Exploring the feasibility of using long-term stored newborn dried blood spots to identify metabolic features for congenital heart disease screening.

Congenital heart disease (CHD) represents a significant contributor to both morbidity and mortality in neonates and children. There's currently no analogous dried blood spot (DBS) screening for CHD immediately after birth. This study was set to assess the feasibility of using DBS to identify reliable metabolite biomarkers with clinical relevance, with the aim to screen and classify CHD utilizing the DBS. We assembled a cohort of DBS datasets from the California Department of Public Health (CDPH) Biobank, encompassing both normal controls and three pre-defined CHD categories. A DBS-based quantitative metabolomics method was developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). We conducted a correlation analysis comparing the absolute quantitated metabolite concentration in DBS against the CDPH NBS records to verify the reliability of metabolic profiling. For hydrophilic and hydrophobic metabolites, we executed significant pathway and metabolite analyses respectively. Logistic and LightGBM models were established to aid in CHD discrimination and classification. Consistent and reliable quantification of metabolites were demonstrated in DBS samples stored for up to 15 years. We discerned dysregulated metabolic pathways in CHD patients, including deviations in lipid and energy metabolism, as well as oxidative stress pathways. Furthermore, we identified three metabolites and twelve metabolites as potential biomarkers for CHD assessment and subtypes classifying. This study is the first to confirm the feasibility of validating metabolite profiling results using long-term stored DBS samples. Our findings highlight the potential clinical applications of our DBS-based methods for CHD screening and subtype classification.

Facile Synthesis of Needle-like Copper Sulfide Structures as an Effective Electrochemical Sensor Material for Neurotransmitter Detection in Biological Samples.

Electrochemical sensors, due to their excellent and unique features, are of high interest nowadays for the detection and monitoring of several biological compounds. In such a case, serotonin (SRN), an important neurotransmitter, was herein studied for its detection in biological fluids since its presence is more crucial to be monitored and detected in clinical and medical applications. Several study strategies have been used to determine the chemical and physical properties. The crystalline size of the constructed copper sulfide (Cu2S) material was measured to be 25.92 nm. The Cu2S was fabricated over the working surface and further analyzed for several sensor parameters to be optimized. The charge transfer resistance of the copper sulfide-modified glassy carbon electrode (Cu2S/GCE) was determined to be about 277.0 Ω. With the linear range from about 0.029 µM to 607.6 µM for SRN, the limit of detection (LOD) was calculated as 3.2 nM, with a good sensitivity of 13.23 µA µM-1 cm2. The sensor experienced excellent repeatability, reproducibility, and long-term stability. The fabricated electrode was selective with the presence of different interfering compounds. The real sample analysis, as determined with the regular addition method with human serum and urine samples, revealed a good recovery percentage. Thus, the employed fabricated electrode material will be highly effective in sensing other analytes of choice.

Two dimensional architectures of graphitic carbon nitride with the substitution of heteroatoms for bifunctional electrochemical detection of nilutamide.

The exploration of graphitic carbon nitride (g-C3N4), a two-dimensional (2D) metal-free polymer semiconducting material, is largely discussed due to its large specific surface area, high electrical conductivity, thermal stability, and adaptable electronic structure. The adaption of sulfur (S) and phosphorous (P) atoms into the layers of g-C3N4 increases the electrochemical performance of detecting nilutamide (NT). The aggregation severity can be decreased by integrating S/P into g-C3N4, thereby improving surface area and electrical conductance. The g-C3N4, S/gC3N4, P/g-C3N4, and S/P/g-C3N4 were studied with X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Field emission scanning electron microscopy (FESEM), Transmission electron microscopy (TEM), Fourier transform infrared (FTIR), Ultraviolet visible spectroscopy (UV), Thermogravimetric analysis (TGA), and Brunauer-Emmett-Teller (BET). The well-assigned S/P/g-C3N4 exhibited a good crystalline structure with more active sites for improved electron transfer toward NT detection. Both differential pulse voltammetry (DPV) and amperometry (IT) was studied for NT detection. The electrochemical studies were done with a linear range of 0.019-1.17 µM to 5.36-1891.98 µM in DPV and 0.01 µM-158.3 µM in IT technique. The attained limit of detection in DPV analysis was 3.2 nM and with IT analysis 2.4 nM. The nanocomposite S/P/g-C3N4 shows good selectivity towards NT. The fabricated electrode showed excellent repeatability, reproducibility, and stability, with a significant recovery range in real sample analysis.

