In the May Issue
• Base Pair Aptamers to Antibiotics
• Discovery of Aptamers to Small Molecule Antibiotics
• Aptamers and Antibiotics: Detection, Monitoring, and Continuous Dosing
Base Pair Aptamers to Antibiotics
Base Pair has selected natural DNA aptamers to the antibiotics ampicillin and tetracycline. Additional DNA aptamers to antibiotics are in development.
Discovery of Aptamers to Small Molecule Antibiotics
Base Pair is collaborating on a DARPA project* to develop a multiplex biosensor for on-site antibiotic drug monitoring and individualized dosing. Base Pair has selected DNA aptamers to the small molecule antibiotics amphotericin, voriconazole, and meropenem. Lead aptamer candidates are being evaluated in an electrochemical sensing platform at Base Pair. Top aptamer candidates will be transferred to collaborators for multiplex biosensor development.
Learn more about selection of aptamers to small molecules at Base Pair.
*This material is based upon work supported by the Defense Advanced Research Projects Agency (DARPA) under Contract No. 140D63-18-C-0026. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the Defense Advanced Research Projects Agency (DARPA) and no official endorsement should be inferred.
Aptamers and Antibiotics: Detection, Monitoring, and Continuous Dosing
Antibiotics include low molecular weight metabolites of bacteria or fungi and synthetic small molecules that are used to fight bacterial infection (4). Because of their small size, antibiotics are non-immunogenic, making it difficult to raise antibodies for use in detection. Aptamers (small, single strands of DNA or RNA) can be selected for binding to small molecule drugs and are being employed in a variety of new detection platforms designed to measure small molecules possessing a single binding site.
Current methods for the detection of antibiotics typically involve chromatography or mass spectrometry. Tests are expensive and time-consuming. They require expensive instrumentation operated by trained personnel (4). Aptamer-based assays and biosensors offer faster, simpler, less expensive methods for detection of antibiotics that can be performed outside of a central laboratory.
Detection of Antibiotics
The overuse of antibiotics in animal care, release of antibiotic contaminants into surface water and ground water, and transfer of residual antibiotics through meat and dairy products are of particular concern in the effort to prevent long-term antibiotic exposure and slow the development of antibiotic resistance (3,4).
Researchers at Beijing University developed an aptamer-based assay for the detection of the antibiotic cefquinome (CFQ) in milk. CFQ is an antibiotic primarily used to treat porcine and bovine infections that can be found at fairly high levels in some animal-sourced food. The assay is based on the use of biotinylated structure-switching CFQ aptamers bound to SA-magnetic nanoparticles and complementary FAM-labeled, 17 base ssDNA probes that are dislodged upon binding of CFQ. Following incubation, FAM-probe in the supernatant, which correlates to the concentration of CFQ in sample, is detected using a fluorescence plate reader. Levels of 5 ng/mL to 8 ng/mL were detected in processed milk samples, well below the maximum residue limit of 20 ng/mL in animal products (5).
Researchers in China and Japan developed an aptamer-based assay for the detection of the antibiotic chloramphenicol in water samples. The team utilized a metal-organic framework (MOF) that oxidizes tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2) for colorimetric detection. Binding of chloramphenicol to aptamer-gold nanoparticles in the presence of the MOF reduced oxidation of TMB and decreased absorbance. TEM images suggest the CAP-aptamer-AuNP conjugates coat the outside of the MOF, restricting electron transfer. The limit of detection of the assay for chloramphenicol was 25 nM, or 8 ng/mL. The method was also demonstrated using aptamers selective for oxytetracycline, tetracycline, and ampicillin (3).
In Vivo Antibiotic Monitoring and Feedback-Controlled Delivery
Dosing is not an exact science. Circulating levels of drug can vary depending upon physiological differences between patients, disease progression, drug-drug interactions, diet, and environmental factors. The ability to better regulate antibiotic levels can maximize effectiveness by ensuring sufficient dosage and minimizing the risk of drug toxicity, particularly in very ill patients (2).
In 2017, researchers at UC-Santa Barbara reported on the use of an electrochemical aptamer-based biosensor for the continuous detection of aminoglycoside antibiotics (kanamycin, gentamycin, and tobramycin) and the chemotherapeutic doxorubicin in Sprague-Dawley rats. Effective monitoring of small molecule drugs was demonstrated in both anesthetized and active rats. Drug levels within a single animal and between animals were easily evaluated over time and showed significant variability (1).
More recently, researchers at Johns Hopkins and UC-Santa Barbara combined in vivo monitoring with automated drug delivery to maintain a consistent level of antibiotic in anesthetized rats. The team used an aminoglycoside sensor and square wave voltammetry to measure binding of the antibiotic tobramycin. A PID-controller, communicating with a pump, evaluated readings versus a calibration curve and reference set point every 7 seconds and adjusted the drug infusion rate accordingly over a 6 hour time period, the maximum time the animals could remain anesthetized. Traditional dosing based on subject weight yielded a 30% variability in circulating drug level between subjects. Feedback-controlled infusions reduced subject-to-subject variability to only 4% (2). The application of this kind of aptamer-based technology can enhance pharmacokinetic and dosing studies for drugs in development and improve the effectiveness of treatment in the clinic.
Custom Aptamer Selection
With Base Pair’s patented multiplex SELEX, aptamers recognizing a family of antibiotics or a specific antibiotic can be selected. Our in-house electrochemical sensing platform can be used to screen aptamers for biosensing applications involving small molecules.
Contact Base Pair today for more information on selection of aptamers to antibiotics and other small molecules.
- Arroyo-Curras, N., et al. Real-time measurement of small molecules directly in awake, ambulatory animals. PNAS. 2017. 114(4):645-650.
- Arroyo-Curras, N., et al. High-precision control of plasma drug levels using feedback-controlled dosing. ACS Pharmacology and Translational Science. 2018. 1(2):110-118.
- Li, J. et al. Novel sensing platform based on gold nanoparticle-aptamer and Fe-metal-organic framework for multiple antibiotic detection and signal amplification. Environment International. 2019. 125: 135-141.
- Mehlhorn, A., et al. Aptamer-based biosensors for antibiotic detection: A review. Biosensors. 2018. 8(2):54
- Wang, L., et al. Selection of DNA aptamers and establishment of an effective aptasensor for highly sensitive detection of cefquinome residues in milk. Analyst. 2018. 143: 3202-3208.