Early-stage diagnosis of bladder cancer using surface-enhanced Raman spectroscopy combined with machine learning algorithms in a rat model.

Early diagnosis and accurate assessment of tumor development facilitate early bladder cancer resection and initiation of drug therapy. This study enabled an early, accurate, label-free, noninvasive diagnosis of bladder tumors by analyzing nano-biomarkers in a single drop of urine through surface-enhanced Raman spectroscopy (SERS). In a standard N-butyl-N-4-hydroxybutyl nitrosamine-induced rat model of bladder cancer, cancer stage and polyp tumor development were monitored using a small endoscope with a diameter of 1.2 mm in a minimally invasive manner without the need to kill the rats. Samples were divided into cancer-free, early-stage, and polyp-form cancer. Training data were classified according to micro-cystoscopic 5-aminolevulinic acid fluorescence diagnosis, and specimens were postmortem verified through histopathological analysis. A drop of urine from each sample group was placed on an Au-coated zinc oxide nanoporous chip to filter nano-biomaterials and selectively enhance the Raman signals of nanoscale analytes via SERS. Principal component analysis was used to reduce the dimensionality of the collected Raman spectra, and partial least squares discriminant analysis was used to find diagnostic clusters based on the labeled samples. The combination of SERS and machine learning achieved an accuracy ≥99.6% in diagnosing both early- and polyp-stage bladder tumors. With an area under the receiver operating characteristic curve greater than 0.996, the accuracy of the diagnosis in the rat model suggests that SERS-based diagnostic methods are promising when coupled with machine learning. Low-cost, label-free, and noninvasive surface-enhanced Raman spectra are ideal for developing clinically relevant point-of-care diagnostic techniques.

Deep Learning Assisted Surface-Enhanced Raman Spectroscopy (SERS) for Rapid and Direct Nucleic Acid Amplification and Detection: Toward Enhanced Molecular Diagnostics.

Surface-enhanced Raman scattering (SERS) has evolved into a robust analytical technique capable of detecting a variety of biomolecules despite challenges in securing a reliable Raman signal. Conventional SERS-based nucleic acid detection relies on hybridization assays, but reproducibility and signal strength issues have hindered research on directly amplifying nucleic acids on SERS surfaces. This study introduces a deep learning assisted ZnO-Au-SERS-based direct amplification (ZADA) system for rapid, sensitive molecular diagnostics. The system employs a SERS substrate fabricated by depositing gold on uniformly grown ZnO nanorods. These nanorods create hot spots for the amplification of the target nucleic acids directly on the SERS surface, eliminating the need for postamplification hybridization and Raman reporters. The limit of detection of the ZADA system was superior to those of the conventional amplification methods. Clinical validation of the ZADA system with coronavirus disease 2019 (COVID-19) samples from human patients yielded a sensitivity and specificity of 92.31% and 81.25%, respectively. The integration of a deep learning program further enhanced sensitivity and specificity to 100% and reduced SERS analysis time, showcasing the potential of the ZADA system for rapid, label-free disease diagnosis via direct nucleic acid amplification and detection within 20 min.

Label-free atherosclerosis diagnosis through a blood drop of apolipoprotein E knockout mouse model using surface-enhanced Raman spectroscopy validated by machine learning algorithm.

The direct preventative detection of flow-induced atherosclerosis remains a significant challenge, impeding the development of early treatments and prevention measures. This study proposes a method for diagnosing atherosclerosis in the carotid artery using nanometer biomarker measurements through surface-enhanced Raman spectroscopy (SERS) from single-drop blood samples. Atherosclerotic acceleration is induced in apolipoprotein E knockout mice which underwent a partial carotid ligation and were fed a high-fat diet to rapidly induce disturbed flow-induced atherosclerosis in the left common carotid artery while using the unligated, contralateral right carotid artery as control. The progressive atherosclerosis development of the left carotid artery was verified by micro-magnetic resonance imaging (micro-MRI) and histology in comparison to the right carotid artery. Single-drop blood samples are deposited on chips of gold-coated ZnO nanorods grown on silicon wafers that filter the nanometer markers and provide strong SERS signals. A diagnostic classifier was established based on principal component analysis (PCA), which separates the resultant spectra into the atherosclerotic and control groups. Scoring based on the principal components enabled the classification of samples into control, mild, and severe atherosclerotic disease. The PCA-based analysis was validated against an independent test sample and compared against the PCA-PLS-DA machine learning algorithm which is known for applicability to Raman diagnosis. The accuracy of the PCA modification-based diagnostic criteria was 94.5%, and that of the machine learning algorithm 97.5%. Using a mouse model, this study demonstrates that diagnosing and classifying the severity of atherosclerosis is possible using a single blood drop, SERS technology, and machine learning algorithm, indicating the detectability of biomarkers and vascular factors in the blood which correlate with the early stages of atherosclerosis development.

