Early disease biomarker detection often requires high sensitivity, achieved through molecular amplification or physical signal enhancement of fluorescence. Fluorescence signal enhancement using plasmonic nanoantennas offers improved sensitivity and enables single-molecule detection on cost-effective, mobile devices. Previous approaches using DNA origami to position quantum emitters in nanoantenna hotspots suffered from limited hotspot accessibility, resulting in moderate fluorescence enhancement insufficient for low-numerical-aperture (NA) optics like those in smartphones. This study aimed to develop a diagnostic single-molecule assay leveraging the signal amplification potential of DNA origami nanoantennas to enable detection with affordable, low-NA optics.

Existing literature highlights the use of plasmonic nanoantennas for fluorescence signal enhancement. Challenges include precise placement of emitters within nanoantenna hotspots and the accessibility of these hotspots for bioassays. DNA origami has been utilized as a construction material to overcome the challenges of positioning quantum emitters within the nanoantenna hotspots. However, prior work showed only moderate fluorescence enhancement, insufficient for single-molecule detection with low-NA optics. The limited accessibility of the hotspot in previous designs restricted the binding of single nanoparticles, reducing the achievable signal amplification.

This research introduced NanoAntennas with Cleared HotSpots (NACHOS), a novel three-dimensional DNA origami structure designed for high fluorescence signal amplification and full addressability. The design incorporates two pillars for attaching silver nanoparticles, creating a plasmonic hotspot between them, leaving the space free for bioassays. The DNA origami structure has a rigid cross-like base for immobilization on a surface. The two pillars provide anchor points for 100 nm silver nanoparticles. The signal amplification was evaluated by incorporating an Alexa Fluor 647-labeled DNA staple strand directly into the hotspot. Single-molecule fluorescence transients were recorded using confocal microscopy for both the DNA origami without nanoparticles and for NACHOS. A sandwich binding assay was designed for detecting a DNA fragment specific to the OXA-48 gene, indicative of antibiotic-resistant *Klebsiella pneumoniae*. Three capture strands were incorporated into the hotspot to hybridize with the target DNA, exposing a region for a dye-labeled imager strand. Confocal microscopy and single-molecule photobleaching analysis were used to quantify the fluorescence enhancement and binding efficiency. The assay was also performed in human blood serum. A portable, battery-powered smartphone microscope was constructed using a monochrome smartphone camera, a low-cost objective lens, and a 638 nm excitation laser. Single-molecule detection with this microscope was performed with and without the sandwich assay, and both in buffer and in serum.

NACHOS demonstrated significantly improved signal-to-background ratios (SBR) compared to the reference structure (361 ± 35 vs. 7.4 ± 0.9). Fluorescence enhancement values of up to 417-fold (average 74 ± 3-fold) were achieved with the new nanoantenna design. In the single-molecule DNA diagnostic assay, fluorescence enhancement values reached up to 461-fold (average 89 ± 7-fold), a significant improvement over previous designs. The sandwich assay showed high binding efficiencies (66% in NACHOS, 84% in the reference) and low false-positive rates (~2.5%). The assay performed similarly well in human blood serum, with fluorescence enhancement values reaching 457-fold (average 70 ± 4-fold). Single-molecule detection was successfully demonstrated using the portable smartphone microscope, showing slow single-molecule blinking and bleaching events, with signal-to-background (SBR) and signal-to-noise (SNR) ratios of 25 ± 2 and 3.8 ± 0.2, respectively. The smartphone microscope also successfully detected single DNA molecules using the sandwich assay in both buffer and serum, with bleaching steps consistent with single-molecule behavior. The robustness of NACHOS in complex biological fluids and their functionality with a low-cost smartphone microscope were clearly demonstrated.

This study successfully addressed the limitations of previous DNA origami nanoantennas by creating NACHOS with a cleared and addressable hotspot. The significantly increased fluorescence enhancement values, achieved through improved hotspot accessibility, enabled single-molecule detection using low-cost optics. The successful demonstration of the assay in human blood serum highlights the potential for real-world applications. The development of the portable smartphone microscope further expands the accessibility and affordability of single-molecule detection, making it suitable for point-of-care diagnostics. The results show that the use of NACHOS significantly improves the sensitivity and applicability of single-molecule assays, paving the way for widespread use in various applications.

This research successfully developed addressable DNA origami nanoantennas (NACHOS) with significantly improved fluorescence enhancement, enabling single-molecule detection with a low-cost smartphone microscope. The high sensitivity, robustness in complex biological fluids, and ease of use suggest considerable potential for point-of-care diagnostics and other applications where cost-effectiveness and portability are crucial. Future work could focus on optimizing the assay for higher throughput, exploring different target molecules, and further miniaturizing the smartphone microscope.

While NACHOS demonstrate significant improvements, limitations remain. The fluorescence enhancement distribution shows some heterogeneity, potentially due to variations in nanoparticle size, shape, and orientation. The assay's selectivity, while demonstrated, might be further improved by optimizing the capture strand sequence and length. The current smartphone microscope prototype is still relatively expensive compared to a standard cellphone, but the authors foresee significant cost reduction with increased production.