Molecular insights into the crystalline nanocellulose and human lysozyme interactions: An experimental and theoretical research.

In the present research, we performed a combination of detailed computational and spectroscopic methods to determine the effect of crystalline nanocellulose (CNC) on the structure and dynamics of human lysozyme (hLyz). Fluorescence spectroscopy revealed static quenching as the major mechanism in forming a stable CNC-hLyz complex, and the binding was energetically favorable. The obtained values of the thermodynamic parameters (∆G, ∆H, and ∆S) proposed that the complex formation between the enzyme and cellulose nanocrystals is driven by electrostatic interactions, which were also confirmed by molecular dynamics (MD) simulation. Additionally, the MD simulation analysis displays that the enzyme's structural elements and tertiary structure were primarily maintained, and only loops regions were affected in the presence of cellulose nanocrystals. At the same time, circular dichroism (CD) outcomes highlighted that higher cellulose nanocrystals concentration caused a reduction in the secondary structure of hLyz. Our observations proved that low cellulose nanocrystals concentrations have no considerable effect on the human lysozyme structure. The current research results provide a valuable opportunity to elucidate the molecular interactions between protein and nanocelluloses, guiding further investigations of CNC-based material for biomedical, pharmaceutical, and food industry applications.

Buried-Gate MWCNT FET-Based Nanobiosensing Device for Real-Time Detection of CRP.

C-reactive protein (CRP), an acute-phase protein synthesized in the liver in response to inflammation, is one of the biomarkers used for the detection of several diseases. Sepsis and cardiovascular diseases are two of the most important diseases for which detection of CRP at very early stages in the clinical range can help avert serious consequences. Here, a CNT-based nanobiosensing system, which is portable and reproducible, is used for label-free, online detection of CRP. The system consists of an aptameric CNT-based field-effect transistor benefiting from a buried gate geometry with Al2O3 as a high dielectric layer and can reflect the pro-cytokine concentration. Test results show that the device responds to CRP changes within 8 min, with a limit of detection as low as 150 pM (0.017 mg L-1). The device was found to have a linear behavior in the range of 0.43-42.86 nM (0.05-5 mg L-1). The selectivity of the device was tested with TNF-α, IL-6, and BSA, to which the nanosensing system showed no significant response compared with CRP. The device showed good stability for 14 days and was completely reproducible during this period. These findings indicate that the proposed portable system is a potential candidate for CRP measurements in the clinical range.

A lectin-coupled porous silicon-based biosensor: label-free optical detection of bacteria in a real-time mode.

Accuracy and speed of detection, along with technical and instrumental simplicity, are indispensable for the bacterial detection methods. Porous silicon (PSi) has unique optical and chemical properties which makes it a good candidate for biosensing applications. On the other hand, lectins have specific carbohydrate-binding properties and are inexpensive compared to popular antibodies. We propose a lectin-conjugated PSi-based biosensor for label-free and real-time detection of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) by reflectometric interference Fourier transform spectroscopy (RIFTS). We modified meso-PSiO2 (10-40 nm pore diameter) with three lectins of ConA (Concanavalin A), WGA (Wheat Germ Agglutinin), and UEA (Ulex europaeus agglutinin) with various carbohydrate specificities, as bioreceptor. The results showed that ConA and WGA have the highest binding affinity for E. coli and S. aureus respectively and hence can effectively detect them. This was confirmed by 6.8% and 7.8% decrease in peak amplitude of fast Fourier transform (FFT) spectra (at 105 cells mL-1 concentration). A limit of detection (LOD) of about 103 cells mL-1 and a linear response range of 103 to 105 cells mL-1 were observed for both ConA-E. coli and WGA-S. aureus interaction platforms that are comparable to the other reports in the literature. Dissimilar response patterns among lectins can be attributed to the different bacterial cell wall structures. Further assessments were carried out by applying the biosensor for the detection of Klebsiella aerogenes and Bacillus subtilis bacteria. The overall obtained results reinforced the conjecture that the WGA and ConA have a stronger interaction with Gram-positive and Gram-negative bacteria, respectively. Therefore, it seems that specific lectins can be suggested for bacterial Gram-typing or even serotyping. These observations were confirmed by the principal component analysis (PCA) model.