SERS Effect on Spin-Coated Seeding of Tilted Au-ZnO Nanorods for Low-Cost Diagnosis.

Uniformly parallel Au-coated ZnO nanorods have previously been shown to amplify local Raman signals, providing increased sensitivity to disease markers in the detection of inflammation and cancer. However, practical and cost-effective fabrication methods of substrates for surface-enhanced Raman spectroscopy (SERS) fail to produce highly uniform surfaces. Here, the feasibility of Raman enhancement on less-uniform substrates is assessed. ZnO nanorod structures were fabricated by hydrothermal synthesis, starting from spin-coated seed substrates. Following analysis, the nanostructures were coated with Au to create stochastically variant substrates. The non-uniformity of the fabricated Au-coated ZnO nanorod structures is confirmed morphologically by FE-SEM and structurally by X-ray diffraction, and characterized by the angular distributions of the nanorods. Monte Carlo finite element method simulations matching the measured angular distributions and separations predicted only moderate increases in the overall Raman enhancement with increasing uniformity. Highly variant substrates exhibited approximately 76% of the Raman enhancement of more uniform substrates in simulations and experiments. The findings suggest that, although highly inhomogeneous Au-coated ZnO nanorod substrates may not attain the same Raman enhancement as more uniform substrates, the relaxation of fabrication tolerances may be economically viable.

Selective Detection of Nano-Sized Diagnostic Markers Using Au-ZnO Nanorod-Based Surface-Enhanced Raman Spectroscopy (SERS) in Ureteral Obstruction Models.

BACKGROUND: This study investigated the diagnosis of renal diseases using a biochip capable of detecting nano-sized biomarkers. Raman measurements from a kidney injury model were taken, and the feasibility of early diagnosis was assessed. MATERIALS AND METHODS: Rat models with mild and severe unilateral ureteral obstructions were created, with the injury to the kidney varying according to the tightness of the stricture. After generating the animal ureteral obstruction models, urine was collected from the kidney and bladder. RESULTS AND DISCUSSION: After confirming the presence of renal injury, urine drops were placed onto a Raman chip whose surface had been enhanced with Au-ZnO nanorods, allowing nano-sized biomarkers that diffused into the nanogaps to be selectively amplified. The Raman signals varied according to the severity of the renal damage, and these differences were statistically confirmed. CONCLUSION: These results confirm that ureteral stricture causes kidney injury and that signals in the urine from the release of nano-biomarkers can be monitored using surface-enhanced Raman spectroscopy.

Stereotaxic endoscopy for the ocular imaging of awake, freely moving animal models.

Stereotaxic instruments are increasingly used in research animals for the study of disease, but typically require restraints and anesthetic procedures. A stereotaxic head mount that enables imaging of the anterior chamber of the eye in alert and freely mobile mice is presented in this study. The head mount is fitted based on computed tomography scans and manufactured using 3D printing. The system is placed noninvasively using temporal mount bars and a snout mount, without breaking the skin or risking suffocation, while an instrument channel stabilizes the ocular probes. With a flexible micro-endoscopic probe and a confocal scanning laser microscopy system, <20 µm resolution is achieved in vivo with a field of view of nearly 1 mm. Discomfort is minimal, and further adaptations for minimally invasive neuroscience, optogenetics and auditory studies are possible.

Optimization of ZnO Nanorod-Based Surface Enhanced Raman Scattering Substrates for Bio-Applications.

