Antibody-based sensors for multiplex point-of-care drug monitoring

Existing methods for drug detection

Urine is considered the gold standard sample source for non-invasive drug testing, but saliva, sweat, and hair are becoming promising sources for testing. Because drugs of abuse have short half lives in the body, methods used for detection need to be sensitive. Commonly used methods for detection include gas chromatography, mass spectrometry, Raman spectroscopy, and nuclear magnetic resonance. However, these instruments are not readily accessible or portable, are costly, and require extensive training to operate.

Therefore, a point-of-care test for drug monitoring needs to be miniaturized, affordable, flexible, easy to use, and highly sensitive. Because the use of a stimulant and a depressant together is becoming one of the most common fatal drug abuse methods, it’s important for a sensor to detect more than one drug simultaneously.

In recent years, scientists have developed a multiplex immunosensor to detect morphine, tetrahydrocannabinol, and benzoylecgonine from urine in 40 minutes2 and electrochemical methods for detecting amphetamine type stimulants3 but no point-of-care assay currently exists to detect all families of commonly abused drugs simultaneously. Lateral flow assays are easy to use and portable but can have low sensitivity and provide only qualitative or semi-quantitative data.

To address these gaps in drug monitoring assays, a global team of scientists created a multiplex point-of-care sensor for simultaneously detecting amphetamine (AMP), cocaine (COC), and benzodiazepine (BZD) from saliva¹. (Figure 1)

Figure 1. Saliva samples added to an antibody-based multiplex drug detection platform provides readouts on a smartphone¹.

Multiplex laser-scribed graphene (LSG) sensing platform

The multiplex sensor designed by these researchers is a laser-scribed graphene platform that contains five electrodes: three working electrodes to detect each of the three drugs, one counter electrode, and one reference electrode. The working electrode surfaces can be modified with antibodies that bind each of the three drugs. For this study, this includes using a benzodiazepine monoclonal antibody and an amphetamine monoclonal antibody from Arista Biologicals, Inc., a Fortis Life Sciences® company, as well as a cocaine monoclonal antibody. The sensor is integrated with a potentiostat and can be operated via a smartphone app.

When an analyte (benzodiazepine, amphetamine, or cocaine) binds to its corresponding antibody, this reduces the oxidation current, which can be measured using differential pulse voltammetry and results are displayed in the smartphone app.

Simultaneous detection of amphetamine, cocaine, and benzodiazepine

With the sensor designed, the researchers ran a series of experiments to understand the analyte concentration it can detect in complex media. When they tested each drug individually, they found the limit of detection of their sensor to be 9.7 ng/ml AMP, 9.0 ng/ml BZD, and 4.3 ng/ml COC. They next tested the sensors with increasing drug concentrations in complex media and found that the oxidation current is further reduced with increasing drug concentrations. Having obtained these foundational results, the scientists moved to detection of each drug in saliva samples spiked with all three drugs together (Figure 2). Similar to the tests in complex media, the sensors displayed a concentration dependent oxidation current difference.

Figure 2. Multiplexed detection of AMP, BZD, and COC on one platform shows the effect of varying drug concentration (BZD) on oxidation current¹.

Selectivity of the multiplex assay

Once the team proved they could detect AMP, BZD, and COC simultaneously in saliva, they then asked whether other drugs that may be present in saliva, such as methamphetamine (MET) or analgesics, would interfere with the assay.

They compared detection of AMP from control saliva or MET patient saliva spiked with AMP and found that the MET patient saliva showed a greater oxidation current difference than control saliva. The authors of the study think that similarities between the molecular structures of AMP and MET could account for this. However, the assay could still detect AMP, even in the presence of MET.

Because illicit drug use in combination with analgesics is increasing, the researchers next tested whether analgesics interfere with AMP, BZD, or COC detection. When testing samples spiked individually with AMP, BZD, and COC in the presence and absence of analgesic, they found that they could still detect AMP, BZD, and COC despite a 10-20% increase in current with the presence of the analgesic. They also tested for any potential interference from delta-9-tetrahydrocannabinol (THC) and codeine, finding that the target drugs have a 3-4.5-fold change in current response compared to the current detected from either THC or codeine.

Based on these results, the researchers concluded that their sensor is selective for AMP, BZD, and COC. A multiplex point-of-care sensor like this one has the potential to be a faster, portable alternative to existing drug monitoring.