The transmission of SARS-CoV-2 coronavirus has led to the COVID-19 pandemic. Nucleic acid testing while specific has limitations for mass surveillance. One alternative is the main protease (M pro ) due to its functional importance in mediating the viral life cycle. Here, we describe a combination of modular substrate and gold colloids to detect M pro via visual readout. The strategy involves zwitterionic peptide that carries opposite charges at the C-/N-terminus to exploit the specific recognition by M pro . Autolytic cleavage releases a positively charged moiety that assembles the nanoparticles with rapid color changes (t < 10 min). We determine a limit of detection for M pro in breath condensate matrices < 10 nM. We further assayed ten COVID-negative subjects and found no false-positive result. In the light of simplicity, our test for viral protease is not limited to an equipped laboratory, but also is amenable to integrating as portable point-of-care devices including those on face-coverings.

Photoacoustic imaging is a rapidly maturing imaging modality in biological research and medicine. This modality uses the photoacoustic effect (“light in, sound out”) to combine the contrast and specificity of optical imaging with the high temporal resolution of ultrasound. The primary goal of image-guided therapy, and theranostics in general, is to transition from conventional medicine to precision strategies that combine diagnosis with therapy. Photoacoustic imaging is well-suited for noninvasive guidance of many therapies and applications currently being pursued in three broad areas. These include the image-guided resection of diseased tissue, monitoring of disease states, and drug delivery. In this review, we examine the progress and strategies for development of photoacoustics in these three key areas with an emphasis on the value photoacoustics has for image-guided therapy.

The gold-standard periodontal probe is an aging tool that can detect periodontitis and monitor gingival health but is highly error-prone, does not fully characterize the periodontal pocket, and causes pain. Photoacoustic imaging is a noninvasive technique that can address these limitations. Here, a range of ultrasound frequencies between 16–40 MHz were used to image the periodontium and a contrast medium based on cuttlefish ink was used to label the pockets. A 40 MHz ultrasound frequency could spatially resolve the periodontal anatomy, including tooth, gum, gingival margin, and gingival thickness of tooth numbers 7–10 and 22–27. The photoacoustic-ultrasound measurements were more precise (0.01 mm) than those taken with physical probes by a dental hygienist. Furthermore, the full geometry of the pockets could be visualized with relative standard deviations of 10% (n = 5). This study shows the potential for non-invasive monitoring of periodontal health with photoacoustic-ultrasound imaging in the dental clinic.

There is a need for surveillance of COVID-19 to identify individuals infected with SARS-CoV-2 coronavirus. Although specific, nucleic acid testing has limitations in terms of point-of-care testing. One potential alternative is the nonstructural protease (nsp5, also known as M pro /3CL pro ) implicated in SARS-CoV-2 viral replication but not incorporated into virions. Here, we report a divalent substrate with a novel design, (Cys) 2 –(AA) x –(Asp) 3 , to interface gold colloids in the specific presence of M pro leading to a rapid and colorimetric readout. Citrate- and tris(2-carboxyethyl)phosphine (TCEP)-AuNPs were identified as the best reporter out of the 17 ligated nanoparticles. Furthermore, we empirically determined the effects of varying cysteine valence and biological media on the sensor specificity and sensitivity. The divalent peptide was specific to M pro , that is, there was no response when tested with other proteins or enzymes. Furthermore, the M pro detection limits in Tris buffer and exhaled breath matrices are 12.2 and 18.9 nM, respectively, which are comparable to other reported methods (i.e., at low nanomolar concentrations) yet with a rapid and visual readout. These results from our work would provide informative rationales to design a practical and noninvasive alternative for COVID-19 diagnostic testing—the presence of viral proteases in biofluids is validated.