Catalytic parameters and thermal stability of chondroitinase ABCI on red porous silicon nanoparticles.

The bacterial enzyme chondroitinase ABC, which digests extracellular chondroitin sulfate proteoglycans, has been shown to enhance axonal regeneration. However, the utilization of this enzyme as therapeutics is notably restricted due to its thermal instability. Therefore, red luminescent porous silicon that hold promise for potential applications in biological/medical imaging was used as a carrying matrix for chondroitinase with the aim of enhancing its stability. Porous Si nanoparticles were prepared by electrochemical etching of silicon wafers in ethanolic HF solution. The size of nanoparticles (210 nm) and the mean pore diameter (8 -20 nm) were determined using dynamic light scattering and scanning electron microscopy. Purified chondroitinase was then incorporated into the silicon pores. Results revealed similar Km and lower Vmax value for the immobilized enzyme when compared with the free enzyme. The immobilized chondroitinase exhibited about a 4 fold increase in stability at 37 °C after 50 min. It is likely possible that, the enzyme was protected inside the pores resulted in higher stability. Moreover, porous silicon was seen to be capable of holding the chondroitinase for repeated cyclic tests for three times. The cell viability assay exhibited no significant cytotoxicity for Psi-chondroitinase up to 24 h.

Controlled Tyrosine Kinase Inhibitor Delivery to Liver Cancer Cells by Gate-Capped Mesoporous Silica Nanoparticles.

Hepatocellular carcinoma is the most common type of primary malignancy in the liver and one of the most common types of cancer worldwide. Its readily increasing mortality rate highlights the urgent need for the development of efficient therapeutic strategies. Tyrosine kinase inhibitors (TKIs) such as sorafenib and sunitinib are used as efficient angiogenesis inhibitors for this purpose. However, despite their pharmacological effects, their transfer into clinical practice is characterized by their poor aqueous solubility and accumulation in off-target tissues, resulting in unfavorable side effects. Here, we report a nanocomposite made of amine-functionalized mesoporous silica nanocomposites (MSNs) that are surface-coated with cerium oxide nanoparticles (CNPs) for the controlled delivery and release of TKIs. Amine-functionalized MSNs were prepared using a sol-gel method and loaded with TKIs. To trap drug molecules into the mesoporous structure, CNPs were covalently conjugated to the surface of MSNs. The synthesis and functionalization steps were controlled using different characterization methods, confirming the desired morphology and structure, the identity of functional groups on the surface, successful coating, and appropriate loading efficiency. Under physiological conditions, CNP-capped MSNs demonstrated a sustained drug release over time as a result of CNPs' gatekeeping effect on the payloads. Strong cellular interactions with different liver cancer cells and enhanced cellular uptake were also observed in vitro for the gate-capped MSNs. Internalization of nanocomposites induced cell death via the production of reactive oxygen species, and subsequent activation of apoptosis pathways. This study demonstrates that gate-capped MSNs are promising chemotherapeutic vehicles characterized by a sustained drug release profile and high cellular internalization.

Label-free discrimination of single nucleotide changes in DNA by reflectometric interference Fourier transform spectroscopy.

Phenotypic variation - such as disease susceptibility and differential drug response - has a strong genetic component. Substantial effort has therefore been made to identify causal genomic variants explaining such variation among humans. Point mutations (PMs), which are single nucleotide changes in the genome, have been identified to be the most abundant form of causal genomic variants, making them useful, reliable diagnostic markers. Methods developed to genotype PMs have moved towards solid-phase assays, which not only show greater sensitivity and specificity, but also enable scalability and faster processing time. Most current assays are, however, based on fluorescent probes, which makes them relatively expensive. To develop a more cost-effective label-free genotyping method, we used a porous silicon (PSi) base as an efficient support for DNA biosensing and coupled it with reflectometric interference Fourier transform spectroscopy (RIFTS). To assess the versatility of this approach, we tested both a single nucleotide substitution in VKORC1 (-1639G > A; rs9923231) and a single nucleotide insertion in BRCA1 (5382insC; rs80357906). We demonstrate that the PSi-RIFTS method can efficiently detect both PM types with high sensitivity where hybridization of complementary DNA can be quantifiably differentiated from mismatch and non-complementary hybridization events. In addition, we show that the PSi base with immobilized DNA not only can be re-used to type further samples, but it also remains stable for 14 days, suggesting its potential for high-throughput applications.

