Highly Sensitive Molecular Diagnostic Platform for Scrub Typhus Diagnosis Using O. tsutsugamushi Enrichment and Nucleic Acid Extraction.

Scrub typhus is caused by the Gram-negative obligate intracellular bacterium Orientia tsutsugamushi, and this tick-borne disease is difficult to distinguish from other acute febrile illnesses as it typically presents with symptoms such as rash, crusting at the bite site, headache, myalgia, lymphadenopathy, and elevated liver transaminases. It can often be diagnosed clinically, but not all patients present with characteristic symptoms, so serological diagnosis and molecular techniques may be required. However, existing diagnostic tests often have low sensitivity and specificity, making early detection difficult. This study presents a nucleic acid extraction method using large volumes of plasma and buffy coat to increase sensitivity, as well as an improved detection method using two target genes. Using the I-PULL device, nucleic acids can be extracted from up to 4 mL of sample in 30 min, avoiding contamination. The extracted DNA detects two genes of O. tsutsugamushi, increasing sensitivity compared to single-gene detection. Clinical validation in 38 patient samples showed 100% specificity and 95.24% sensitivity for the single target gene, with specificity and sensitivity rising to 100% when both genes are analyzed. This molecular diagnostic platform can be useful for distinguishing scrub typhus from similar diseases.

Transferrin-conjugated magnetic nanoparticles for the isolation of brain-derived blood exosomal MicroRNAs: A novel approach for Parkinson's disease diagnosis.

BACKGROUND: Brain-derived exosomes circulate in the bloodstream and other bodily fluids, serving as potential indicators of neurological disease progression. These exosomes present a promising avenue for the early and precise diagnosis of neurodegenerative conditions. Notably, miRNAs found in plasma extracellular vesicles (EVs) offer distinct diagnostic benefits due to their stability, abundance, and resistance to breakdown. RESULTS: In this study, we introduce a method using transferrin conjugated magnetic nanoparticles (TMNs) to isolate these exosomes from the plasma of patients with neurological disorders. This TMNs technique is both quick (<35 min) and cost-effective, requiring no high-priced ingredients or elaborate equipment for EV extraction. Our method successfully isolated EVs from 33 human plasma samples, including those from patients with Parkinson's disease (PD), Multiple Sclerosis (MS), and Dementia. Using quantitative polymerase chain reaction (PCR) analysis, we evaluated the potential of 8 exosomal miRNA profiles as biomarker candidates. Six exosomal miRNA biomarkers (miR-195-5p, miR-495-3p, miR-23b-3P, miR-30c-2-3p, miR-323a-3p, and miR-27a-3p) were consistently linked with all stages of PD. SIGNIFICANCE: The TMNs method provides a practical, cost-efficient way to isolate EVs from biological samples, paving the way for non-invasive neurological diagnoses. Furthermore, the identified miRNA biomarkers in these exosomes may emerge as innovative tools for precise diagnosis in neurological disorders including PD.

CSMP: A Self-Assembled Plant Polysaccharide-Based Hydrofilm for Enhanced Wound Healing.

Wound management remains a critical healthcare issue due to the rising incidence of chronic diseases leading to persistent wounds. Traditional dressings have their limitations, such as potential for further damage during changing and suboptimal healing conditions. Recently, hydrogel-based dressings have gained attention due to their biocompatibility, biodegradability, and ability to fill wounds. Particularly, polysaccharide-based hydrogels have shown potential in various medical applications. This study focuses on the development of a novel hydrofilm wound dressing produced from a blend of chia seed mucilage (CSM) and polyvinyl alcohol (PVA), termed CSMP. While the individual properties of CSM and PVA are well-documented, their combined potential in wound management is largely unexplored. CSMP, coupled with sorbitol and glycerin, and cross-linked using ultraviolet light, results in a flexible, adhesive, and biocompatible hydrofilm demonstrating superior water absorption, moisturizing, and antibacterial properties. This hydrofilm promotes epithelial cell migration, enhanced collagen production, and outperforms existing commercial dressings in animal tests. The innovative CSMP hydrofilm offers a promising, cost-effective approach for improved wound care, bridging existing gaps in dressing performance and preparation simplicity. Future research can unlock further applications of such polysaccharide-based hydrofilm dressings.

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.

Magnetic transferrin nanoparticles (MTNs) assay as a novel isolation approach for exosomal biomarkers in neurological diseases.