Strategic orchestration of MoSe2 microspheres on ß-cd functionalized rGO: A sustainable electrocatalyst for detection of rifampicin in real samples.

The ill effects of prolonged use of rifamycin antibiotics such as rifampicin accentuates its need for detection in the environment as well as in biological fluids. Antibiotics in water and soil are long-lasting, bio-accumulative, and hazardous to aquatic species as well as human health. To address this issue, a sensing platform has been developed using Molybdenum diselenide (MoSe2) embedded on reduced graphene oxide (rGO) functionalized with ß-cyclodextrin (ß-cd) polymer. The formation of hybrid composite was validated with X-ray diffraction analysis (XRD), Raman spectroscopy, fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and field emission scanning electron microscopy (FE-SEM) with EDX analysis. The formation of microspheres were observed with hexagonal crystal system and P63/mmc space group. Furthermore, the composite was employed to fabricate an efficient electrochemical sensor for detecting the widely used antibiotic, rifampicin (RIF). The results reveal excellent activity of the sensor with a limit of detection (LOD) of 28 nM in a linear working range from 0.019 to 374.5 µM. The sensor also exhibited a high sensitivity of 11.64 µA µM-1 cm-2. Additionally, the sensor showed appreciable recovery range when monitored in real-samples such as human serum and urine, and industrial water, and fish samples.

Self-assembled nanorods with reduced graphene oxide as efficient nano-catalyst for dual modality sensing of hazardous phenolic compound.

The modern development in the agricultural production has huge influential factors being highly beneficial and also includes some health hazards. Under the class of chlorophenols, 2,4,6-trichlorophenol is a widely used chemical which remains as a major pollutant in the environment. The detection of 2,4,6-trichlorophenol was initiated as a controlling measure to decrease the seriousness prevailing in the ecosystem. The electrochemical and UV-vis absorption sensing platform are simple and low-cost detection techniques with precise and sensitive analysis. Cadmium tin oxide integrated with the reduced graphene oxide was employed as a nanohybrid for the construction of the working electrode. The structural and morphological analysis confirmed the high degree of crystallinity of the nanocomposite with nanorod formation. The high surface area, with high charge carrier mobility, and increased electrical conductivity of the material boosted the 2,4,6-trichlorophenol detection. The active surface area was calculated to be 0.068 cm-1, 0.089 cm-1, 0.118 cm-1 and 0.146 cm-1 for all the modified electrodes. The resistance of the electrodes was about 91.4 Ω, 72.9 Ω, 48.8 Ω and 41.6 Ω. The linear range of 2,4,6-trichlorophenol was 0.019 µM-0.299 µM and 1.299 µM-1678.97 µM in electrochemical sensing and 10.99 µM-24.84 µM in UV detection. The obtained limit of detection with the formulation 3σ/SD was about 3.05 nM and 80 nM with sensitivity about 14.01 µA µM-1 cm-2. The real sample detection in environmental real samples showed good recovery results. The specific selectivity, good repeatability, reproducibility and stability analysis proves the good sensing parameters. Thus, the fabricated electrode is highly sufficient of sensing 2,4,6-trichlorophenol. These excellent features of the material can be applied for several other applications which will provide good performances.

Investigation of T site variation in spinel aluminates TAl2O4 (T= Mg, Zn & Cu), and formation of electrocatalyst CuAl2O4/carbon for efficient sensing application.