Nanorods based on ZnO for surface enhanced Raman spectroscopy are promising for the non-invasive and rapid detection of biomarkers and diagnosis of disease. However, optimization of nanorod and coating parameters is essential to their practical application. With the goal of establishing a baseline for early detection in biological applications, gold-coated ZnO nanorods were grown and coated to form porous structures. Prior to gold deposition, the grown nanorods were 30⁻50 nm in diameter and 500⁻600 nm in length. Gold coatings were grown on the nanorod structure to a series of thicknesses between 100 and 300 nm. A gold coating of 200 nm was found to optimize the Rhodamine B model analyte signal, while performance for rat urine depended on the biomarkers to be detected. These results establish design guidelines for future use of Au-ZnO nanorods in the study and early diagnosis of inflammatory diseases.

Diagnosis in a Preclinical Model of Bladder Pain Syndrome Using a Au/ZnO Nanorod-based SERS Substrate.

To evaluate the feasibility of ZnO nanorod-based surface enhanced Raman scattering (SERS) diagnostics for disease models, particularly for interstitial cystitis/bladder pain syndrome (IC/BPS), ZnO-based SERS sensing chips were developed and applied to an animal disease model. ZnO nanorods were grown to form nano-sized porous structures and coated with gold to facilitate size-selective biomarker detection. Raman spectra were acquired on a surface enhanced Raman substrate from the urine in a rat model of IC/BPS and analyzed using a statistical analysis method called principal component analysis (PCA). The nanorods grown after the ZnO seed deposition were 30 to 50 nm in diameter and 500 to 600 nm in length. A volume of gold corresponding to a thin film thickness of 100 nm was deposited on the grown nanorod structure. Raman spectroscopic signals were measured in the scattered region for nanometer biomarker detection to indicate IC/BPS. The Raman peaks for the control group and IC/BPS group are observed at 641, 683, 723, 873, 1002, 1030, and 1355 cm-1, which corresponded to various bonding types and compounds. The PCA results are plotted in 2D and 3D. The Raman signals and statistical analyses obtained from the nano-sized biomarkers of intractable inflammatory diseases demonstrate the possibility of an early diagnosis.

Polarity-inverted lateral overgrowth and selective wet-etching and regrowth (PILOSWER) of GaN.

On an SiO2-patterned c-plane sapphire substrate, GaN domains were grown with their polarity controlled in accordance with the pattern. While N-polar GaN was grown on hexagonally arranged circular openings, Ga-polar GaN was laterally overgrown on mask regions due to polarity inversion occurring at the boundary of the circular openings. After etching of N-polar GaN on the circular openings by H3PO4, this template was coated with 40-nm Si by sputtering and was slightly etched by KOH. After slight etching, a thin layer of Si left on the circular openings of sapphire,but not on GaN, was oxidized during thermal annealing and served as a dielectric mask during subsequent regrowth. Thus, the subsequent growth of GaN was made only on the existing Ga-polar GaN domains, not on the circular openings of the sapphire substrate. Transmission electron microscopy analysis revealed no sign of threading dislocations in this film. This approach may help fabricating an unholed and merged GaN film physically attached to but epitaxially separated from the SiO2-patterned sapphire.

The determining factor of a preferred orientation of GaN domains grown on m-plane sapphire substrates.

Epitaxial lateral overgrowth in tandem with the first-principles calculation was employed to investigate the determining factor of a preferred orientation of GaN on SiO2-patterned m-plane sapphire substrates. We found that the (1100)-orientation is favored over the (1103)-orientation in the region with a small filling factor of SiO2, while the latter orientation becomes preferred in the region with a large filling factor. This result suggests that the effective concentration determines the preferred orientation of GaN: the (1100)- and (1103)-orientations preferred at their low and high concentrations, respectively. Our computational study revealed that at a low coverage of Ga and N atoms, the local atomic arrangement resembles that on the (1103) surface, although the (1100) surface is more stable at their full coverage. Such a (1103)-like atomic configuration crosses over to the local structure resembling that on the (1100) surface as the coverage increases. Based on results, we determined that high effective concentration of Ga and N sources expedites the growth of the (1103)-orientation while keeping from transition to the (1100)-orientation. At low effective concentration, on the other hand, there is a sufficient time for the added Ga and N sources to rearrange the initial (1103)-like orientation to form the (1100)-orientation.