BACKGROUND: Brain-derived exosomes released into the blood are considered a liquid biopsy to investigate the pathophysiological state, reflecting the aberrant heterogeneous pathways of pathological progression of the brain in neurological diseases. Brain-derived blood exosomes provide promising prospects for the diagnosis of neurological diseases, with exciting possibilities for the early and sensitive diagnosis of such diseases. However, the capability of traditional exosome isolation assays to specifically isolate blood exosomes and to characterize the brain-derived blood exosomal proteins by high-throughput proteomics for clinical specimens from patients with neurological diseases cannot be assured. We report a magnetic transferrin nanoparticles (MTNs) assay, which combined transferrin and magnetic nanoparticles to isolate brain-derived blood exosomes from clinical samples. METHODS: The principle of the MTNs assay is a ligand-receptor interaction through transferrin on MTNs and transferrin receptor on exosomes, and electrostatic interaction via positively charged MTNs and negatively charged exosomes to isolate brain-derived blood exosomes. In addition, the MTNs assay is simple and rapid (< 35 min) and does not require any large instrument. We confirmed that the MTNs assay accurately and efficiently isolated exosomes from serum samples of humans with neurodegenerative diseases, such as dementia, Parkinson's disease (PD), and multiple sclerosis (MS). Moreover, we isolated exosomes from serum samples of 30 patients with three distinct neurodegenerative diseases and performed unbiased proteomic analysis to explore the pilot value of brain-derived blood protein profiles as biomarkers. RESULTS: Using comparative statistical analysis, we found 21 candidate protein biomarkers that were significantly different among three groups of neurodegenerative diseases. CONCLUSION: The MTNs assay is a convenient approach for the specific and affordable isolation of extracellular vesicles from body fluids for minimally-invasive diagnosis of neurological diseases.

A novel platform using homobifunctional hydrazide for enrichment and isolation of urinary circulating RNAs.

Changes in specific circulating RNA (circRNA) expressions can serve as diagnostic noninvasive biomarkers for prostate cancer (PCa). However, there are still unmet needs, such as unclear types and roles of circRNAs, PCa detection in benign prostatic hyperplasia (BPH) by unstandardized methods, and limitations of sample volume capacity and low circRNA concentrations. This study reports a simple and rapid circRNA enrichment and isolation technique named "HAZIS-CirR" for the analysis of urinary circRNAs. The method utilizes homobifunctional hydrazides with amine-modified zeolite and polyvinylidene fluoride (PVDF) syringe filtration for combining electrostatic and covalent coupling and size-based filtration, and it offers instrument-free isolation of circRNAs in 20 min without volume limitation, thermoregulation, and lysis. HAZIS-CirR has high capture efficiency (82.03%-92.38%) and a 10-fold more sensitive detection limit (20 fM) than before enrichment (200 fM). The clinical utility of HAZIS-CirR is confirmed by analyzing circulating mRNAs and circulating miRNAs in 89 urine samples. Furthermore, three miRNA panels that differentiate PCa from BPH and control, PCa from control, and BPH from control, respectively, are established by comparing miRNA levels. HAZIS-CirR will be used as an optimal and established method for the enrichment and isolation of circRNAs as diagnostic, prognostic, and predictive biomarkers in human cancers.

Gene-Based Diagnosis of Tuberculosis from Oral Swabs with a New Generation Pathogen Enrichment Technique.

