Picomolar-Level Sensing of Cannabidiol by Metal Nanoparticles Functionalized with Chemically Induced Dimerization Binders

Abstract

Simple and fast detection of small molecules is critical for health and environmental monitoring. Methods for chemical detection often use mass spectrometers or enzymes; the former relies on expensive equipment, and the latter is limited to those that can act as enzyme substrates. Affinity reagents like antibodies can target a variety of small-molecule analytes, but the detection requires the successful design of chemically conjugated targets or analogs for competitive binding assays. Here, we developed a generalizable method for the highly sensitive and specific in-solution detection of small molecules, using cannabidiol (CBD) as an example. Our sensing platform uses gold nanoparticles (AuNPs) functionalized with a pair of chemically induced dimerization (CID) nanobody binders (nanobinders), where CID triggers AuNP aggregation and sedimentation in the presence of CBD. Despite moderate binding affinities of the two nanobinders to CBD (equilibrium dissociation constants K_D of ∼6 and ∼56 μM), a scheme consisting of CBD-AuNP preanalytical incubation, centrifugation, and electronic detection (ICED) was devised to demonstrate a high sensitivity (limit of detection of ∼100 picomolar) in urine and saliva, a relatively short sensing time (∼2 h), a large dynamic range (5 logs), and a sufficiently high specificity to differentiate CBD from its analog, tetrahydrocannabinol. The high sensing performance was achieved with the multivalency of AuNP sensing, the ICED scheme that increases analyte concentrations in a small assay volume, and a portable electronic detector. This sensing system is readily applicable for wide molecular diagnostic applications.

Keywords: cannabidiol; chemically induced dimerization; metal nanoparticles; rapid electronic detection; small-molecule sensing.

Figure 1.. Overview of small molecule detection.

(a) Schematic showing key steps in enhanced CBD detection, including functionalization of AuNPs with nano-binders (anchor binder: blue; dimerization binder, purple), pre-incubation of CBD sample with anchor-binder on AuNPs, and mixing the CBD-anchored AuNPs with dimerization-binder-functionalized AuNPs for reaction. (b) Schematics showing CBD molecule signal readout. Here AuNP clusters form at the presence of CBD in the target sample. The LED serves as light source to probe the floating AuNPs and the photodetector converts the transmitted light into electronic signals.

Figure 2.. ICED detection of CBD molecule in PBS buffer.

(a-b) Optical images of microcentrifuge tubes of CBD detection: (a) CBD mixed with CA14- and DB21-functionalized AuNPs, and then 4-hour incubation was applied (protocol in Figure S1). (b) CBD mixed with functionalized AuNPs, then centrifuged, incubated for 20 min and vortexed (protocol in Figure S3). (c) Calculated molecule and AuNP distributions across liquid height after centrifugation. (d) Schematic showing process flow of ICED detection of CBD molecule. Here CA14-functionalized AuNPs was preincubated with to-be-tested CBD samples prior to mixing with DB21-functionalized AuNPs for sensing (protocol in Figure S5). (e-f) Optical images of CBD detection using ICED method: (e) microcentrifuge tubes; (f) PDMS plate image of extracted top liquid from Figure e. (g) Extinction spectra of AuNPs from PDMS plate in Figure f. (h) Extinction peak values (at 559 nm) for ICED method (extracted from plot Figure g, Black triangle) and after 4-hour incubation (red squares).

Figure 3.. Modular analytic model for ICED assay.

(a-b) Schematics showing the enhancement in effective binding affinity with AuNPs: (a) a typical sandwich assay with an effective binding affinity of 56 nM using a pair of anchor binder (CBD binding affinity of 6 μM) and dimerization binder (CBD binding affinity of 56 μM); (b) the use of multivalent AuNPs effectively improves the binding affinity by a factor of C_f. (c) Model framework showing modular simulation strategy comprising of individual physical models used to simulate reaction mechanism for multivalent AuNPs aggregation and sedimentation based bio-sensing system.

Figure 4.. Experimental and simulation analyses of nanoparticle size effect on CBD detection.

(a) Optical images of CBD detection in PBS for AuNP sizes of 40 nm, 60 nm, 80 nm and 100 nm. The pictures were taken after all reactions. Initial and intermediate-stage images were provided in supplementary figures. (b) Extracted optical extinction peak values for CBD detection in PBS for different nanoparticle sizes. (c) Simulated extinction peak values for CBD detection for dimerization system CA14-CBD-DB21.

Figure 5.. Impact of nano-binder and reagent filtration on CBD detection.

(a) Extracted optical extinction peak for CBD detection in PBS for dimerization system CA14-CBD-DB21 (Black triangle with solid line) and CA14-CBD-DB21 (red square with dashed line). (b) Extracted optical extinction peak values for CBD detection in PBS for standard ICED approach (sensors filtered, black triangle with solid line) and simplified ICED approach (not filtrated, red square with dashed line) assay.

Figure 6. Rapid and portable electronic detection of CBD molecule in urine and saliva.

(a) Schematic and optical image of PED system, consisting mainly of a LED circuit, a photodiode circuit, and a 3D printed tube holder. A coin with its size marked is pictured as a reference. (b) Optical image of detecting CBD spiked in urine. (c) Optical image of detecting CBD spiked in saliva. (d-e) CBD and THC sensing in: (d) urine and (e) saliva. The CBD-containing, urine and saliva solutions are 20% prior to mixing with AuNP sensors, but 5% in the final mixture. Optical extinction peak values (559 nm) in CBD sensing (black squares and fitted dash-dot line) and THC sensing (red open-circle and dash line) were extracted from their optical spectra (Figure S21 e and S22 e for CBD, and Figure S23 e and S24 e for THC). The electronic voltage signals of CBD (blue triangles and solid line) are directly measured from the tube samples shown in Figure 6b and 6c.