Spinel structured aluminates TAl2O4 (T = Mg, Zn, and Cu) were synthesized by a facile hydrothermal method. The resultant enhancement in the electrochemical behavior was achieved due to the covalent synergism among the elements coexisting together. Structural and morphological characterizations were performed by X-ray diffraction, Fourier transform infrared spectroscopy, Raman spectroscopy, and field emission scanning electron microscopy. MgAl2O4, ZnAl2O4 and CuAl2O4 has displayed same space group Fd3m of Laue class lattice type of the cubic structure as they were synthesized at same temperature (600 °C). CuAl2O4 spinel structure displayed a nanoneedle like structure along with the small sized cylindrical particles alongside to which CuAl2O4 spinel is combined with activated carbon (CuAl/C) and was applied to develop a facile sensor for the electrochemical detection of Acetaminophen (ACAP) using cyclic voltammetry (CV) and differential pulse voltammetry (DPV), which exhibited maximum conductivity, and a substantial electroactive surface area. Finally, the defect-rich composite, CuAl/C, showed excellent sensor performance towards DPV with 21.5 nM limit of detection (LOD) in a wide linear working range of 0.199 µM-165.88 µM ACAP concentration, with a high sensitivity of 19.1221 µA µM-1cm2. Additionally, the sensor showed excellent recovery results in real-time analysis for environmental aquatic samples like industrial wastewater and Tuna Fish.

Serological Phenotyping Analysis Uncovers a Unique Metabolomic Pattern Associated With Early Onset of Type 2 Diabetes Mellitus.

Background: Type 2 diabetes mellitus (T2DM) is a multifaceted disorder affecting epidemic proportion at global scope. Defective insulin secretion by pancreatic ß-cells and the inability of insulin-sensitive tissues to respond effectively to insulin are the underlying biology of T2DM. However, circulating biomarkers indicative of early diabetic onset at the asymptomatic stage have not been well described. We hypothesized that global and targeted mass spectrometry (MS) based metabolomic discovery can identify novel serological metabolic biomarkers specifically associated with T2DM. We further hypothesized that these markers can have a unique pattern associated with latent or early asymptomatic stage, promising an effective liquid biopsy approach for population T2DM risk stratification and screening. Methods: Four independent cohorts were assembled for the study. The T2DM cohort included sera from 25 patients with T2DM and 25 healthy individuals for the biomarker discovery and sera from 15 patients with T2DM and 15 healthy controls for the testing. The Pre-T2DM cohort included sera from 76 with prediabetes and 62 healthy controls for the model training and sera from 35 patients with prediabetes and 27 healthy controls for the model testing. Both global and targeted (amino acid, acylcarnitine, and fatty acid) approaches were used to deep phenotype the serological metabolome by high performance liquid chromatography-high resolution mass spectrometry. Different machine learning approaches (Random Forest, XGBoost, and ElasticNet) were applied to model the unique T2DM/Pre-T2DM metabolic patterns and contrasted with their effectiness to differentiate T2DM/Pre-T2DM from controls. Results: The univariate analysis identified unique panel of metabolites (n = 22) significantly associated with T2DM. Global metabolomics and subsequent structure determination led to the identification of 8 T2DM biomarkers while targeted LCMS profiling discovered 14 T2DM biomarkers. Our panel can effectively differentiate T2DM (ROC AUC = 1.00) or Pre-T2DM (ROC AUC = 0.84) from the controls in the respective testing cohort. Conclusion: Our serological metabolite panel can be utilized to identifiy asymptomatic population at risk of T2DM, which may provide utility in identifying population at risk at an early stage of diabetic development to allow for clinical intervention. This early detection would guide ehanced levels of care and accelerate development of clinical strategies to prevent T2DM.

Preparation of substrates for microarray protein chips with different ending functional groups.