A rapid and sensitive diagnosis is crucial for the management of tuberculosis (TB). A simple and label-free approach via homobifunctional imidoesters with a microfluidic platform (SLIM) assay showed a higher sensitivity than the Xpert MTB/RIF assay in the diagnosis of pulmonary TB (PTB). Here, we evaluated the efficacy of the SLIM assay for oral swab samples from cases of suspected PTB. Patients with clinically suspected PTB were prospectively enrolled and oral swab samples were processed using the SLIM assay and the attending physicians were blinded to the results of the SLIM assay. TB cases were defined as those treated with anti-TB chemotherapy for at least 6 months at the discretion of the specialists based on their clinical features and conventional laboratory results, including the Xpert assay. A total of 272 patients (with TB, n = 128 [47.1%]; without TB, n = 144 [52.9%]; mean age, 59.8 years) were enrolled. Overall, the sensitivity of the oral swab-based SLIM assay (65.6%) was higher than that of the sputum-based Xpert assay (43.4%; P = 0.001). Specifically, the SLIM oral swab assay showed a notably higher sensitivity in culture-negative TB cases compared with the Xpert assay (69.0% [95% CI: 49.2 to 84.7%] versus 7.4% [95% CI: 0.9 to 24.3%]; P = 0.001). The specificity of the SLIM and the Xpert assays was 86.1% (95% CI: 79.3 to 91.3%) and 100% (95% CI: 97.2 to 100%), respectively. When only culture-confirmed cases were analyzed, the SLIM oral swab was comparable to sputum Xpert in sensitivity (64.7% versus 54.3%, P = 0.26). The oral swab-based SLIM assay showed a superior sensitivity for TB diagnosis over the sputum-based Xpert assay, especially for culture-negative cases. IMPORTANCE The development of a rapid, accessible, and highly sensitive diagnostic tool is a major challenge in the control and management of tuberculosis. Gene-based diagnostics is recommended for the rapid diagnosis of pulmonary tuberculosis (PTB), but its sensitivity, such as Xpert MTB/RIF assay (Xpert), drops in cases with a low bacterial load. It can only be applied to sputum samples, and it is quite difficult for some patients to produce an adequate amount of sputum. We evaluated the clinical validity of an oral swab-based microfluidic system, i.e., the SLIM assay. The SLIM assay showed a significantly higher sensitivity than the Xpert assay, especially in smear-negative TB cases. This non-sputum-based SLIM assay can be a useful diagnostic test by overcoming the limitations of conventional sputum-based tests in pulmonary TB.

Chimeric nanocomposites for the rapid and simple isolation of urinary extracellular vesicles.

Cancer cell-derived extracellular vesicles (EVs) are promising biomarkers for cancer diagnosis and prognosis. However, the lack of rapid and sensitive isolation techniques to obtain EVs from clinical samples at a sufficiently high yield limits their practicability. Chimeric nanocomposites of lactoferrin conjugated 2,2-bis(methylol)propionic acid dendrimer-modified magnetic nanoparticles (LF-bis-MPA-MNPs) are fabricated and used for simple and sensitive EV isolation from various biological samples via a combination of electrostatic interaction, physically absorption, and biorecognition between the surfaces of the EVs and the LF-bis-MPA-MNPs. The speed, efficiency, recovery rate, and purity of EV isolation by the LF-bis-MPA-MNPs are superior to those obtained by using established methods. The relative expressions of exosomal microRNAs (miRNAs) from isolated EVs in cancerous cell-derived exosomes are verified as significantly higher than those from noncancerous ones. Finally, the chimeric nanocomposites are used to assess urinary exosomal miRNAs from urine specimens from 20 prostate cancer (PCa), 10 benign prostatic hyperplasia (BPH), patients and 10 healthy controls. Significant up-regulation of miR-21 and miR-346 and down-regulation of miR-23a and miR-122-5p occurs in both groups compared to healthy controls. LF-bis-MPA-MNPs provide a rapid, simple, and high yield method for human excreta analysis in clinical applications.

Rapid COVID-19 Molecular Diagnostic System Using Virus Enrichment Platform.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus (SARS-CoV)-2, is rapidly spreading and severely straining the capacities of public health communities and systems around the world. Therefore, accurate, rapid, and robust diagnostic tests for COVID-19 are crucial to prevent further spread of the infection, alleviate the burden on healthcare and diagnostic facilities, and ensure timely therapeutic intervention. To date, several detection methods based on nucleic acid amplification have been developed for the rapid and accurate detection of SARS-CoV-2. Despite the myriad of advancements in the detection methods for SARS-CoV-2, rapid sample preparation methods for RNA extraction from viruses have rarely been explored. Here, we report a rapid COVID-19 molecular diagnostic system that combines a self-powered sample preparation assay and loop-mediated isothermal amplification (LAMP) based naked-eye detection method for the rapid and sensitive detection of SARS-CoV-2. The self-powered sample preparation assay with a hydrophilic polyvinylidene fluoride filter and dimethyl pimelimidate can be operated by hand, without the use of any sophisticated instrumentation, similar to the reverse transcription (RT)-LAMP-based lateral flow assay for the naked-eye detection of SARS-CoV-2. The COVID-19 molecular diagnostic system enriches the virus population, extracts and amplifies the target RNA, and detects SARS-CoV-2 within 60 min. We validated the accuracy of the system by using 23 clinical nasopharyngeal specimens. We envision that this proposed system will enable simple, facile, efficient, and inexpensive diagnosis of COVID-19 at home and the clinic as a pre-screening platform to reduce the burden on the medical staff in this pandemic era.

