Probing the mutation independent interaction of DNA probes with SARS-CoV-2 variants through a combination of surface-enhanced Raman scattering and machine learning.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution has been characterized by the emergence of sets of mutations impacting the virus characteristics, such as transmissibility and antigenicity, presumably in response to the changing immune profile of the human population. The presence of mutations in the SARS-CoV-2 virus can potentially impact therapeutic and diagnostic test performances. We design and develop here a unique set of DNA probes i.e., antisense oligonucleotides (ASOs) which can interact with genetic sequences of the virus irrespective of its ongoing mutations. The probes, developed herein, target a specific segment of the nucleocapsid phosphoprotein (N) gene of SARS-CoV-2 with high binding efficiency which do not mutate among the known variants. Further probing into the interaction profile of the ASOs reveals that the ASO-RNA hybridization remains unaltered even for a hypothetical single point mutation at the target RNA site and diminished only in case of the hypothetical double or triple point mutations. The mechanism of interaction among the ASOs and SARS-CoV-2 RNA is then explored with a combination of surface-enhanced Raman scattering (SERS) and machine learning techniques. It has been observed that the technique, described herein, could efficiently discriminate between clinically positive and negative samples with ∼100% sensitivity and ∼90% specificity up to 63 copies/mL of SARS-CoV-2 RNA concentration. Thus, this study establishes N gene targeted ASOs as the fundamental machinery to efficiently detect all the current SARS-CoV-2 variants regardless of their mutations.

Multimodal Label-Free Monitoring of Adipogenic Stem Cell Differentiation Using Endogenous Optical Biomarkers.

Stem cell-based therapies carry significant promise for treating human diseases. However, clinical translation of stem cell transplants for effective treatment requires precise non-destructive evaluation of the purity of stem cells with high sensitivity (<0.001% of the number of cells). Here, a novel methodology using hyperspectral imaging (HSI) combined with spectral angle mapping-based machine learning analysis is reported to distinguish differentiating human adipose-derived stem cells (hASCs) from control stem cells. The spectral signature of adipogenesis generated by the HSI method enables identifying differentiated cells at single-cell resolution. The label-free HSI method is compared with the standard techniques such as Oil Red O staining, fluorescence microscopy, and qPCR that are routinely used to evaluate adipogenic differentiation of hASCs. HSI is successfully used to assess the abundance of adipocytes derived from transplanted cells in a transgenic mice model. Further, Raman microscopy and multiphoton-based metabolic imaging is performed to provide complementary information for the functional imaging of the hASCs. Finally, the HSI method is validated using matrix-assisted laser desorption/ionization-mass spectrometry imaging of the stem cells. The study presented here demonstrates that multimodal imaging methods enable label-free identification of stem cell differentiation with high spatial and chemical resolution.

Printed Electrode for Measuring Phosphate in Environmental Water.

Phosphate is a major nonpoint source pollutant in both the Louisiana local streams as well as in the Gulf of Mexico coastal waters. Phosphates from agricultural run-off have contributed to the eutrophication of global surface waters. Phosphate environmental dissemination and eutrophication problems are not yet well understood. Thus, this study aimed to monitor phosphate in the local watershed to help identify potential hot spots in the local community (Mississippi River, Louisiana) that may contribute to nutrient loading downstream (in the Gulf of Mexico). An electrochemical method using a physical vapor deposited cobalt microelectrode was utilized for phosphate detection using cyclic voltammetry and amperometry. The testing results were utilized to evaluate the phosphate distribution in river water and characterize the performance of the microsensor. Various characterizations, including the limit of detection, sensitivity, and reliability, were conducted by measuring the effect of interferences, including dissolved oxygen, pH, and common ions. The electrochemical sensor performance was validated by comparing the results with the standard colorimetry phosphate detection method. X-ray photoelectron spectroscopy (XPS) measurements were performed to understand the phosphate sensing mechanism on the cobalt electrode. This proof-of-concept sensor chip could be utilized for on-field monitoring using a portable, hand-held potentiostat.

Characterization of fibrillar collagen isoforms in infarcted mouse hearts using second harmonic generation imaging.

We utilized collagen specific second harmonic generation (SHG) signatures coupled with correlative immunofluorescence imaging techniques to characterize collagen structural isoforms (type I and type III) in a murine model of myocardial infarction (MI). Tissue samples were imaged over a four week period using SHG, transmitted light microscopy and immunofluorescence imaging using fluorescently-labeled collagen antibodies. The post-mortem cardiac tissue imaging using SHG demonstrated a progressive increase in collagen deposition in the left ventricle (LV) post-MI. We were able to monitor structural morphology and LV remodeling parameters in terms of extent of LV dilation, stiffness and fiber dimensions in the infarcted myocardium.

Optical Identification of Middle Ear Infection.