Optimization of Porous Silicon Conditions for DNA-based Biosensing via Reflectometric Interference Spectroscopy.

OBJECTIVE: Substantial effort has been put into designing DNA-based biosensors, which are commonly used to detect presence of known sequences including the quantification of gene expression. Porous silicon (PSi), as a nanostructured base, has been commonly used in the fabrication of optimally transducing biosensors. Given that the function of any PSi-based biosensor is highly dependent on its nanomorphology, we systematically optimized a PSi biosensor based on reflectometric interference spectroscopy (RIS) detecting the high penetrance breast cancer susceptibility gene, BRCA1. MATERIALS AND METHODS: In this experimental study, PSi pore sizes on the PSi surface were controlled for optimum filling with DNA oligonucleotides and surface roughness was optimized for obtaining higher resolution RIS patterns. In addition, the influence of two different organic electrolyte mixtures on the formation and morphology of the pores, based on various current densities and etching times on doped p-type silicon, were examined. Moreover, we introduce two cleaning processes which can efficiently remove the undesirable outer parasitic layer created during PSi formation. Results of all the optimization steps were observed by field emission scanning electron microscopy (FE-SEM). RESULTS: DNA sensing reached its optimum when PSi was formed in a two-step process in the ethanol electrolyte accompanied by removal of the parasitic layer in NaOH solution. These optimal conditions, which result in pore sizes of approximately 20 nm as well as a low surface roughness, provide a considerable RIS shift upon complementary sequence hybridization, suggesting efficient detectability. CONCLUSION: We demonstrate that the optimal conditions identified here makes PSi an attractive solid-phase DNA-based biosensing method and may be used to not only detect full complementary DNA sequences, but it may also be used for detecting point mutations such as single nucleotide substitutions and indels.

Rapid optimization of liposome characteristics using a combined microfluidics and design-of-experiment approach.

Liposomes have attracted much attention as the first nanoformulations entering the clinic. The optimization of physicochemical properties of liposomes during nanomedicine development however is time-consuming and challenging despite great advances in formulation development. Here, we present a systematic approach for the rapid size optimization of liposomes. The combination of microfluidics with a design-of-experiment (DoE) approach offers a strategy to rapidly screen and optimize various liposome formulations, i.e., up to 30 liposome formulations in 1 day. Five representative liposome formulations based on clinically approved lipid compositions were formulated using systematic variations in microfluidics flow rate settings, i.e., flow rate ratio (FRR) and total flow rate (TFR). Interestingly, flow rate-dependent DoE models for the prediction of liposome characteristics could be grouped according to lipid-phase transition temperature and surface characteristics. For all formulations, the FRR had a significant impact (p < 0.001) on hydrodynamic diameter and size distribution of liposomes, while the TFR mainly affected the production rate. Liposome characteristics remained constant for TFRs above 8 mL/min. The stability study revealed an influence of lipid:cholesterol ratio (1:1 and 2:1 ratio) and presence of PEG on liposome characteristics during storage. To validate our DoE models, we formulated liposomes incorporating hydrophobic dodecanethiol-coated gold nanoparticles. This proof-of-concept step showed that flow rate settings predicted by DoE models successfully determined the size of resulting empty liposomes (109.3 ± 15.3 nm) or nanocomposites (111 ± 17.3 nm). This study indicates that a microfluidics-based formulation approach combined with DoE is suitable for the routine development of monodisperse and size-specific liposomes in a reproducible and rapid manner.

The Molecular Basis of the Sodium Dodecyl Sulfate Effect on Human Ubiquitin Structure: A Molecular Dynamics Simulation Study.

To investigate the molecular interactions of sodium dodecyl sulfate (SDS) with human ubiquitin and its unfolding mechanisms, a comparative study was conducted on the interactions of the protein in the presence and absence of SDS at different temperatures using six independent 500 ns atomistic molecular dynamics (MD) simulations. Moreover, the effects of partial atomic charges on SDS aggregation and micellar structures were investigated at high SDS concentrations. The results demonstrated that human ubiquitin retains its native-like structure in the presence of SDS and pure water at 300 K, while the conformation adopts an unfolded state at a high temperature. In addition, it was found that both SDS self-assembly and the conformation of the resulting protein may have a significant effect of reducing the partial atomic charges. The simulations at 370 K provided evidence that the SDS molecules disrupted the first hydration shell and expanded the hydrophobic core of ubiquitin, resulting in complete protein unfolding. According to these results, SDS and temperature are both required to induce a completely unfolded state under ambient conditions. We believe that these findings could be useful in protein folding/unfolding studies and structural biology.

