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.

Multicenter Testing of a Simple Molecular Diagnostic System for the Diagnosis of Mycobacterium Tuberculosis.

Mycobacterium tuberculosis (MTB) is a communicable disease and still remains a threat to common health. Thus, early diagnosis and treatment are required to prevent the spread of infection. Despite the recent advances in molecular diagnostic systems, the commonly used MTB diagnostic tools are laboratory-based assays, such as mycobacterial culture, MTB PCR, and Xpert MTB/RIF. To address this limitation, point-of-care testing (POCT)-based molecular diagnostic technologies capable of sensitive and accurate detection even in environments with limited sources are needed. In this study, we propose simple tuberculosis (TB) molecular diagnostic assay by combining sample preparation and DNA-detection steps. The sample preparation is performed using a syringe filter with amine-functionalized diatomaceous earth and homobifunctional imidoester. Subsequently, the target DNA is detected by quantitative PCR (polymerase chain reaction). The results can be obtained within 2 h from samples with large volumes, without any additional instruments. The limit of detection of this system is 10 times higher than those of conventional PCR assays. We validated the clinical utility of the proposed method in 88 sputum samples obtained from four hospitals in the Republic of Korea. Overall, the sensitivity of this system was superior to those of other assays. Therefore, the proposed system can be useful for MTB diagnosis in limited-resource settings.

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.

Self-directed molecular diagnostics (SdMDx) system for COVID-19 via one-pot processing.

Rapid, sensitive, and specific detection of the severe acute respiratory syndrome coronavirus (SARS-CoV)- 2 during early infection is pivotal in controlling the spread and pathological progression of Coronavirus Disease 2019 (COVID-19). Thus, highly accurate, affordable, and scalable point-of-care (POC) diagnostic technologies are necessary. Herein, we developed a rapid and efficient self-directed molecular diagnostic (SdMDx) system for SARS-CoV-2. This system combines the sample preparation step, including virus enrichment and extraction processes, which involve dimethyl suberimidate dihydrochloride and diatomaceous earth functionalized with 3-aminopropyl(diethoxy)methylsilane, and the detection step using loop-mediated isothermal amplification-lateral flow assay (LAMP-LFA). Using the SdMDx system, SARS-CoV-2 could be detected within 47 min by hand without the need for any larger instruments. The SdMDx system enabled detection as low as 0.05 PFU in the culture fluid of SARS-CoV-2-infected VeroE6 cells. We validated the accuracy of the SdMDx system on 38 clinical nasopharyngeal specimens. The clinical utility of the SdMDx system for targeting the S gene of SARS-CoV-2 showed 94.4% sensitivity and 100% specificity. This system is more sensitive than antigen and antibody assays, and it minimizes the use of complicated processes and reduces contamination risks. Accordingly, we demonstrated that the SdMDx system enables a rapid, accurate, simple, efficient, and inexpensive detection of SARS-CoV-2 at home, in emergency facilities, and in low-resource sites as a pre-screening platform and POC testing through self-operation and self-diagnosis.

Hot-Spot-Specific Probe (HSSP) for Rapid and Accurate Detection of KRAS Mutations in Colorectal Cancer.

Detection of oncogene mutations has significance for early diagnosis, customized treatment, treatment progression, and drug resistance monitoring. Here, we introduce a rapid, sensitive, and specific mutation detection assay based on the hot-spot-specific probe (HSSP), with improved clinical utility compared to conventional technologies. We designed HSSP to recognize KRAS mutations in the DNA of colorectal cancer tissues (HSSP-G12D (GGT→GAT) and HSSP-G13D (GGC→GAC)) by integration with real-time PCR. During the PCR analysis, HSSP attaches to the target mutation sequence for interference with the amplification. Then, we determine the mutation detection efficiency by calculating the difference in the cycle threshold (Ct) values between HSSP-G12D and HSSP-G13D. The limit of detection to detect KRAS mutations (G12D and G13D) was 5-10% of the mutant allele in wild-type populations. This is superior to the conventional methods (≥30% mutant allele). In addition, this technology takes a short time (less than 1.5 h), and the cost of one sample is as low as USD 2. We verified clinical utility using 69 tissue samples from colorectal cancer patients. The clinical sensitivity and specificity of the HSSP assay were higher (84% for G12D and 92% for G13D) compared to the direct sequencing assay (80%). Therefore, HSSP, in combination with real-time PCR, provides a rapid, highly sensitive, specific, and low-cost assay for detecting cancer-related mutations. Compared to the gold standard methods such as NGS, this technique shows the possibility of the field application of rapid mutation detection and may be useful in a variety of applications, such as customized treatment and cancer monitoring.

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.

Utility of plasma cell-free DNA detection using homobifunctional imidoesters using a microfluidic system for diagnosing active tuberculosis.