Ear infection is one of the most commonly occurring inflammation diseases in the world, especially for children. Almost every child encounters at least one episode of ear infection before he/she reaches the age of seven. The typical treatment currently followed by physicians is visual inspection and antibiotic prescription. In most cases, a lack of improper treatment results in severe bacterial infection. Therefore, it is necessary to design and explore advanced practices for effective diagnosis. In this review paper, we present the various types of ear infection and the related pathogens responsible for middle ear infection. We outline the conventional techniques along with clinical trials using those techniques to detect ear infections. Further, we highlight the need for emerging techniques to reduce ear infection complications. Finally, we emphasize the utility of Raman spectroscopy as a prospective non-invasive technique for the identification of middle ear infection.

Multiplexed Paper Microfluidics for Titration and Detection of Ingredients in Beverages.

Food safety and access to systematic approaches for ensuring detection of food hazards is an important issue in most developing countries. With the arrival of paper-based analytical devices (µPADs) as a promising, rapid, easy-to-use, and low-cost analytical tool, we demonstrated a simple microfluidic-based titration study for the analysis of packaged fruit juices. Similar, to the titration experiments using traditional glassware in chemistry laboratories, in this study the titration experiments were developed using paper microfluidics for the analysis of several analytes such as pH, vitamin C, sugars, and preservatives present in the packaged fruit juices. The allergen found commonly in dairy based mixtures and the non-pathogenic biochemical component responsible for food spoilage in cider based fruit juices were also determined. The results obtained using paper microfluidics were compared with those obtained using a conventional spectrophotometric technique. Finally, a paper microfluidics based multiplexed sensor was developed for the analysis of common nutritional ingredients, an allergen, and a non-pathogenic byproduct present in packaged fruit juices on a single platform. Overall, the results presented in this study reveal that the proposed paper microfluidic assisted colorimetric multiplexed sensor offers a quick and reliable tool for on-spot routine analysis for food safety applications.

Nanohole array plasmonic biosensors: Emerging point-of-care applications.

Point-of-care (POC) applications have expanded hugely in recent years and is likely to continue, with an aim to deliver cheap, portable, and reliable devices to meet the demands of healthcare industry. POC devices are designed, prototyped, and assembled using numerous strategies but the key essential features that biosensing devices require are: (1) sensitivity, (2) selectivity, (3) specificity, (4) repeatability, and (5) good limit of detection. Overall the fabrication and commercialization of the nanohole array (NHA) setup to the outside world still remains a challenge. Here, we review the various methods of NHA fabrication, the design criteria, the geometrical features, the effects of surface plasmon resonance (SPR) on sensing as well as current state-of-the-art of existing NHA sensors. This review also provides easy-to-understand examples of NHA-based POC biosensing applications, its current status, challenges, and future prospects.

Improvement of Tribological and Biocompatibility Properties of Orthopedic Materials Using Piezoelectric Direct Discharge Plasma Surface Modification.

Various types of alloys and polymers are utilized in orthopedic implants. However, there are still several issues accompanied by the use of prosthetic materials, such as low wear performance and catastrophic failure. Surface enhancement of biomaterials is a promising method that can improve the success rate of prosthetic operations without negatively affecting their bulk properties while improving the biocompatibility of implants and reducing infections. Nonthermal plasma treatment has become a ubiquitous surface modification method in sterilization and healthcare applications. However, the clinical applications of such an approach have been limited due to the lack of detailed studies delineating the wear behavior and biocompatibility of implants after plasma treatment. In this study, we have employed a handheld piezoelectric direct discharge (PDD) plasma generator to modify the surface of two common metallic (Ti6Al4V) and nonmetallic (GUR1020 polymer) biomaterials used typically in joint and disc replacements. We have observed an approximately 60-fold reduction in tribological wear rate along with a 2- to 3-fold increase in the biocompatibility properties of plasma coated samples compared to noncoated (untreated) surfaces, respectively. Our study introduces a novel application of nonthermal PDD plasma technology that is capable of increasing the quality and success rate of joint and disc replacements.

Current and future functional imaging techniques for post-traumatic stress disorder.

