In This Issue:
- New Aptamer Development Manager, Robert (Rob) Batchelor
- New Grant Award: “Aptamers and BSI for Sub-microliter Analysis of Pharmaceuticals in Neonatal Urine”
- Targeting Circulating Cancer Biomarkers
- Aptamers in CTC Enrichment and Exosome Detection and Characterization
- Product Highlights: Aptamers to CD63, a Common Exosome Marker
New Aptamer Development Manager
Continuing the evolution of Base Pair Biotechnologies, we are delighted to announce the newest addition to our company: Robert (Rob) Batchelor, Base Pair’s new Aptamer Development Manager, who will be joining us effective May 1, 2016. Rob brings a wealth of assay and product development experience to Base Pair’s team. He led or participated in the development of more than fifty products while at Molecular Probes, Invitrogen, Life Technologies, and Marker Gene Technologies, including Amplex® and Amplex UltraRed kits, RediPlate™, EnzChek™, FluoReporter®, Antibody Beacon, Vybrant® and Quant-iT™ assays, BacMam™ and CellLights™ bacculovirus constructs, the Qubit™ minifluorometer, Tali™ and Countess™ cell counting platforms and related kits, and more. His extensive experience in optimizing fluorescence-based assays is immediately applicable to designing microscale thermophoresis binding assays, array-based binding assays, flow cytometry probes, fluorogenic sensor aptamers, enzyme-linked aptamer sorbent assays, microscopy-based detection probes, and many more.
Rob will be leading Base Pair’s aptamer discovery and assay development projects, managing the aptamer discovery laboratory team, ensuring a high degree of scientific rigor, coupled with timely delivery of results to meet the needs of you, our valued customers. Rob will also be assisting Base Pair’s Chief Scientist, Dr. Bill Jackson, in designing our aptamer discovery and assay development projects. We and Rob look forward to working with you to design and deliver on a new aptamer discovery or assay development project for you.
New Grant Awarded to Base Pair
Base Pair Biotechnologies is pleased to announce that it has just been awarded a new Phase I Small Business Technology Transfer grant from the National Institutes of Health, entitled “Aptamers and BSI for Sub-Microliter Analysis of Pharmaceuticals in Neonatal Urine.” In this project, Base Pair will collaborate extensively with the laboratory of Dr. Darryl Bornhop of Vanderbilt University to use Backscattering Interferometry (BSI) for very sensitive, label-free detection of pharmaceutical compounds that can pass from mother to child during pregnancy.
Neonatal and pediatric intensive care still involves performing painful needle punctures to obtain blood samples for routine clinical monitoring. Pain management in the neonatal setting is often achieved by long term, continuous intravenous infusion of morphine or related compounds. It has become increasingly clear that long term use of opioid compounds in neonates is likely to have significant deleterious neurological effects [1,2]. The very nature of neonates (low weight and incomplete and/or varied metabolic development ) complicates pharmacokinetic studies and modeling . Unfortunately, quantitative analysis of such compounds generally requires expensive, time-consuming mass spectrometry (typically LC-MS/MS) methods [5–9]. New analytical platforms are needed to enable studies of commonly used compounds that may transfer over to newborns. Non- or minimally-invasive microfluidic platforms with the ability to address multiple analytes in a flexible manner have great promise in improving neonatal pain management and patient outcomes.
Why aptamers are well suited for this purpose:
The immunogenicity of molecules smaller than a few thousand Daltons, such as hormones, antibiotics, opioids, chemodrugs, anesthesia agents, and other bioactive compounds is generally low. Therefore, obtaining antibodies that recognize small molecules usually requires a carrier protein, such as keyhole limpet hemocyanin (KLH). The resulting KLH-conjugated target molecules may not be the strongest immunogen moiety in the resulting conjugates, requiring multiple screens and separation steps to remove linker-directed antibodies as well as carrier-directed antibodies from animal bleeds. In addition, even in conjugated form, many small molecules are not highly immunogenic. Finally, toxic and otherwise bioactive compounds can themselves adversely affect immune system function as well as the health and/or viability of the immunized animal, resulting in poor antibody titers and a diminished immune response.
Base Pair’s aptamers, which are highly specific and sensitive affinity agents derived from nucleic acids, are selected and synthesized entirely in vitro and are therefore very well-suited for targeting small molecules. Additionally, unlike conventional immunization for antibody development, aptamers can be selected in non-blood backgrounds such as urine and saliva. Selection and counter-selection steps can be easily combined in a well-designed development project to obtain highly specific affinity agents.
Base Pair scientists routinely select aptamers to small molecules and are experts in their characterization. We also can help with the incorporation of aptamers into functional assays and biosensors. Please contact us for more information about these capabilities.