BACKGROUND: It is difficult to diagnose tuberculosis (TB), particularly sputum-scarce pulmonary TB and extrapulmonary TB, using conventional diagnostic tests. Since these cases require additional invasive procedures to obtain appropriate specimens, new non-invasive diagnostic tests are needed. Plasma cell-free DNA (cfDNA) detection has gained interest as a novel diagnostic test for TB as it is convenient and less invasive. Therefore, we investigated the performance of enriched cfDNA for diagnosing pulmonary TB and extrapulmonary TB. METHODS: All patients suspected to have TB, who consented to the use of blood for detecting cfDNA, were prospectively enrolled from January 2019 to June 2020. We categorised the patients as confirmed, probable, possible TB, and not-TB. We compared the performance of cfDNA with those of conventional diagnostic tests. RESULTS: Among the 96 patients enrolled, 40 (41.7%) had TB, including 34 with confirmed TB and six probable TB, and 41 (42.7%) did not have TB. Acid-fast bacilli microscopy, Xpert MTB/RIF, and mycobacterial culture results were positive in 12 (31.6%), 22 (61.1%), and 25 (65.8%) patients, respectively. The sensitivity and specificity of cfDNA were 80.0% and 78.1%, respectively. While the sensitivity and specificity of cfDNA were similar to those of interferon-gamma releasing assay (IGRA) (sensitivity 80.6% and specificity 71.4%), the combined sensitivity and specificity of the two assays were 94.4% and 64.3%, respectively, which can be used to rule out TB. CONCLUSIONS: Plasma cfDNA assay seems to be a useful adjunct to the current tests for diagnosing TB, especially when used in combination with IGRA for ruling out TB.AbbreviationsTBtuberculosiscfDNAcell-free DNAPCRpolymerase chain reactionAFBacid-fast bacilliIGRAinterferon-gamma releasing assayCTcomputed tomographyHIVhuman immunodeficiency virus.

Molecular diagnosis of Coxiella burnetii in culture negative endocarditis and vascular infection in South Korea.

BACKGROUND: Q fever endocarditis is a major cause of culture-negative endocarditis. The role of Coxellia burnetii is underestimated because it is difficult to diagnose. We investigated the significance of C. burnetii as the cause of culture-negative endocarditis and vascular infection by examining blood and tissue specimens using serological testing and polymerase chain reaction (PCR). METHODS: All patients with infective endocarditis or large vessel vasculitis were prospectively enrolled at a tertiary-care hospital from May 2016 through September 2020. Q fever endocarditis and vascular infection were diagnosed based on: (1) positive PCR for a cardiac valve or vascular tissue, (2) positive PCR for blood or phase I immunoglobulin G (IgG) ≥ 6400, or (3) phase I IgG ≥ 800 and < 6400 with morphologic abnormality. PCR targeted C. burnetii transposase gene insertion element IS1111a. RESULTS: Of the 163 patients, 40 (25%) had culture-negative endocarditis (n = 35) or vascular infection (n = 5). Of the 40 patients, 24 (60%) were enrolled. Eight (33%) were diagnosed with Q fever endocarditis or vascular infection. Of these 8 patients, 6 had suspected acute Q fever endocarditis or vascular infection with negative phase I IgG. Six patients were not treated for C. burnetii, 4 were stable after surgery. One patient died due to surgical site infection after 5 months post-operatively and one died due to worsening underlying disease. CONCLUSIONS: Approximately one-third of patients with culture-negative endocarditis and vascular infection was diagnosed as Q fever. Q fever endocarditis and vascular infection may be underestimated in routine clinical practice in South Korea.KEY MESSAGEQ fever endocarditis and vascular infection may be underestimated in routine clinical practice, thus, try to find evidence of C. burnetti infection in suspected patients by all available diagnostic tests including PCR.

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.

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.

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.

Bis(sulfosuccinimidyl)suberate-Based Helix-Shaped Microchannels as Enhancers of Biomolecule Isolation from Liquid Biopsies.

Most studies of ultrasensitive diagnosis of biomolecules from liquid specimens are limited by problems during sample preparation steps, including enrichment and isolation of biomolecules. Here we report a novel platform combining bis(sulfosuccinimidyl)suberate (BS3) and helix-shaped microchannels (BSH) to change the sample preparation paradigm. This BSH system is composed of BS3 for pathogen enrichment and nucleic acid isolation by electrostatic and covalent interaction, and helix-shaped microchannels to minimize sample loss and remove bubbles in large liquid specimens without pH change. The system detected Mycobacterium tuberculosis following enrichment and isolation of 10 mL of liquefied sputum from 11 patients with tuberculosis. Moreover, the system identified KRAS mutations following cell-free DNA isolation of blood plasma from 10 patients with colorectal cancer. This system allows ultrasensitive diagnosis in various disease applications with large volumes of liquid samples.

The reliability and validity of gait speed with different walking pace and distances against general health, physical function, and chronic disease in aged adults.

PURPOSE: Gait speed is an important objective values associated with several health-related outcomes including functional mobility in aging people. However, walking test methodologies and descriptions are not standardized considering specific aims of research. This study examine the reliability and validity of gait speed measured at various distances and paces in elderly Koreans. METHODS: Fifty-four female participants ≥70 years of age were recruited from a local retirement community. Gait speed was assessed at 4, 6 and 10 meters, and at usual- and fast-pace walking mode. The short physical performance battery (SPPB) that estimates senior fitness includes three tests of lower-body function. Data concerning for the chronic conditions and self-perceived health of the participants was collected using questionnaires. Concurrent validity of gait speed using the aforementioned test protocols was determined by calculating the Pearson correlation coefficients. RESULTS: Significant positive correlations were evident between skeletal muscle mass and maximal pace walking regardless of distance (r=.301~.308; p<.05), but not with body fat. All gait tests significantly positively correlated with self-rated health (normal pace r=.328~.346, p<.05; maximal pace r=.427~.472, p<.001) and depression (normal pace r=.279~.430, p<.05; maximal pace r=.413~.456, p<.001). CONCLUSION: Walking test at the normal pace appears suitable for estimating physical function and deterioration due to chronic disease. Walking test at a maximum pace might be useful for estimating subjective general health and skeletal muscle mass.