Protein microarray chips are composed of three components, these are pre-treatment substrates, surface chemical modification, and immobilizing protein on substrate surfaces. In this study, self-assembly monolayers are used for surface chemical modification. Using this method, silanization on a glass and silicon chip is achieved, forming the terminal group substrates. Modification of the substrate surface to provide COOH and NH2 terminal functional groups provides a mechanism to proteins to immobilize on the substrate surface. To observe immobilized proteins on the substrate surface, they are first labeled with Cy5 fluorescent dye before analysis using a GenePix 4000B Microarray Scanner. The scanner induces fluorescence in the labelling dye and the resulting light is analyzed to provide information concerning both the quantity of immobilized protein, and the orientation of attachment. The antigen of the HSV-1 virus, a common human virus, was used in this study, performing an antigen-antibody analysis to determine the efficacy of the method under test for clinical diagnosis.

Vanadium selenide decorated reduced graphene oxide nanocomposite: A co-active catalyst for the detection of 2,4,6 - Trichlorophenol.

Transition metal chalcogenides (TMCs) have great potential in diverse electrochemical technologies owing to their unique characteristics. In the present work, we portray the design and synthesis of Vanadium selenide (V2Se9)/reduced graphene oxide (rGO) forming a two-dimensional (2D) hybrid nanocomposite via a simple hydrothermal method. The successfully synthesized nanocomposite underwent in-depth surface and morphological characterizations by XRD, Raman spectroscopy, XPS, TEM, STEM and its potential as an electro catalyst was investigated by using glassy carbon electrode (GCE) for the detection of 2,4,6-trichlorophenol (TCP). The structural features favored a high charge transfer ratio, high surface area as well as excellent conductivity and catalytic activity. The V2Se9/rGO/GCE modified electrode showed a low charge transfer resistance (Rct) of 54.057 Ω cm2, a decent detection limit (LOD) of 35.07 nM and a very high sensitivity of 22 µA µM-1 cm-2 in a working range of 0.001 µM-1150 µM. This is due to the active proton interaction, surface enhancement, and positive synergistic effect between rGO and V2Se9. The proposed sensor has good detection potential in agricultural soil, river water, fish, and beverage samples like wine and apple juice. The obtained results from our investigation would elucidate the application of the catalyst in electrochemical sensors.

Facile Synthesis of Ag/AgVO3/N-rGO Hybrid Nanocomposites for Electrochemical Detection of Levofloxacin for Complex Biological Samples Using Screen-Printed Carbon Paste Electrodes.

Silver vanadate nanorods (ß-AgVO3) with silver nanoparticles (Ag-NPs) decorated on the surface of the rods were synthesized by using simple hydrothermal technique and later anchored onto nitrogen-doped reduced graphene oxide (N-rGO) to make a novel nanocomposite. Experimental analyses were carried out to identify the electronic configuration by X-ray diffraction analysis, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy analysis, which revealed monoclinic patterns of the C12/m1 space group with Wulff construction forming beta silver vanadate (ß-AgVO3) crystals with optical density and phase transformations. Ag nucleation showed consistent results with metallic formation and electronic changes occurring in [AgO5] and [AgO3] clusters. Transmission electron microscopy and field-emission scanning electron microscopy with elemental mapping and EDX analysis of the morphology reveals the nanorod structure for ß-AgVO3 with AgNPs on the surface and sheets for N-rGO. Additionally, a novel electrochemical sensor is constructed by using Ag/AgVO3/N-rGO on screen-printed carbon paste electrodes for the detection of antiviral drug levofloxacin (LEV) which is used as a primary antibiotic in controlling COVID-19. Using differential pulse voltammetry, LEV is determined with a low detection limit of 0.00792 nm for a linear range of 0.09-671 µM with an ultrahigh sensitivity of 152.19 µA µM-1 cm-2. Furthermore, modified electrode performance is tested by real-time monitoring using biological and river samples.

High-throughput quantitation of serological ceramides/dihydroceramides by LC/MS/MS: Pregnancy baseline biomarkers and potential metabolic messengers.

