DNA Aptamer Drug Sensors Instantly Detect Cocaine, Heroin and Fentanyl – Even When Combined With Other Drugs

Newswise — Researchers from North Carolina State University have developed a new generation of high-performance DNA aptamers and highly accurate drug sensors for cocaine and other opioids. The sensors are drug specific and can detect trace amounts of fentanyl, heroin, and cocaine – even when these drugs are mixed with other drugs or with cutting agents and adulterants such as caffeine, sugar, or procaine. The sensors could have far-reaching benefits for health care workers and law enforcement agencies.

“This work can provide needed updates to currently used tests, both in health care and law enforcement settings,” says Yi Xiao, associate professor of chemistry at NC State and corresponding author of two studies describing the work.

“For example, drug field testing currently used by law enforcement still relies on chemical tests developed a century ago that are poorly specific, which means they react to compounds that may not be the drug they’re looking for,” Xiao says.

“And the existing aptamer test for cocaine isn’t sensitive and specific enough to detect clinically relevant amounts of the drug in biological samples, like blood. The sensors we developed can detect cocaine in blood at nanomolar, rather than micromolar, levels, which represents a 1,000-fold improvement in sensitivity.”

In a pair of studies appearing in the Journal of the American Chemical Society (JACS) and JACS Au, Xiao led a team that developed aptamer-based sensors for cocaine, heroin, codeine, fentanyl and other illicit drugs.

An aptamer is a short single strand of DNA or RNA that will bind to a specific molecule with high affinity, meaning that it won’t bind to molecules other than the one of interest. The researchers begin the search by adding the molecule of interest – cocaine, for example – to a mixture of hundreds of trillions of randomized DNA sequences. Then they see which aptamer binds to the molecule.

“We refer to the process as ‘bio-panning,’ since it is a lot like sifting through river sediment to find gold,” says Obtin Alkhamis, NC State graduate student and co-author of both papers. “Once we separate the target-bound strands from non-bound strands, we rigorously test that aptamer against other interfering compounds to ensure that it is specific only to the compound of interest.”

The researchers then tested the compound-specific aptamers against pharmaceutical mixtures, tablets and blood, to determine whether they could identify certain drugs in the samples.

Xiao’s team used the cocaine aptamer to detect cocaine in blood serum at levels of 10 nanomolar (equivalent to 30 nanograms per milliliter or 30 parts-per-billion), a 1,000-fold improvement over the best prior aptamer test which can only detect 10 micromolar cocaine in 50% serum.

Additionally, collaborators at the University of California Santa Barbara were able to incorporate the aptamer into an electrode that could measure drug concentration in situ in the blood (in a vein) of rats in real time every 10 seconds over a two-hour time period. This is the first study able to measure the pharmacology of a drug of abuse with time resolution measured in seconds.

The opioid-specific aptamers were incorporated into colorimetric assays that can specifically detect opioids like heroin and oxycodone in solution at levels as low as 0.5 micromolar (μM). A colorimetric assay is a test that changes color when the compound of interest is detected. These assays were also able to detect opioids in complex chemical matrices within seconds – including pharmaceutical tablets and drug mixtures.

For comparison, the “Marquis test,” a standard test used by law enforcement officials and forensic laboratories, could not detect opioids in chemical mixtures.

The researchers believe that these aptamer sensors have useful applications for health and public safety.

“The aptamers can be mass produced, have a long shelf life and are easily chemically modified, which means they can be utilized for any type of sensor you develop,” Xiao says. “So they could be incorporated into testing strips for officers in the field, for use at home or for human patients in a physician’s office.”

“Since they detect drugs at clinically relevant levels, you could potentially do a blood drop test in the ER to immediately determine what a patient may have taken, without a full blood draw and lab testing,” Alkhamis says. “The possible uses are really exciting.”

The work was supported by the National Institute of Justice (awards 2019-DU-BX-0024 and 2022-GG-04440-RESS), the National Science Foundation (grant CHE-2135005), and the National Institutes of Health (grant R01DA051100). Nicole Emmons, Yuting Wang, Kevin Honeywell, Kevin Plaxco and Tod Kippin, all from the University of California Santa Barbara, contributed to the development of the electrochemical aptamer-based sensor for in vivo cocaine testing. NC State graduate students Juan Canoura, Yuyang Wu, Matthew Venzke and Phuong Ly also contributed to the opioid work.

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Note to editors: Abstracts follow.

“High-Affinity Aptamers for In Vitro and In Vivo Cocaine Sensing”

DOI: 10.1021/jacs.3c11350

Authors: Obtin Alkhamis, Juan Canoura, Yuyang Wu, Yi Xiao, North Carolina State University; Nicole A. Emmons, Yuting Wang, Kevin M. Honeywell, Kevin W. Plaxco, Tod E. Kippin, University of California Santa Barbara
Published: Jan. 30, 2024 in the Journal of the American Chemical Society

Abstract:
The ability to quantify cocaine in biological fluids is crucial for both the diagnosis of intoxication and overdose in the clinic as well as investigation of the drug’s pharmacological and toxicological effects in the laboratory. To this end, we have performed high-stringency in vitro selection to generate DNA aptamers that bind cocaine with nanomolar affinity and clinically relevant specificity, thus representing a dramatic improvement over the current-generation, micromolar-affinity, low-specificity cocaine aptamers. Using these novel aptamers, we then developed two sensors for cocaine detection. The first, an in vitro fluorescent sensor, successfully detects cocaine at clinically relevant levels in 50% human serum without responding significantly to other drugs of abuse, endogenous substances, or a diverse range of therapeutic agents. The second, an electrochemical aptamer-based sensor, supports the real-time, seconds-resolved measurement of cocaine concentrations in vivo in the circulation of live animals. We believe the aptamers and sensors developed here could prove valuable for both point-of-care and on-site clinical cocaine detection as well as fundamental studies of cocaine neuropharmacology.

“Developing Aptamer-Based Colorimetric Opioid Tests”

DOI: 10.1021/jacsau.3c00801

Authors: Juan Canoura, Obtin Alkhamis, Matthew Venzke, Phuong T. Ly and Yi Xiao, North Carolina State University
Published: March 1, 2024 in the JACS Au

Abstract:
Opioids collectively cause over 80,000 deaths in the United States annually. The ability to rapidly identify these compounds in seized drug samples on-site will be essential for curtailing trafficking and distribution. Chemical reagent-based tests are fast and simple but also notorious for giving false results due to poor specificity, whereas portable Raman spectrometers have excellent selectivity but often face interference challenges with impure drug samples. In this work, we develop on-site sensors for morphine and structurally related opioid compounds based on in vitro-selected oligonucleotide affinity reagents known as aptamers. We employ a parallel-and-serial selection strategy to isolate aptamers that recognize heroin, morphine, codeine, hydrocodone, and hydromorphone, along with a toggle-selection approach to isolate aptamers that bind oxycodone and oxymorphone. We then utilize a new high-throughput sequencing-based approach to examine aptamer growth patterns over the course of selection, and a high-throughput exonuclease-based screening assay to identify optimal aptamer candidates. Finally, we use two high-performance aptamers with KD of ~1 µM to develop colorimetric dye-displacement assays that can specifically detect opioids like heroin and oxycodone at concentrations as low as 0.5 µM with a linear range of 0–16 µM. Importantly, our assays can detect opioids in complex chemical matrices, including pharmaceutical tablets and drug mixtures; in contrast, the conventional Marquis test completely failed in this context. These aptamer-based colorimetric assays enable naked-eye identification of specific opioids within seconds and will play an important role in combatting opioid abuse.