Diatomaceous earth/zinc oxide micro-composite assisted antibiotics in fungal therapy.

As the second wave of COVID-19 hits South Asia, an increasing deadly complication 'fungal infections (such as Mycosis, Candida and Aspergillus) outbreak' has been raised concern about the insufficient technologies and medicals for its diagnosis and therapy. Biosilica based nano-therapy can be used for therapeutic efficacy, yet their direct role as antibiotic agent with biocompatibility and stability remains unclear. Here, we report that a diatomaceous earth (DE) framework semiconductor composite conjugated DE and in-house synthesized zinc oxide (DE-ZnO), as an antibiotic agent for the enhancement of antibiotic efficacy and persistence. We found that the DE-ZnO composite had enhanced antibiotic activity against fungi (A. fumigatus) and Gram-negative bacteria (E. coli, S. enterica). The DE-ZnO composite provides enhancing large surface areas for enhancement of target pathogen binding affinity, as well as produces active ions including reactive oxygen species and metal ion for breaking the cellular network of fungi and Gram-negative bacteria. Additionally, the toxicity of DE-ZnO with 3 time less amount of dosage is 6 times lower than the commercial SiO2-ZnO. Finally, a synergistic effect of DE-ZnO and existing antifungal agents (Itraconazole and Amphotericin B) showed a better antifungal activity, which could be reduced the side effects due to the antifungal agents overdose, than a single antibiotic agent use. We envision that this DE-ZnO composite can be used to enhance antibiotic activity and its persistence, with less-toxicity, biocompatibility and high stability against fungi and Gram-negative bacteria which could be a valuable candidate in medical science and industrial engineering.

Facile Homobifunctional Imidoester Modification of Advanced Nanomaterials for Enhanced Antibiotic Synergistic Effect.

Resistance to antibiotics because of misuse and overuse is one of the greatest public health challenges worldwide. Despite the introduction of advanced nanotechnology in the production of antibiotics, the choice of appropriate medicines is limited due to side effects such as blood coagulation, toxicity, low efficacy, and low biocompatibility; therefore, novel nanomaterial composites are required to counter these repercussions. We first introduce a facile method for synthesizing a homobifunctional imidoester-coated nanospindle (HINS) zinc oxide composite for enhancement of antibiotic efficacy and reduction of toxicity and blood coagulation. The antibiotic efficacy of the composites is twice that of commercialized zinc nanoparticles; in addition, they have good biocompatibility, have increased surface charge and solubility owing to the covalent acylation groups of HI, and produce a large number of Zn+ ions and defensive reactive oxygen species (ROS) that effectively kill bacteria and fungi. The synergistic effect of a combination therapy with the HINS composite and itraconazole shows more than 90% destruction of fungi in treatments with low dosage with no cytotoxicity or coagulation evident in intravenous administration in in vitro and in vivo experiments. Thus, HINS composites are useful in reducing the effect of misuse and overuse of antibiotics in the medical field.

Multi-Sample Preparation Assay for Isolation of Nucleic Acids Using Bio-Silica with Syringe Filters.

The spin-column system for the isolation of nucleic acids (NAs) from multiple samples presents the inconvenience of repeated experimentation, time-consumption, and the risk of contamination in the process of the spin-column exchange. Herein, we propose a convenient and universal assay that can be used to diagnose multiple pathogens using a multi-sample preparation assay. The multi-sample preparation assay combines a 96-well filter/membrane plate, a bio-micromaterial lattice-like micro amine-functional diatomaceous earth (D-APDMS), and homobifunctional imidoesters (HI) for the processing of pathogen enrichment and extraction for multiple samples simultaneously. The purity and quantity of the extracted NAs from pathogens (E. coli and Brucella) using the proposed assay is superior to that of the commercialized spin-column kit. The assay also does not require the replacement of several collection tubes during the reaction processing. For the multi-sample testing, we used as many as six samples simultaneously with the proposed assay. This assay can simultaneously separate up to 96 NAs from one plate, and the use of multichannel pipettes allows faster and simpler experimentation. Therefore, we believe it is a convenient and easy process, and can be easily integrated with other detection methods for clinical diagnostics.