Introduction:: Electrochemical biosensors are favorable for point-of-care (POC) diagnostics in low-resource settings (LRS) due to their sensitivity and portability. Gold is a common substrate for electrochemical biosensors due to strong gold-thiol coupling. However, traditional gold electrode fabrication techniques are too costly and laborious for use in LRS. In order to make gold electrodes more broadly accessible, we made affordable gold leaf electrodes using an equipment-free manufacturing technique. Each electrode cost $0.50 to make, which is nearly an order of magnitude cheaper than their commercially available counterparts. To demonstrate their functionality, electrodes were paired with a CRISPR-based assay to detect human papillomavirus (HPV) from clinical samples. This is a platform technology that can be designed to detect any pathogen in resource-limited settings.
Materials and Methods:: Electrode fabrication, adapted from (Zamani et al., 2021): An oil-based gold leaf adhesive (DUX, DUX-QUIC) was applied to the glossy side of a self-adhesive sheet (Fellowes, FEL5221502) and left to dry for 2.5 h. 24K Gold leaf (L.A. Gold Leaf, DBL2400-BK25T) was transferred to the dried adhesive by manually applying pressure. The working (4 mm diameter) and counter electrodes were cut using a flatbed cutter plotter (Graphtec, FCX2000) to make peelable gold leaf stickers. Gold leaf sticker electrodes were applied to the transparency sheet. A reference electrode was painted on using silver conductive paint (SPI, 05001-AB). Silver paint was painted on the leads of the working and counter electrodes and used to glue folded aluminum foil (Fisherbrand, 01-213-101) contacts to the ends of the electrodes.
Electrode pretreatment Electrodes underwent cyclic voltammetry in 0.5 M sulfuric acid for 10 cycles from -0.1 V to 1.5 V at 100 mV/sec.
Electrode Functionalization: After pretreatment, electrodes were functionalized with methylene -blue tagged single stranded DNA oligonucleotides via standard gold-thiol coupling.
LAMP assay Loop-mediated isothermal amplification of HPV-18 DNA was performed. Six primers were used with a standard LAMP reaction mix (NEB) to generate HPV-18 amplicons.
CRISPR-Cas12a assay A CRISPR-Cas12a assay was designed to detect HPV 18 DNA LAMP amplicons. A standard Cas12a reaction was used (NEB) to activate the Cas12a.
Electrochemical measurements Square wave voltammetry was used to measure the methylene blue signal from the DNA-modified electrodes. The electrodes were measured before and after treatment with Cas12a.
Results, Conclusions, and Discussions:: We fabricated novel gold leaf electrodes and demonstrated that they could be used for biosensing for the first time. Not only were these electrodes nearly an order of magnitude cheaper than their commercially available counterparts, they outperformed commercially available screen-printed gold electrodes, due to the lack of interfering dopants used in the leaf electrode fabrication. To demonstrate their viability as a diagnostic tool, we paired the electrodes with a CRISPR -based assay to detect HPV DNA from clinical samples.
We used CRISPR-Cas12a, an enzyme that exhibits endonuclease activity upon binding to a specific sequence of DNA, to detect HPV-18 DNA. In the presence of activated Cas12a enzyme, the methylene-blue tagged, single stranded oligos on the electrode surface are cleaved, resulting in a decrease in signal from the methylene blue. In the presence of inactivated Cas12a enzyme, the oligos on the electrode surface are not cleaved, resulting in no signal change from the methylene blue.
We were able to use our electrochemical, CRISPR-based assay to detect HPV-18 LAMP amplicons. Due to the specificity of the CRISPR portion of the assay, our assay did not detect commonly observed, non-specific LAMP amplification and exhibited 100% assay specificity. Our assay was able to detect clinically relevant viral loads of HPV-18. As a proof of concept, we demonstrated detection of HPV-18 DNA extracted from clinical samples with 100% specificity and 89% sensitivity.
Given the versatility of CRISPR assays as well as the vast possibilities of gold-thiol coupling, the leaf electrodes are a platform technology that can be interfaced with different assays to detect any pathogen in LRS, while also achieving multiplexed diagnostic capabilities in the future.
