In The January Issue
• New! BLI Affinity Determination Service from Base Pair
• Advantages of Biomarker Detection with Aptamers
BLI Affinity Determination Service
The determination of aptamer-target binding characteristics is an important tool in the aptamer discovery process. Bio-Layer Interferometry (BLI) calculates an association constant, dissociation constant, and affinity constant for evaluation of lead aptamer candidates and identification of complementary aptamer sequences. BLI affinity testing is available as part of Base Pair aptamer discovery projects OR as a stand-alone service. Whether you have discovered aptamers with Base Pair, in your own laboratory, or with another research team, Base Pair can assist with affinity determination.
Learn more about BLI and affinity testing at Base Pair.
Advantages of Biomarker Detection with Aptamers
Antibodies have been the go-to affinity reagent for biomarker detection for many years, but antibodies are not an ideal solution for all targets and applications. Aptamers (small strands of ssDNA or ssRNA with selective binding) offer some unique advantages that are increasingly being applied to detect difficult targets. Because aptamers are selected in vitro, they can be raised against toxic compounds, toxic metabolites, or infected cells that would not be tolerated in a biological system.
Aptamer discovery is often faster and less expensive than antibody development.Once selected, aptamers are chemically synthesized, yielding excellent lot-to-lot reproducibility. Whether selected for binding to a specific target (protein, peptide, toxin, small molecule, etc.) or a specific cell type, aptamers can be selected to differentiate between highly similar molecules. From diagnostic imaging to histology to blood/urine sample analysis, aptamers offer specific advantages in biomarker detection.
In Vivo and In Vitro Imaging for Detection of Cell Surface Biomarkers
In vitro tissue analysis (IHC, IF) is currently the primary method for tumor diagnosis (1). While many oncology biomarkers have been identified, there is a critical need for biomarkers for earlier detection of many forms of cancer along with more sensitive non-invasive screening methods. While antibody development typically starts with a purified target, aptamers can be selected against a purified target or a cell type of interest, without prior identification or isolation of a specific cell surface target, accelerating the process of biomarker discovery. (Learn more about Cell-SELEX and biomarker discovery.)
Selected aptamers can be labeled and used to detect target cells in vivo and in vitro. Aptamers have been used to selectively detect tumor-related tissue versus normal tissue in immunohistochemistry (IHC) and immunofluorescence (IF) (1,4). They have been used to assess fresh post-operative tissue, frozen tissue samples, and formalin-fixed, paraffin-embedded (FFPE) tissues, though not all aptamers have worked for all sample types. Labeled aptamers have been applied in near-infrared (NIR) imaging, MRI, and CT-scans, showing an enhanced ability to access targets in dense tumor tissue (5,6,7). (Read more about aptamers for in vivo imaging.)
- Because aptamers are non-immunogenic, there is no interference with processes being studied
- Small aptamer size ofers enhanced penetration of tumor tissue and access to cell surface targets for improved imaging
- Aptamers can be labeled with a wide range of molecules with no loss of selectivity or function
- Several aptamer modifications are commonly used to delay clearance, extending stability in vivo
Biosensors for Point-of-Care Testing
Rapid detection and treatment is important in many clinical areas. Existing laboratory tests can often take several hours and sometimes days, with additional time for processing through a central laboratory. Point-of-care tests to identify high-risk patients and accurately diagnose disease would expedite treatment and improve outcomes. Small aptamer size, stability, and rapid in vitro development make aptamers ideal affinity reagents for biosensor development.
An international team of researchers has developed aptamer-based electrochemical biosensors for the detection of two cardiac biomarkers, brain natriuretic peptide (BNP-32) and troponin I (cTnI), in serum. Both sensors demonstrated sensitive detection (1 pg/mL) and a broad dynamic range (≥ 4 logs). Biosensor detection was completed in just 30 minutes and biosensors were successfully regenerated with a 20 minute incubation in NaOH, enabling repeated testing over an extended time using a single biosensor. These particular biosensors showed comparable sensitivity to current antibody-based tests and successful analysis of undiluted serum (3). Development of multi-electrode systems for testing of a panel of biomarkers will enable rapid point-of-care diagnosis and extended treatment monitoring.
- Small aptamer size enables close proximity to sensor surface for enhanced detection
- Easily immobilized on a wide range of surfaces or conjugated for detection without loss of activity
- Thermal and chemical aptamer srability enables extended monitoring and re-generation of aptamer-based biosensors
Plate-Based and Lateral Flow Assays for Detection of Circulating Biomarkers
Antibodies are widely used for the detection of circulating biomarkers via ELISA and other plate-based assays and lateral flow tests. While antibody-based assays are very sensitive, there are sometimes concerns related to non-specific binding, antibody instability, and batch-to-batch variability. Developing antibodies to non-immunogenic or toxic compounds is also a huge challenge. For these reasons, researchers are increasingly turning to aptamers.
Researchers at the University of Illinois have developed a lateral flow assay (LFA) for detection of cortisol, a small steroid hormone (362 Da) that is elevated in response to stress. The lateral flow assay utilizes aptamer binding to gold nanoparticles. Cortisol in the sweat sample causes dissociation of the anti-cortisol aptamers from the gold nanoparticles, enabling gold nanoparticles to bind cysteamine at the test line. If no cortisol is present, the gold nanoparticles are shielded by anti-cortisol aptamers and cannot bind at the test line. The lateral flow assay was designed for detection in sweat. The lower limit of detection is 1 ng/mL, well below the normal range of 8 – 140 ng/mL (2). Selective aptamers can be designed for use in competitive LFAs and plate-based assays for a wide range of small molecule biomarkers and metabolites.
- Aptamers are easily selected for binding to non-immunogenic small molecules, toxic compounds
- No antibody-related non-specific binding, including heterophilic antibodies and anti-species antibodies
- Thermal stability enables field-based detection in a wide range of climates
- Chemical synthesis offers improved batch-to-batch reproducibility and reduced regulatory concerns versus bioproduction
Custom Aptamer Discovery
Base Pair has experience discovering aptamers to a wide range of targets, including proteins, peptides, small molecule drugs, metabolites, toxins, and live cells. With Base Pair’s patented multiplex SELEX, aptamers to multiple targets can be discovered in a single SELEX protocol.
Contact Base Pair today to learn more about selection of aptamers for biomarker detection.
- Bauer, M. et al. The application of aptamers for immunohistochemistry. Nucleic Acid Therapeutics. 2016. 26(3):120-6.
- Dalirirad, Shima and Andrew Steckl. Aptamer-based lateral flow assay for point-of-care cortisol detection in sweat. Sensors and Acuators B: Chemical. 2019. 283: 79-86
- Grabowska, I, et al. Electrochemical aptamer-based biosensors for the detection of cardiac biomarkers. ACS Omega. 2018. 3:12010-12018.
- Zamay, G.S., et al. DNA aptamers for the characterization of histological structure of lung adenocarcinoma. 2017. 6:150-162.
- Zhao, M., et al. In vivo fluorescence imaging of hepatocellular carcinoma using a novel GPC3-specific aptamer probe. Quantitative Imaging in Medicine and Surgery. 2018. 8(2) :151-160.
- Zhang, Y. et al. Aptamer-targeted magnetic resonance imaging contrast agents and their applications. Journal of Neuroscience and Nanotechnology. 2018. 18:3759-3774.
- Zhu, H., et al. Aptamer-PEG-modified Fe3O4@Mn as a novel T1- and T2- dual-model MRI contrast agent targeting hypoxia-induced cancer stem cells. Nature: Scientific Reports. 2016. 6:39245.