monitor the consumption of cocoa in other epidemiological studies.” Combined with other ‘omics’ this type of metabolomics study can provide valuable information about the complex relationships between diet and health, thus reducing the distance from nutrition to personalized nutrition.
The current study was on healthy volunteers; the next step is to apply this strategy in “non-healthy” volunteers. Rafael Llorach explained to Bioanalysis: “In fact, we are involved in a study focused on the application of metabolomics to evaluate the impact on health after one month of regular cocoa consumption in patients at high risk of cardiovascular disease.” Targeted analysis of this kind of clinical trial has been recently published in American Journal of Clinical Nutrition by the same research group; the results of this study suggest a positive influence of cocoa polyphenols on the modulation of inflammatory mediators in human subjects at high risk of cardiovascular heart disease. The next step is the application of metabolomics in a context of “free-living” people studies, where the diet and others variables are not controlled.
Sources: Llorach R, Urpi-Sarda M, Jauregui O, Monagas M, Andres-Lacueva C. An LC-MS-Based Metabolomics Approach for Exploring Urinary Metabolome Modifications after Cocoa Consumption. J. Proteome Res. Epub ahead of print. DOI: 10.1021/pr900470a (2009); Monagas M, Khan N, Andres-Lacueva C et al. Effect of cocoa powder on the modulation of inflammatory biomarkers in patients at high risk of cardiovascular disease. Am. J. Clin. Nutr. 90(5), 1144–1150 (2009); Llorach-Asunción R, Jauregui O, Urpi-Sarda M, Andres-Lacueva C. Methodological aspects for metabolome visualization and characterization: a metabolomic evaluation of the 24 h evolution of human urine after cocoa powder consumption. J. Pharm. Biomed. Anal. 51(2), 373–381 (2010).
A new collaboration between Agilent Technologies Inc. and the Institute for Systems Biology aims to create a database of all the, approximately, 20,000 human proteins, opening new possibilities for large-scale proteomics research.
The work will see researchers from the Institute for Systems Biology and ETH Zurich provide quantitative data on up to four peptides for every protein-coding gene. It is funded, in part, by the National Human Genome Research Institute of the NIH and the European Research Council.
“We believe this will be a revolutionary development in protein analysis, one that will accelerate and catalyze the routine use of protein quantitation for immensely important breakthroughs in the understanding, early detection and monitoring of human disease,” commented Rob Moritz from the Institute for Systems Biology.
The work will provide scientists with in-depth verified MS data from Agilent’s triple quadropole TOF–LC–MS system. “Agilent is pleased to share leadership in creating the Human MRM Atlas and MRM-based methods to support quantitative protein research,” said Ken Miller from Agilent, “the combination of our triple quadrupole instrumentation, software tools specific for protein analysis, and unique HPLC–chip–MS technologies creates a stable, sensitive platform for the analysis of these large sample sets.”
The data from the Human Multiple Reaction Monitoring Atlas has many potential uses, including aiding research into proteomic diagnostics and biomarker discovery and validation.
Source: Institute for Systems Biology, Agilent Technologies to build MRM atlas for quantification of all human proteins, Agilent press release www.agilent.com/about/newsroom/presrel/2009/21oct-ca09062.html
A novel lab-on-a-chip approach to identifying the most effective anticancer therapy for each individual patient has been developed by researchers at the Heinz Nixdorf Chair for Medical Electronics at the Technische Universitaët Muënchen.
The device consists of a number of bioelectric sensors that monitor the metabolic activity and viability of tumor cells in microtiter plates. After a few hours, a robot attached to the chip applies an anti-tumor therapy to each sample and monitors the cells’ progress over the next few days. The effects of 24 therapies on tumor cells can be tested simultaneously.
Personalized cancer therapy is an important goal in medicine, as it is estimated that, for a given patient, only a third of available anticancer drugs will be effective. The lab-on-a-chip system could not only speed up diagnosis but also improve the accuracy of the therapy prognosis.
Helmut Grothe, from the Technische Universitaët Muënchen, commented on the potential of the chip to also reduce anticancer drug resistance, by identifying those therapies to which the tumors show signs of resistance: “treatment with an ineffective cancer drug sometimes leads to the development of resistance to other drugs in the patient.”
The team are also working on a sensor chip that could control tumor growth, releasing a therapy only when the tumor becomes larger.
Source: Sensor biochips could aid in cancer diagnosis and treatment, AAAS press release www.eurekalert.org/pub_releases/2009-10/tum-sbc102109.php