Ceramides and dihydroceramides are sphingolipids that present in abundance at the cellular membrane of eukaryotes. Although their metabolic dysregulation has been implicated in many diseases, our knowledge about circulating ceramide changes during the pregnancy remains limited. In this study, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of 16 ceramides and 10 dihydroceramides in human serum within 5 min. by using stable isotope-labeled ceramides as internal standards. This method employs a protein precipitation method for high throughput sample preparation, reverse phase isocratic elusion for chromatographic separation, and Multiple Reaction Monitoring for mass spectrometric detection. To qualify for clinical applications, our assay has been validated against the FDA guidelines for Lower Limit of Quantitation (1 nM), linearity (R2>0.99), precision (imprecision<15 %), accuracy (inaccuracy<15 %), extraction recovery (>90 %), stability (>85 %), and carryover (<0.01 %). With enhanced sensitivity and specificity from this method, we have, for the first time, determined the serological levels of ceramides and dihydroceramides to reveal unique temporal gestational patterns. Our approach could have value in providing insights into disorders of pregnancy.

Rational design and preparation of copper vanadate anchored on sulfur doped reduced graphene oxide nanocomposite for electrochemical sensing of antiandrogen drug nilutamide using flexible electrodes.

Copper vanadate nanoparticles (Cu2V2O7) are synthesized by using a simple hydrothermal method and later anchored with sulfur-doped reduced graphene oxide (S-rGO) by using ultrasonication to form a hybrid nanocomposite. The synthesized composite underwent characterizations like X-ray diffraction analysis (XRD), Raman spectroscopy, Fourier transform infrared spectroscopy (FT-IR), Dynamic ray scattering-Ultra violet-visible spectroscopy (DRS-UV-visible) and X-ray photoelectron spectroscopically revealed the triclinic pattern of the P 1̅ space group of α-Cu2V2O7 and the reduced oxygen deficiency state of metal centers (Cu+ or V4+) resulting with oxides of mixed-valence oxidative states and forming of Cu-O bond. Morphological analysis was carried out by using transmission electron microscopy (TEM) and Field emission scanning electron microscopy (FE-SEM) with elemental mapping and EDX analysis. Furthermore, a novel electrochemical sensor is prepared by using the hybrid sCu2V2O7/S-rGO nanocomposite on to a disposable screen-printed carbon paste electrode (SPCE) for electrochemical sensing of antiandrogen drug nilutamide (NLT). This report reveals excellent activity in determining NLT with a low detection limit of 0.00459 nM for the linear range of 0.001-15 µM with high sensitivity of 26.2605 µA µM-1 cm-2. Further, electrode performance showed appreciable performance in real-time monitoring of biological samples like human blood serum, urine samples.

Synthesis and cytotoxic analysis of thiolated xylose derivatives decorated on gold nanoparticles.

The rapid development of metal nanoparticles capped by an organic monolayer offers the possibility to create a whole new variety of products with novel characteristic, functions and applications. Among these, nanoparticles covered with carbohydrates (glyconanoparticles) constitute a good bio-mimetic model of carbohydrate presentation at the cell surface and are currently centered on many glycobiological and biomedical applications. In this study, a series of novel D-xylose gold nanoparticles (AuNPs) with linkages of alkyl or polyethylene glycol have been synthesized via D-xylosethiols, forming self-assembled monolayers on gold nanoparticles. The nano-gold solution, two carbohydrate derivatives and modified nano-gold solution were tested for cytotoxicity to check the biocompatibility. The MTT assay on NIH 3T3 cell lines confirmed that all the test materials showed no toxicity with the more than 90 % of cell viability in both low concentration (1 µM) and high concentration (100 µM), compared with the control.

Kinetics of SARS-CoV-2 positivity of infected and recovered patients from a single center.

Recurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive detection in infected but recovered individuals has been reported. Patients who have recovered from coronavirus disease 2019 (COVID-19) could profoundly impact the health care system. We sought to define the kinetics and relevance of PCR-positive recurrence during recovery from acute COVID-19 to better understand risks for prolonged infectivity and reinfection. A series of 414 patients with confirmed SARS-Cov-2 infection, at The Second Affiliated Hospital of Southern University of Science and Technology in Shenzhen, China from January 11 to April 23, 2020. Statistical analyses were performed of the clinical, laboratory, radiologic image, medical treatment, and clinical course of admission/quarantine/readmission data, and a recurrence predictive algorithm was developed. 16.7% recovered patients with PCR positive recurring one to three times, despite being in strict quarantine. Younger patients with mild pulmonary respiratory syndrome had higher risk of PCR positivity recurrence. The recurrence prediction model had an area under the ROC curve of 0.786. This case series provides characteristics of patients with recurrent SARS-CoV-2 positivity. Use of a prediction algorithm may identify patients at high risk of recurrent SARS-CoV-2 positivity and help to establish protocols for health policy.

Influence of temperature variation on spinel-structure MgFe2O4 anchored on reduced graphene oxide for electrochemical detection of 4-cyanophenol.

The effect of annealing temperature variance on magnesium ferrites (MgFe2O4) later anchored on reduced graphene oxide (rGO) forming hybrid nanocomposite is demonstrated and its electrochemical performance investigated by using a screen-printed carbon paste electrode (SPCE) for detection of the environmental hazardous phenolic compound 4-cyanophenol (4-CY). The MgFe2O4 (MFO-600 °C) displayed an enhanced charge transfer ratio with high conductivity and electrocatalytic activity. To confirm the structural and morphological parameters of the rGO-MFO-2 hybrid micro/nanocomposite, X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron microscopy (XPS), and field-emission electron microscopy (FE-EM) with EDX mapping have been utilized. The rGO/MFO-2/SPCE electrode displayed high catalytic performance in detecting 4-CY with good sensitivity of 6.836 µA µM-1 cm-2 in a working range 0.001 to 700 µM with a limit of detection of 0.0012 µM by using differential pulse voltammetry (DPV). This is achieved for the active interaction between rGO and MFO-2 active surface site areas resulting in good electrochemical activity and high electron transfer rate. Moreover, 4-CY detection has been performed in the presence of various interferents and through real-time analysis in samples like tap water, industrial river water, and fish which resulted in admirable recovery.Graphical abstract.

Nano assembly of NiFe spheres anchored on f-MWCNT for electrocatalytic reduction and sensing of nitrofurantoin in biological samples.

The current study reports a facile simple, low-cost electrochemical sensor in the detection of nitrofurantoin (NFT) by using NiFe/f-MWCNT hybrid composite as a promising electrocatalyst. NFT is an antibiotic drug that is extensively using in pharmaceuticals and also in animal food production which causes a severe threat for both human and animal environments. Extending the residues of NFT are left into rivers, soils, lakes, and groundwaters either found or discharged leading health issues. To this NiFe/f-MWCNT composite was synthesized using a hydrothermal mechanism and then ultrasonicated to form a hybrid composite for catalytic evaluation and electrochemical detection of NFT for the very first time. Furthermore, the physicochemical properties of NiFe nanospheres conjugated on f-MWCNT are scrutinized using various analytical and spectroscopical techniques. Resulting transmission electron microscopy (TEM) displays a chain like NiFe nanospheres anchored on f-MWCNT with a well-defined spherical shape, without any comprehensive agglomeration. The NiFe/f-MWCNT screen printed carbon paste electrode (SPCE) displayed an excellent electrocatalytic activity for NFT with a LOD of 0.03 µM and a sensitivity of 11.45 µA µM-1 cm-2. establishing a new selectivity and with the existence of co-interfering compounds. To enhance the practical abilities analysis were performed in Human serum and urine samples which resulted in satisfactory recoveries with high precision and linear accuracy illustrated in Scheme 1.

Ultrasonic synthesis of CuO nanoflakes: A robust electrochemical scaffold for the sensitive detection of phenolic hazard in water and pharmaceutical samples.

