We recently reported a real-time method to measure heparin in blood based on photoacoustic (PA) signal from methylene blue (MB). The PA enhancement was surprisingly accompanied by a decrease in absorbance. Here, we describe a mechanistic study of the MB-heparin binding in water and phosphate buffered saline. The addition of 0.79 mg/mL heparin decreased the nuclear magnetic resonance (NMR) magnitude of 0.90 mg/mL MB by 63% with a 0.25 ppm downshift—this indicated formation of MB aggregates due to π-π staking of MB. We also observed nanoscale MB/heparin aggregates under transmission electron microscopy (TEM). Spectroscopic analysis of the isolated aggregates found that the percentage of MB inside the MB-heparin aggregate increased from 3.6% to 82.5% when heparin concentration was increased from 0.16 mg/mL to 0.79 mg/mL. Meanwhile, the photoacoustic intensity increased 25-fold. The signal increase was largely due to the aggregates rather than free MB in the solution. These trends suggested that the MB-heparin aggregation was responsible for the PA enhancement likely due to the decreased degrees of freedom for MB. Molecular dynamics simulations revealed MB dimer formation on heparin and indicated that electrostatic binding occurred between the central thiazine ring of MB and the sulfates and glucosamines in heparin via electrostatic interaction. These findings elucidate the binding process of MB and heparin and provide strategies for immobilizing MB-like molecules on implantable devices for intravascular heparin sensing.
The interaction of methylene blue (MB) and sodium dodecyl sulfate (SDS) leads to a reversible spectral shift during SDS micellization, but the underlying mechanism has remained unclear. Here, we measured photoacoustic (PA) intensity, micelle concentration, and spectral shift of MB-SDS complex to elucidate this interaction mechanism. We observed a switchable PA effect of MB, which is sensitive to critical micelle concentration (CMC) of SDS (i.e. 8 mM). The addition of 3.47 mM SDS increased the PA intensity of 0.05 mM MB 492-fold because of fluorescence quenching. Then, the PA intensity decreased by 54-fold when the SDS concentration was increased above the CMC at 8.67 mM due to decrease of MB aggregation. Meanwhile, we observed increased number of non-micellar MB-SDS clusters, ranging from 80 to 400 nm, as the SDS concentration approaching to CMC and then the number decreased once the SDS concentration was above CMC. The correlation between PA intensity and nanoparticle number indicated that the formation of MB-SDS cluster was responsible for the PA enhancement. Further controlled studies using MB/hexadecyltrimethylammonium bromide, MB/sodium octyl sulfate, and MB/sodium chloride showed that the binding between MB and SDS occurred at the sulfate moiety of SDS. They also found that MB-SDS clusters disassociated to micelles MB-SDS monomers at the SDS micellar concentrations. These findings further elucidate the binding mechanism of MB and SDS and presented the potential for developing an activatable MB PA contrast agent.
We recently described a technique to monitor heparin anticoagulation therapy in real-time using methylene blue and photoacoustic imaging. The photoacoustic signal of methylene blue was significantly amplified in the presence of heparin, but the exact mechanism underlying this novel photoacoustic behavior remains unclear. Here, we showed that the signal amplification was due to the aggregation of methylene blue. Methylene blue formed different aggregates in water and phosphate buffered saline (PBS). In water, the absorbance maximum of methylene blue with heparin from 0 to 3 U/mL blue shifted from 660 to 570 nm and the corresponding fluorescence intensity decreased 6-fold, which indicated the methylene blue aggregated from monomer to dimer and eventually to high order aggregates. Furthermore, the corresponding 0.04 ppm chemical shift of the proton in the phenothiazine ring of methylene blue from the nuclear magnetic resonance spectrum suggested the electron delocalization and self-aggregation of methylene blue. The coupling of methylene blue molecules results in extra vibrational relaxations within the split exciton states, and this causes enhanced photoacoustic signal. In PBS, we observed the aggregation of methylene blue/heparin complex using transmission electron microscopy (size=150.5 nm), but the absorbance maximum reversed back to 660 nm. This suggested the methylene blue formed monomer bound to heparin—the heparin could not self-aggregate due to electrostatic repulsive forces. The methylene blue bound monomers experienced less degree of freedom than free monomers and therefore caused excess photoacoustic signal.
