Current method for monitoring patients for cancer recurrence after treatment requires patients to travel to a centralized laboratory, causing time in scheduling appointments/waiting for results, financial burden in travel costs to clinics, and invasive procedures (i.e., biopsies) leading to discomfort in patients. To improve convenience, outcomes, and enable more frequent monitoring of cancer recurrence, we propose using an implantable hydrogel sensor for remote cancer surveillance. Gold nanostars (GNS), efficient plasmonic nanomaterials, embedded in hydrogels, enhance Raman scattering signals of cancer biomarkers. A handheld Raman spectroscopy probe collects these signals, representing the unique vibrational molecular fingerprint. Toward this effort, this study demonstrates the performance of a GNS-embedded hydrogel for discriminating serum in two preclinical mouse prostate cancer models: NSG and C57BL/6J mice. GNS labeled with 4- mercaptobenzoic acid (4-MBA) were embedded in 70μL hydrogels. Six serum samples from NSG mice (3 with LNCaP subcutaneous tumors, 3 normal) and eight serum samples from C57BL/6J mice (3 wild type, 5 transgenic modified– TRAMP with prostate cancer) were obtained. Serum (70μL) was incubated overnight (4°C) with the hydrogel sample. Raman spectra were collected at five distinct locations using the Raman handheld probe. Spectral analysis involved intensity normalization, principal component analysis (PCA) for dimension reduction, and linear discriminate analysis (LDA) for classification with leave-one-spectra-out cross-validation. NSG mice exhibited band differences at 775-825 cm-1, 1202-1249 cm-1, and 1430-1478 cm-1 (LDA ROC AUC = 0.83), while C57BL/6J mice showed differences at 1152-1245 cm-1 and 1362-1407 cm-1 (LDA ROC AUC = 0.98). Successful discrimination of serum in mouse models demonstrates the presence of biomarkers that differentiate cancer-bearing mice and the potential for remote cancer monitoring.
Early cancer detection is critical for successful treatment. Current cancer detection methods require travel to a centralized laboratory for testing which can be time-consuming, costly, invasive, and infrequent. Patients could benefit from a less invasive method to monitor recurrence which could be performed more frequently from the comfort of their home. We propose an implanted hydrogel sensor for remote cancer monitoring. Gold nanostars (GNS) embedded within the hydrogel produce surface enhanced Raman scattering signals of cancer biomarkers collected remotely using a handheld probe, with the results being sent to their provider. Here, we present results demonstrating the ability to discriminate human prostate cancer plasma. GNS were labeled with 4-mercaptobenzoic acid (4-MBA) and embedded into 70μL hydrogels. Four prostate cancer samples and five non-prostate cancer samples were obtained from a biobank. 70μL of each sample were combined with one hydrogel per sample and incubated overnight at 4°C. A handheld probe was used to collect Raman spectra at 5 different locations across each hydrogel face. The classification algorithm included intensity normalization based on intensity of the 4MBA signal, principal component analysis (PCA) for dimension reduction, and linear discriminate analysis (LDA) or logistic regression for classification with leave-one-sample- out cross validation. Comparison of cancer and non-cancer spectra shows relative peak intensity differences between the two groups including at 726cm-1 and 1450cm-1. The area under the ROC curve was up to 0.94 for logistic regression. Results show the potential of remote cancer monitoring with a hydrogel SERS sensor.
Tissue biopsy and histological evaluation is the gold standard for disease diagnosis including cancer. For example, a punch several millimeters in diameter is often used to biopsy suspicious skin sites. The biopsy is then formalin fixed, paraffin embedded, sectioned, stained with hematoxylin and eosin (H&E), and examined by a pathologist. While this process has been the gold standard for decades, two limitations are recognized. First, the biopsy is invasive with a limited number that can be reasonably tolerated by the patient. Second, the tissue processing steps are slow. We report an alternative approach consisting of a laser microbiopsy for harvest of sub-microliter (<1 mm3) tissue sections combined with rapid virtual H&E staining methods. A Ho:YAG laser (Lumenis P120) was shaped into an annular beam and focused onto ex vivo porcine skin. The epidermis and dermis were laser cut and the tissue section in the center of the annulus was ejected and collected by an overlying glass coverslip. Tetrafluoroethane (R134A) was sprayed at the ablation site prior to ablation and at the collected tissue section post ablation to limit thermal damage and preserve histological features. Two virtual H&E imaging methods were tested with confocal microscopy. The first combined acridine orange fluoresce with reflectance. The second combined acridine orange and sulforhodamine 101 fluorescence. For each method, the two channels were false colored and combined to create virtual H&E images. Virtual H&E images show histological features, including cell nuclei. Laser microbiopsy is minimally invasive, harvesting tissue sections on the order of 0.01 to 0.1 mm3, and tissue processing is rapid requiring 2 min or less for staining. Laser microbiopsy is a promising candidate technique for rapid minimally invasive diagnosis.
Lasers are commonly employed in surgery for hard and soft tissues due to their precise space-time energy delivery and compatibility with optical fibers for delivery into body cavities, including for treatment of urological diseases. Infrared laser ablation in tissues can result in non-specific heating and thermal injury. Methods that maximize ablation efficiency, or tissue volume removed per unit energy, while minimizing non-specific thermal injury can improve surgical workflows and outcomes. We report a novel approach for increased ablation efficiency by modifying the beam shape. Specifically, a Ho:YAG laser is shaped into a converging annular beam. Ablation efficiency was measured on a hard tissue phantom (BegoStone) and soft tissue (porcine kidney). An annular beam ~800 μm in diameter was used to ablate each sample at 10 different locations using a single 1 J pulse per location. The procedure was repeated using a circular beam with similar diameter by placing a 200 μm fiber 1 mm from the tissue surface. Each ablation crater was imaged with optical coherence tomography and the crater volumes calculated from recorded images. For hard tissue phantoms, ablation efficiency increased 183% for annular vs. circular beams (0.065±0.013 vs. 0.023 ± 0.003 mm3 /J). For soft tissue, ablation efficiency increased 69% for annular vs. circular beams (0.098±0.021 vs 0.058 ± 0.018 mm3 /J). Hard and soft tissue ablation with an annular beam is a promising technique for increasing the speed and safety of laser surgery.
Tissue sampling is required for disease diagnostics and research. Traditional tissue sampling tools often remove more tissue than required, causing unnecessary pain and morbidity. We report a minimally invasive laser microbiopsy system to sample sub-microliter (<1mm3 ) tissue sections. A focused annular beam cuts conical-shaped tissue sections. Compatible diagnostic methods may include standard histopathology, rapid point-of-care microscopy, and genetic testing. An objective of the approach is to harvest a minimal volume while limiting damage to retain tissue integrity for diagnosis. The laser microbiopsy system consists of a Ho:YAG laser (Lumenis P120), a ZnSe aspheric collimator (ISP optics, f = 50.8 mm), two fused silica axicons (Thorlabs, α = 20°), and an aspheric focusing lens (ISP optics, f = 25.4 mm). The system is characterized to evaluate the required laser dosimetry, range of harvested tissue volumes, and residual thermal damage. We first characterized the system in silico using raytracing (Zemax) and a heat transfer modeling. We then characterized the system experimentally performing micro-biopsies in ex vivo porcine skin. Raytracing and experimental measurements show agreement in annular beam shape with an inner and outer radius of 300 μm and 400 μm near the beam focus. Experimental results show ability to harvest tissue sections ranging from 0.009 to 0.10 mm3 using pulse energies from 1 - 2 J.
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