Targeting Circulating Cancer Biomarkers
Two types of material circulating in the bloodstream have attracted significant attention as diagnostic targets for cancer diagnosis. Circulating tumor cells (CTCs) and exosomes are quite different in their size and abundance, yet they represent similar potential for diagnostics – that is, they exist in easily accessible blood and carry a large diversity of molecular information . A major attraction of both CTCs and exosomes is that they might be easily and repeatedly obtained for minimally invasive cancer diagnosis and monitoring . While the idea of a “liquid biopsy” wherein purely molecular signatures supplant more invasive, local histological information is enticing, the approach still has its challenges. CTCs are found in concentrations that are very dilute at approximately 5 CTCs per 7.5 ml . Furthermore, the half-life of CTCs in the bloodstream is only 1–2.4 hours . Because of these challenges, a number of technologies have been developed to enrich CTCs. Epithelial cell adhesion molecule (EPCAM) is the cell surface marker that is most frequently used for positive enrichment of epithelial CTCs and members of the family of cytokeratins (that is, CK8, CK18 and CK19) have become ‘gold standard’ markers for the detection of CTCs with an epithelial phenotype in patients with carcinoma .
Exosomes – small “buds” from cells — have been shown to be far more abundant than CTCs at 100’s of billions per milliliter of serum , yet they are much smaller and present unique challenges for routine diagnostics. Exosomes are nano-vesicles of 50–140 nanometers in size that contain proteins, mRNA, and microRNAs (miRNAs) protected by a lipid bilayer [15–17]. A number of studies exemplified by Melo et al. demonstrate that exosome levels in circulation are elevated versus normal patient populations. More importantly, and somewhat amazingly, cancer-associated exosomes carry microRNAs (miRNAs) and other RNA silencing machinery (RISC, Dicer, etc) and can “mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome … Exosomes derived from cells and sera of patients with breast cancer instigate non-tumorigenic epithelial cells to form tumors in a Dicer-dependent manner” .
Aptamers in CTC enrichment and exosome detection and characterization
Aptamers hold tremendous promise in helping to solve the aforementioned challenges above. Because they are nucleic acids, aptamers will not contribute to protein signature if the final workflow involves mass spectrometry (proteomics). Which respect to universal enrichment of CTCs, unfortunately, EpCAM is not always expressed on CTCs, and in general it has been demonstrated that CTCs display significant heterogeneity depending on tumor type [13,18].
In a 2009 publication from Xu et al. at the University of Florida, aptamers to 3 different leukemia CTC types were demonstrated in a microfluidic device for developed CTC enrichment . To generate the necessary aptamers, “whole cell SELEX” was utilized with positive selection against cells of interest and negative selection against healthy cells. The authors point out the advantage of the aptamer approach in that the exact markers on the cell surface need not be known a priori.
While exosomes are more abundant than CTCs, by virtue of being smaller, their content is more varied. Therefore enrichment and isolation of “healthy” exosomes from cancer-cell-derived exosomes or so-called “oncosomes” is still an important goal in upstream processing of exosomes for subsequent analysis . For instance, Dr. Shona Pedersen of Aalborg University Hospital in Denmark is using aptamers in a novel, resistive-pulse sensing nanopore device, the qNano™ by Izon Science (Christchurch, New Zealand) to quantify and differentiate various markers on exosomes . The impact of various pre-analytical variables including sample dilution, freezing, day to day variation and fasting vs. non-fasting healthy subjects have been evaluated on plasma samples. At the 4th fourth annual meeting of the American Society for Exosomes and Microvesicles (ASEMV), Dr. Amy Phillips (also of Izon Sciences) presented a conference paper entitled, “Advantages of iZON qNano System; Trial of Aptamer-Based Exosome Isolation” describing an aptamer-based method of exosome isolation that is currently being verified in a worldwide trial .
Product Highlight – Aptamers to CD63, a Common Exosome Marker
CD63 is the most commonly used marker for antibody-based enrichment of exosomes as well as verification of various exosome isolation procedures . In a recent study by the group of Dr. Alexander Revzin of the Department of Biomedical Engineering, UC Davis, a Base Pair aptamer to CD63 was utilized to develop a novel electrochemical sensor for exosomes . In this microfluidic sensor, a thiol-modified version of the Base Pair aptamer was readily immobilized on the gold sensor surface, while a short complementary probe having a methylene blue redox center was pre-hybridized to the aptamer. Upon introduction of the exosomes displaying CD63, the probe was released, resulting in reduced electrode current.
Figure 1. From: Zhou Q, Rahimian A, Son K, Shin D-S, Patel T, Revzin A. Development of an aptasensor for electrochemical detection of exosomes. Methods. 2016;97:88–93.
Base Pair offers two unique CD63 aptamers in its online Catalog. As described above, Base Pair scientists frequent consult with customers to provide the proper chemical modifications as well as bioconjugation services to enable novel biosensor development.
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19. Xu Y, Phillips JA, Yan J, Li Q, Fan ZH, Tan W. Aptamer-Based Microfluidic Device for Enrichment, Sorting, and Detection of Multiple Cancer Cells. Anal. Chem. 2009;81:7436–42.
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24. Zhou Q, Rahimian A, Son K, Shin D-S, Patel T, Revzin A. Development of an aptasensor for electrochemical detection of exosomes. Methods. 2016;97:88–93.