Label-free live cell imaging was performed using a custom-built high-speed confocal Raman microscopy system. For various cell types, cell-intrinsic Raman bands were monitored. The high-resolution temporal Raman images clearly delineated the intracellular distribution of biologically important molecules such as protein, lipid, and DNA. Furthermore, optical phase delay measured using quantitative phase microscopy shows similarity with the image reconstructed from the protein Raman peak. This reported work demonstrates that Raman imaging is a powerful label-free technique for studying various biomedical problems in vitro with minimal sample preparation and external perturbation to the cellular system.
Transcutaneous Measurement of Essential Vitamins Using Near-Infrared Fluorescent Single-Walled Carbon Nanotube Sensors
Vitamins such as riboflavin and ascorbic acid are frequently utilized in a range of biomedical applications as drug delivery targets, fluidic tracers, and pharmaceutical excipients. Sensing these biochemicals in the human body has the potential to significantly advance medical research and clinical applications. In this work, a nanosensor platform consisting of single-walled carbon nanotubes (SWCNTs) with nanoparticle corona phases engineered to allow for the selective molecular recognition of ascorbic acid and riboflavin, is developed. The study provides a methodological framework for the implementation of colloidal SWCNT nanosensors in an intraperitoneal SKH1-E murine model by addressing complications arising from tissue absorption and scattering, mechanical perturbations, as well as sensor diffusion and interactions with the biological environment. Nanosensors are encapsulated in a polyethylene glycol diacrylate hydrogel and a diffusion model is utilized to validate analyte transport and sensor responses to local concentrations at the boundary. Results are found to be reproducible and stable after exposure to 10% mouse serum even after three days of in vivo implantation. A geometrical encoding scheme is used to reference sensor pairs, correcting for in vivo optical and mechanical artifacts, resulting in an order of magnitude improvement of p-value from 0.084 to 0.003 during analyte sensing.
2020 Beckman Symposium: From biochemical nanosensors to imaging to informatics to COVID-19 convalescent plasma – developing diagnostics and therapies for clinical medicine
2017 Arnold O. Beckman Postdoctoral Fellow Freddy Nguyen, MD, PhD from MIT, presents his research at the 2020 Beckman Symposium.
Implantable Nanosensors for Human Steroid Hormone Sensing In Vivo Using a Self-Templating Corona Phase Molecular Recognition
Dynamic measurements of steroid hormones in vivo are critical, but steroid sensing is currently limited by the availability of specific molecular recognition elements due to the chemical similarity of these hormones. In this work, a new, self‐templating synthetic approach is applied using corona phase molecular recognition (CoPhMoRe) targeting the steroid family of molecules to produce near infrared fluorescent, implantable sensors. A key limitation of CoPhMoRe has been its reliance on library generation for sensor screening. This problem is addressed with a self‐templating strategy of polymer design, using the examples of progesterone and cortisol sensing based on a styrene and acrylic acid copolymer library augmented with an acrylated steroid. The pendant steroid attached to the corona backbone is shown to self‐template the phase, providing a unique CoPhMoRE design strategy with high efficacy. The resulting sensors exhibit excellent stability and reversibility upon repeated analyte cycling. It is shown that molecular recognition using such constructs is viable even in vivo after sensor implantation into a murine model by employing a poly (ethylene glycol) diacrylate (PEGDA) hydrogel and porous cellulose interface to limit nonspecific absorption. The results demonstrate that CoPhMoRe templating is sufficiently robust to enable a new class of continuous, in vivo biosensors.
A Fiber Optic Interface Coupled to Nanosensors: Applications to Protein Aggregation and Organic Molecule Quantification
Fluorescent nanosensors hold promise to address analytical challenges in the biopharmaceutical industry. The monitoring of therapeutic protein critical quality attributes such as aggregation is a longstanding challenge requiring low detection limits and multiplexing of different product parameters. However, general approaches for interfacing nanosensors to the biopharmaceutical process remain minimally explored to date. Herein, we design and fabricate a integrated fiber optic nanosensor element, measuring sensitivity, response time, and stability for applications to the rapid process monitoring. The fiber optic-nanosensor interface, or optode, consists of label-free nIR fluorescent single-walled carbon nanotube transducers embedded within a protective yet porous hydrogel attached to the end of the fiber waveguide. The optode platform is shown to be capable of differentiating the aggregation status of human immunoglobulin G, reporting the relative fraction of monomers and dimer aggregates with sizes 5.6 and 9.6 nm, respectively, in under 5 min of analysis time. We introduce a lab-on-fiber design with potential for at-line monitoring with integration of 3D-printed miniaturized sensor tips having high mechanical flexibility. A parallel measurement of fluctuations in laser excitation allows for intensity normalization and significantly lower noise level (3.7-times improved) when using lower quality lasers, improving the cost effectiveness of the platform. As an application, we demonstrate the capability of the fully-integrated lab-on-fiber system to rapid monitoring of various bioanalytes including serotonin, norepinephrine, adrenaline, and hydrogen peroxide, in addition to proteins and their aggregation states. These results in total constitute an effective form factor for nanosensor based transducers for applications in industrial process monitoring.