Hydroquinone (HQ), a phenolic compound is expansively used in many industrial applications and due to the utilization of HQ, water pollution tragedies frequently found by the improper handling and accidental outflows. When HQ is adsorbed directly through the skin that create toxic effects to human by affecting kidney, liver, lungs, and urinary tract and hence, a highly selective and sensitive technique is required for its quantification. Herein, we have developed the ultrasonic synthesis of copper oxide nanoflakes (CuO-NFs) using ultrasonic bath (20 kHz, 100 W) and successfully employed for the sensitive detection of the environmental hazardous pollutant HQ. The formed CuO-NFs were confirmed by X-ray diffraction, field emission scanning electron microscopy (FE-SEM), FT-IR spectroscopy and UV-visible spectroscopy and fabricated with the screen-printed carbon electrode (SPCE). The SEM images exhibited the uniform CuO-NFs with an average width of 85 nm. The linker-free CuO-NFs fabricated electrode showed the appropriate wide range of concentrations from 0.1 to 1400 µM and the limit of detection was found to be 10.4 nM towards HQ. The fabricated sensor having long term stability and sensitivity was successfully applied for the environmental and commercial real sample analysis and exhibited good recovery percentage, implying that the SPCE/CuO-NFs is an economically viable and benign robust scaffold for the determination of HQ.

Analyzing polymeric matrix for fabrication of a biodegradable microneedle array to enhance transdermal delivery.

Traditional drug delivery systems, using invasive, transdermal, and oral routes, are limited by various factors, such as the digestive system environment, skin protection, and sensory nerve stimulation. To improve the drug delivery system, we fabricated a polysaccharide-based, dissolvable microneedle-based array, which combines the advantages of both invasive and transdermal delivery systems, and promises to be an innovative solution for minimally invasive drug delivery. In this study, we designed a reusable aluminum mold that greatly improved the efficiency and convenience of microneedle fabrication. Physical characterization of the polysaccharides, individual or mixed at different ratios, was performed to identify a suitable molecule to fabricate the dissolvable microneedle. We used a vacuum deposition-based micro-molding method at low temperature to fabricate the model. Using a series of checkpoints from material into product, a systematic feedback mechanism was built into the "all-in-one" fabrication step, which helped to improve production yields. The physical properties of the fabricated microneedle were assessed. The cytotoxicity analysis and animal testing of the microneedle demonstrated the safety and compatibility of the microneedle, and the successful penetration and effective release of a model protein.

Exchange of active site residues alters substrate specificity in extremely thermostable ß-glycosidase from Thermococcus kodakarensis KOD1.

ß-Glycosidase from Thermococcus kodakarensis KOD1 is a hyperthermophilic enzyme with ß-glucosidase, ß-mannosidase, ß-fucosidase and ß-galactosidase activities. Sequence alignment with other ß-glycosidases from hyperthermophilic archaea showed two unique active site residues, Gln77 and Asp206. These residues were represented by Arg and Asp in all other hyperthermophilic ß-glycosidases. The two active site residues were mutated to Q77R, D206N and D206Q, to study the role of these unique active site residues in catalytic activity and to alter the substrate specificity to enhance its ß-glucosidase activity. The secondary structure analysis of all the mutants showed no change in their structure and exhibited in similar conformation like wild-type as they all existed in dimer form in an SDS-PAGE under non-reducing conditions. Q77R and D206Q affected the catalytic activity of the enzyme whereas the D206N altered the catalytic turn-over rate for glucosidase and mannosidase activities with fucosidase activity remain unchanged. Gln77 is reported to interact with catalytic nucleophile and Asp206 with axial C2-hydroxyl group of substrates. Q77R might have made some changes in three dimensional structure due to its electrostatic effect and lost its catalytic activity. The extended side chains of D206Q is predicted to affect the substrate binding during catalysis. The high-catalytic turn-over rate by D206N for ß-glucosidase activity makes it a useful enzyme in cellulose degradation at high temperatures.