Posttraumatic stress disorder (PTSD) is a trauma and stressor related psychiatric disorder associated with structural, metabolic, and molecular alternations in several brain regions including diverse cortical areas, neuroendocrine regions, the striatum, dopaminergic, adrenergic and serotonergic pathways, and the limbic system. We are in critical need of novel therapeutics and biomarkers for PTSD and a deep understanding of cutting edge imaging and spectroscopy methods is necessary for the development of promising new approaches to better diagnose and treat the disorder. According to the Diagnostic and Statistical Manual of Mental Disorders (DSM-V) criterion, all forms of traumatic stress-induced disorder are considered acute stress disorder for the first month following the stressor. Only after symptoms do not remit for one month can the disorder be deemed PTSD. It would be particularly useful to differentiate between acute stress disorder and PTSD during the one month waiting period so that more intensive treatments can be applied early on to patients with a high likelihood of developing PTSD. This would potentially enhance treatment outcomes and/or prevent the development of PTSD. Comprehension of the qualities and limitations of currently applied methods as well as the novel emerging techniques provide invaluable knowledge for fast paced development. Conventional methods of studying PTSD have proven to be insufficient for diagnosis, measurement of treatment efficacy, and monitoring disease progression. As the field currently stands, there is no diagnostic biomarker available for any psychiatric disease, PTSD included. Currently, emerging and available technologies are not utilized to their full capacity and in appropriate experimental designs for the most fruitful possible studies in this area. Therefore, there is an apparent need for improved methods in PTSD research. This review demonstrates the current state of the literature in PTSD, including molecular, cellular, and behavioral indicators, possible biomarkers and clinical and pre-clinical imaging techniques relevant to PTSD, and through this, elucidate the void of current practical imaging and spectroscopy methods that provide true biomarkers for the disorder and the significance of devising new techniques for future investigations. We are unlikely to develop a single biomarker for any psychiatric disorder however. As psychiatric disorders are incomparably complex compared to other medical diagnoses, its most likely that transcriptomic, metabolomic and structural and connectomic imaging data will have to be analyzed in concert in order to produce a dependable non-behavioral marker of PTSD. This can explain the necessity of bridging conventional approaches to novel technologies in order to create a framework for further discoveries in the treatment of PTSD.

Raman Spectroscopy and Microscopy Applications in Cardiovascular Diseases: From Molecules to Organs.

Noninvasive and label-free vibrational spectroscopy and microscopy methods have shown great potential for clinical diagnosis applications. Raman spectroscopy is based on inelastic light scattering due to rotational and vibrational modes of molecular bonds. It has been shown that Raman spectra provide chemical signatures of changes in biological tissues in different diseases, and this technique can be employed in label-free monitoring and clinical diagnosis of several diseases, including cardiovascular studies. However, there are very few literature reviews available to summarize the state of art and future applications of Raman spectroscopy in cardiovascular diseases, particularly cardiac hypertrophy. In addition to conventional clinical approaches such as electrocardiography (ECG), echocardiogram (cardiac ultrasound), positron emission tomography (PET), cardiac computed tomography (CT), and single photon emission computed tomography (SPECT), applications of vibrational spectroscopy and microscopy will provide invaluable information useful for the prevention, diagnosis, and treatment of cardiovascular diseases. Various in vivo and ex vivo investigations can potentially be performed using Raman imaging to study and distinguish pathological and physiological cardiac hypertrophies and understand the mechanisms of other cardiac diseases. Here, we have reviewed the recent literature on Raman spectroscopy to study cardiovascular diseases covering investigations on the molecular, cellular, tissue, and organ level.

DNA microarray analysis using a smartphone to detect the BRCA-1 gene.

DNA microarrays are used to examine changes in gene expression of a large number of genes simultaneously by fluorescent labeling of complementary DNAs (cDNAs). The major bottleneck in implementing microarray technology in resource-limited settings lies in the detection instrument used for generating images of spotted oligonucleotides post-hybridization. While various methods such as a lateral flow assay have been presented to accomplish point-of-care disease detection, there is no simple and effective instrument available to gather spot images maintaining the standard microarray procedures. Nanotechnology based sensors connected with a portable smartphone readout system have the potential to be implemented in microarray technology. Here, we describe a portable fluorescence microarray based imaging system connected to a smartphone for detecting breast cancer gene expression (BRCA-1) from exon 11. This is based on the interactive binding of probe DNA to Cy3-target DNA. A paper-based microfluidics approach was used to demonstrate the DNA hybridization assay. The imaging principles of the assembled device named "FluoroZen" are similar to those of a fluorescence microscope. It uses two light spectrum filters, one to excite the fluorescent dye and the other to capture the emission spectrum. The images were acquired by using CCD cameras from FluoroZen. The smartphone integrated paper microfluidics platform presented here could be translated into clinical settings to perform point-of-care testing.

Zein Nanoparticles Uptake and Translocation in Hydroponically Grown Sugar Cane Plants.

The main objective of this study was to investigate the uptake and translocation of positively charged zein nanoparticles (ZNPs) in hydroponically grown sugar cane plants. Fluorescent ZNPs (spherical and measuring an average diameter 135 ± 3 nm) were synthesized by emulsion-diffusion method from FITC-tagged zein. Fluorescent measurement following digestion of plant tissue indicated that sugar cane roots had a significant adhesion of ZNPs, 342.5 ± 24.2 µg NPs/mg of dry matter, while sugar cane leaves contained a very limited amount, 12.9 ± 1.2 µg NPs/mg dry matter for high dose(1.75 mg/ml) after 12 h. Confocal microscopy studies confirmed presence of fluorescent ZNPs in the epidermis and endodermis of the root system. Given their ability to adhere to roots for extended periods of time, ZNPs are proposed as effective delivery systems for agrochemicals to sugar cane plants, but more studies are needed to identify effect of nanoparticle exposure to health of the plant.