Category: Assay Development and Screening
The United States is in the midst of an opioid epidemic of unprecedented scope. Heroin use is resurging, but the pervasive and increasing abuse of prescription opioids, including methadone, oxycodone and hydrocodone is even more troubling, as is increasing abuse of illegally obtained fentanyl and high potency derivatives, such as carfentanil.
Compensated Backscattering interferometry (CBSI) is a method for detection of molecular interactions that holds promise for translation to the near-patient setting. CBSI is quantitative at femtomolar (fM) sensitivity, requires nanoliter sample volumes and is “label free." CBSI is also uniquely suited to the near-patient setting consisting of a diode laser, sample-containing capillary and camera.
The free solution response function (Free-SRF) described recently in PNAS, provides a framework for understanding how CBSI can function as the only free-solution, label-free technique with sensitivity that rivals fluorescence assays. FreeSRF is compatible a wide range of analytes, can be used with complex matrices, and is quantitative. CBSI transduces FreeSRF assays by measuring changes in structure and hydration when molecules interact. Here we show that CBSI used in concert with FreeSRF is uniquely suited for use with DNA or RNA aptamers probes because these probes undergo significant structural and hydration changes upon binding. Next, we will describe the principles of operation for CBSI and demonstrate how our compensated interferometric approach constrains environmental noise, enabling baseline refractive index (RI) noise levels of a few parts in 10-7 in the presence of thermal perturbations corresponding to 10-4 RIU (8oC). The simplicity and low cost of the technology is anticipated to lead to a miniaturized, bench-top, molecular interaction photometer that can be used by the unskilled operator
Here we will also demonstrate the capability enabled by FreeSRF and CBSI to rapidly develop quantitative assays with detection limits in the 100pM (10’s pg/mL) range for small molecule targets and CV’s < 15% which are typically difficult to quantify using existing techniques. Capitalizing on unique transduction mechanism in FreeSRF and aptamer probes, we will demonstrate quantitative assays for oxycodone, hydrocodone, & fentanyl and their derivatives using CBSI that are 1000-fold more sensitive than the existing MS-MS assays. It will also be shown that these assays are highly specific for the target species and function in both serum and urine. Finally, it will be illustrated that our aptamer-CBSI assay methodology has the potential to function in the near-patient setting, allowing heath care providers the opportunity to accurately determine if a neonate has been exposed to these important opiates.
Michael Kammer– Student, Vanderbilt University, Nashville, TN
Michael is a PhD candidate in the department of Biomedical Engineering at Vanderbilt University in Nashville, TN. His research focus is on development of interferometric methods for measuring free-solution molecular interactions.