DNA-SWCNT Biosensors Allow Real-Time Monitoring of Therapeutic Responses in Pancreatic Ductal Adenocarcinoma
Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic cancer with limited treatment options. There is an urgent need for tools that monitor therapeutic responses in real time. Drugs such as gemcitabine and irinotecan elicit their therapeutic effect in cancer cells by producing hydrogen peroxide (HO). In this study, specific DNA-wrapped single-walled carbon nanotubes (SWCNT), which precisely monitor HO, were used to determine the therapeutic response of PDAC cells and tumors . Drug therapeutic efficacy was evaluated by monitoring HO differences using reversible alteration of Raman G-bands from the nanotubes. Implantation of the DNA-SWCNT probe inside the PDAC tumor resulted in approximately 50% reduction of Raman G-band intensity when treated with gemcitabine versus the pretreated tumor; the Raman G-band intensity reversed to its pretreatment level upon treatment withdrawal. In summary, using highly specific and sensitive DNA-SWCNT nanosensors, which can determine dynamic alteration of hydrogen peroxide in tumor, can evaluate the effectiveness of chemotherapeutics. SIGNIFICANCE: A novel biosensor is used to detect intratumoral hydrogen peroxide, allowing real-time monitoring of responses to chemotherapeutic drugs.
Implanted Nanosensors in Marine Organisms for Physiological Biologging: Design, Feasibility, and Species Variability
In recent decades, biologists have sought to tag animals with various sensors to study aspects of their behavior otherwise inaccessible from controlled laboratory experiments. Despite this, chemical information, both environmental and physiological, remains challenging to collect despite its tremendous potential to elucidate a wide range of animal behaviors. In this work, we explore the design, feasibility, and data collection constraints of implantable, near-infrared fluorescent nanosensors based on DNA-wrapped single-wall carbon nanotubes (SWNT) embedded within a biocompatible poly(ethylene glycol) diacrylate (PEGDA) hydrogel. These sensors are enabled by Corona Phase Molecular Recognition (CoPhMoRe) to provide selective chemical detection for marine organism biologging. Riboflavin, a key nutrient in oxidative phosphorylation, is utilized as a model analyte in in vitro and ex vivo tissue measurements. Nine species of bony fish, sharks, eels, and turtles were utilized on site at Oceanogràfic in Valencia, Spain to investigate sensor design parameters, including implantation depth, sensor imaging and detection limits, fluence, and stability, as well as acute and long-term biocompatibility. Hydrogels were implanted subcutaneously and imaged using a customized, field-portable Raspberry Pi camera system. Hydrogels could be detected up to depths of 7 mm in the skin and muscle tissue of deceased teleost fish ( Sparus aurata and Stenotomus chrysops) and a deceased catshark ( Galeus melastomus). The effects of tissue heterogeneity on hydrogel delivery and fluorescence visibility were explored, with darker tissues masking hydrogel fluorescence. Hydrogels were implanted into a living eastern river cooter ( Pseudemys concinna), a European eel ( Anguilla anguilla), and a second species of catshark ( Scyliorhinus stellaris). The animals displayed no observable changes in movement and feeding patterns. Imaging by high-resolution ultrasound indicated no changes in tissue structure in the eel and catshark. In the turtle, some tissue reaction was detected upon dissection and histopathology. Analysis of movement patterns in sarasa comet goldfish ( Carassius auratus) indicated that the hydrogel implants did not affect swimming patterns. Taken together, these results indicate that this implantable form factor is a promising technique for biologging using aquatic vertebrates with further development. Future work will tune the sensor detection range to the physiological range of riboflavin, develop strategies to normalize sensor signal to account for the optical heterogeneity of animal tissues, and design a flexible, wearable device incorporating optoelectronic components that will enable sensor measurements in moving animals. This work advances the application of nanosensors to organisms beyond the commonly used rodent and zebrafish models and is an important step toward the physiological biologging of aquatic organisms.
We describe a label-free approach based on Raman spectroscopy, to study drug-induced apoptosis in vivo. Spectral-shifts at wavenumbers associated with DNA, proteins, lipids, and collagen have been identified on breast and melanoma tumor tissues. These findings may enable a new analytical method for rapid readout of drug-therapy with miniaturized probes.