We report a novel yet simple 3D-printed tubing holder for characterizing photoacoustic contrast agents. This device supports up to 12 plastic tubing with sample-to-sample spacing as low as 0.3 mm and provides a consistent distance (± 0.12 mm) between the tubing and the transducer, which is critical for validating photoacoustic contrast agents. An immersion media containing both 40% India ink and lipid that mimics tissue scattered the incident irradiation. We further studied different types of tubing and distance between tubing and transducer. Statistical analysis shows that tubing with a larger outside diameter has more inherent signal, and the signal decayed following a linear relationship (R2=0.997) with respect to distance from the laser focal point. We finally provide a computer-assisted drafting code for the community to customize and print their own phantoms.
Chorioretinal imaging has a crucial role for the patients with chorioretinal vascular diseases, such as neovascular age-related macular degeneration. Imaging oxygen gradients in the eye could better diagnose and treat ocular diseases. Here, we describe the use of photoacoustic ocular imaging (PAOI) in measuring chorioretinal oxygen saturation (CR − sO2) gradients in New Zealand white rabbits (n = 5) with ocular ischemia. We observed good correlation (R2 = 0.98) between pulse oximetry and PAOI as a function of different oxygen percentages in inhaled air. We then used an established ocular ischemia model in which intraocular pressure is elevated to constrict ocular blood flow, and notice a positive correlation (R2 = 0.92) between the injected volume of phosphate buffered saline (PBS) and intraocular pressure (IOP) as well as a negative correlation (R2 = 0.98) between CR − sO2 and injected volume of PBS. The CR − sO2 was measured before (baseline), during (ischemia), and after the infusion (600-μL PBS). The ischemia-reperfusion model did not affect the measurement of the sO2 using a pulse oximeter on the animal’s paw, but the chorioretinal PAOI signal showed a nearly sixfold decrease in CR − sO2 (n = 5, p = 0.00001). We also observe a sixfold decrease in CR − sO2 after significant elevation of IOP during ischemia, with an increase close to baseline during reperfusion. These data suggest that PAOI can detect changes in chorioretinal oxygenation and may be useful for application to imaging oxygen gradients in ocular disease.
Heparin is used broadly in cardiac, pulmonary, surgical, and vascular medicine to treat thrombotic disorders with over 500 million doses per year globally. Despite this widespread use, it has a narrow therapeutic window and is one of the top three medication errors. The active partial thromboplastin time (PTT) monitors heparin, but this blood test suffers from long turnaround times, a variable reference range, and limited utility with low molecular weight heparin. Here, we describe an imaging technique that can monitor heparin concentration and activity in real time using photoacoustic spectroscopy via methylene blue as a simple and Federal Drug Agency-approved contrast agent. We found a strong correlation between heparin concentration and photoacoustic signal measured in phosphate buffered saline (PBS) and blood (R2>0.90). Clinically relevant concentrations were detected in blood with a heparin detection limit of 0.28 U/mL and a low molecular weight heparin (enoxaparin) detection limit of 72 μg/mL. We validated this imaging approach by correlation to the PTT (Pearson’s r = 0.86; p<0.05) as well as with protamine sulfate treatment. To the best of our knowledge, this is the first report to use imaging data to monitor anticoagulation.
Ultrasound is broadly used in the clinics yet is limited in early cancer detection because of its poor contrast between healthy and diseased tissues. Photoacoustic imaging can improve this limitation and has been extensively studied in pre-clinical models. Contrast agents can help improve the accuracy of diagnosis. We recently reported a novel copper sulfide (CuS) nanodisk with strong directionally-localized surface plasmon resonance in the near infrared region. This plasmonic resonance of nanodisks is tunable by changing the size and aspect ratio of CuS nanodisk. Here, we demonstrate this CuS nanodisk is a strong photoacoustic contrast agent. We prepared CuS nanodisks via a solvent-based synthesis followed by surface modification of poly(ethylene glycol) methyl ether thiol for in vivo applications. These CuS nanodisks can be detected at a concentration as low as 26 pM at 920 nm. Their nanosize and strong photoacoustic response make this novel CuS nanodisk a strong candidate for photoacoustic cancer imaging.
In this paper, we demonstrate a wiregrid polarizer for the near-IR spectrum fabricated by nanoimprint techniques. High
resolution grating structures with 215nm in linewidth, 375nm pitch and 235nm total height were patterned on silicon by
deep-UV interference lithography followed by reactive ion etching. The grating structures were transferred to a SU-8
thin film by nanoimprint. Then a glancing angle deposition was performed to build the wiregrids. The extinction ratio
was measured to be over 90:1 at 1064nm.
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