Photoacoustic (PA) imaging holds great promise as a noninvasive imaging modality. Gold nanorods (GNRs) with absorption in the second near-infrared (NIR-II) window have emerged as excellent PA probes because of their tunable optical absorption, surface modifiability, and low toxicity. However, pristine GNRs often undergo shape transition upon laser illumination due to thermodynamic instability, leading to a reduced PA signal after a few seconds of imaging. Here, we report monodisperse GNR−melanin nanohybrids where a tunable polydopamine (PDA) coating was conformally coated on GNRs. GNR@PDAs showed a threefold higher PA signal than pristine GNRs due to the increased optical absorption, cross-sectional area, and thermal confinement. More importantly, the PA signal of GNR@PDAs only decreased by 29% during the 5 min of laser illumination in the NIR-II window, while significant attenuation (77%) was observed for GNRs. The GNR@PDAs maintained 87% of its original PA signal in vivo even after 10 min of laser illumination. This PDA-enabled strategy affords a rational design for robust PA imaging probes and provides more opportunities for other types of photomediated biomedicines, such as photothermal and photodynamic regimens.

The etiology of peri-implant crestal bone loss is today better understood and certain factors proposed in the past have turned out to not be of concern. Regardless, the incidence of crestal bone loss remains higher than necessary and this paper reviews current theory on the etiology with a special emphasis on traditional and innovative methods to assess the level of crestal bone around dental implants that will enable greater sensitivity and specificity and significantly reduce variability in bone loss measurement.

The interaction between methylene blue (MB) and sodium dodecyl sulfate (SDS) has been widely studied spectroscopically, but details about their interactions remain unclear. Here, we combined photoacoustic (PA) imaging with nanoparticle tracking analysis (NTA) and spectroscopy to further elucidate this interaction. PA imaging of 0.05 mM MB showed a 492-fold increase in intensity upon the addition of 3.47 mM SDS. Higher concentrations above SDS’s critical micelle concentration (CMC) at 8.67 mM decreased the PA intensity by 54 times. Relative quantum yield measurements indicated that PA intensity increased as a result of fluorescence quenching. Meanwhile, NTA indicated an increased number of nonmicellar MB/SDS clusters at SDS concentrations below the CMC varying in size from 80 to 400 nm as well as a decreased number above the CMC. This trend suggested that MB/SDS clusters are responsible for the PA intensity enhancement. Comparison of PA intensities and spectral shifts with MB/hexadecyltrimethylammonium bromide, MB/sodium octyl sulfate, and MB/sodium chloride demonstrated that MB was bound to the sulfate moiety of SDS before and after micellization. Our observations suggest that MB forms aggregates with SDS at premicellar concentrations, and the MB aggregates disassociate as monomers that are bound to the sulfate moiety of SDS at micellar concentrations. These findings further clarify the process by which MB and SDS interact and demonstrate the potential for developing MB-/SDS-based contrast agents.

Activatable contrast agents are of ongoing research interest because they offer low background and high specificity to the imaging target. Engineered sensitivity to protease activity is particularly desirable because proteases are critical biomarkers in cancer, infectious disease, inflammatory disorders, and so forth. Herein, we developed and characterized a set of peptide-linked cyanine conjugates for dual-modal detection of protease activity via photoacoustic (PA) and fluorescence imaging. The peptide−dye conjugates were designed to undergo contact quenching via intramolecular dimerization and contained n dyes (n = 2, 3, or 4) with n − 1 cleavable peptide substrates. The absorption peaks of the conjugates were blue-shifted 50 nm relative to the free dye and had quenched fluorescence. This effect was sensitive to solvent polarity and could be reversed by solvent switching from water to dimethyl sulfoxide. Employing trypsin as a model protease, we observed a 2.5-fold recovery of the peak absorbance, 330−4600-fold fluorescent enhancement, and picomolar detection limits following proteolysis. The dimer probe was further characterized for PA activation. Proteolysis released single dye−peptide fragments that produced a 5-fold PA enhancement through the increased absorption at 680 nm with nanomolar sensitivity to trypsin. The peptide substrate could also be tuned for protease selectivity; as a proof-of-concept, we detected the main protease (M pro ) associated with the viral replication in SARS-CoV-2 infection. Last, the activated probe was imaged subcutaneously in mice and signal was linearly correlated to the cleaved probe. Overall, these results demonstrate a tunable scaffold for the PA molecular imaging of protease activity with potential value in areas such as disease monitoring, tumor imaging, intraoperative imaging, in vitro diagnostics, and point-of-care sensing.