Design and Fabrication a Gold Nanoparticle-DNA Based Nanobiosensor for Detection of microRNA Involved in Alzheimer's Disease.

MicroRNAs are small RNAs which regulate gene expression by translational repression or degradation of messenger RNAs. Regards to important role of these biomolecules in human disease progress, to produce sensitive, simple and cost-effective assays for microRNAs are in urgent demand. miR-137 in Alzheimer's patients has demonstrated its potential as non-invasive biomarkers in blood for Alzheimer's disease diagnosis and prognosis. This paper describes a novel, sensitive and specific microRNA assay based on Colorimetric detection of gold nanoparticles and hybridization chain reaction amplification (HCR). The new strategy eliminates the need for enzymatic reactions, chemical changes, separation processes and sophisticated equipment. The detection process is visible with the naked eyes and detection limit for this method is 0.25nM which is less than or at least comparable with the previous methods based on colorimetric of AuNPs. The important features of this method are high sensitivity and specificity to differentiate between perfectly matched, mismatched and non-complementary target microRNAs and also decent response in the real sample analysis with blood plasma. In conclusion, the simple and fast nanobiosensor can clinically be used for the early detection of Alzheimer's disease by direct detection of the plasma miR-137 in real clinical samples, without a need for sample preparation, RNA extraction and/or amplification.

A novel approach for osteocalcin detection by competitive ELISA using porous silicon as a substrate.

In this study, porous silicon (PSi) was utilized instead of prevalent polystyrene platforms, and its capability in biomolecule screening was examined. Here, two types of porous structure, macroporous silicon (Macro-PSi) and mesoporous silicon (Meso-PSi), were produced on silicon wafers by electrochemical etching using different electrolytes. Moreover, both kinds of fresh and oxidized PSi samples were investigated. Next, osteocalcin as a biomarker of the bone formation process was used as a model biomarker, and the colorimetric detection was performed by competitive enzyme-linked immunosorbent assay (ELISA). Both Macro-PSi and Meso-PSi substrates in the oxidized state, specifically the Meso-porous structure, were reported to have higher surface area to volume ratio, more capacitance of surface-antigen interaction, and more ability to capture antigen in comparison with the prevalent platforms. Moreover, the optical density signal of osteocalcin detected by the ELISA technique was notably higher than the common platforms. Based on the findings of this study, PSi can potentially be used in the ELISA to achieve better results and consequently more sensitivity. A further asset of incorporating such a nanometer structure in the ELISA technique is that the system response to analyte concentration could be maintained by consuming lower monoclonal antibody (or antigen) and consequently reduces the cost of the experiment.

Porous silicon nanoparticle as a stabilizing support for chondroitinase.

Chondroitinase ABCI (cABCI) from Proteus vulgaris is a drug enzyme that can be used to treat spinal cord injuries. One of the main problems of chondroitinase ABC1 is its low thermal stability. The objective of the current study was to stabilize the enzyme through entrapment within porous silicon (pSi) nanoparticles. pSi was prepared by an electrochemical etch of p-type silicon using hydrofluoric acid/ethanol. The size of nanoparticles were determined 180nm by dynamic light scattering and the mean pore diameter was in the range of 40-60nm obtained by scanning electron microscopy. Enzymes were immobilized on porouse silicon nanoparticles by entrapment. The capacity of matrix was 35µg enzyme per 1mg of silicon. The immobilized enzyme displayed lower Vmax values compared to the free enzyme, but Km values were the same for both enzymes. Immobilization significantly increased the enzyme stability at various temperatures (-20, 4, 25 and 37°C). For example, at 4°C, the free enzyme (in 10mM imidazole) retained 20% of its activity after 100min, while the immobilized one retained 50% of its initial activity. Nanoparticles loading capacity and the enzyme release rate showed that the selected particles could be a pharmaceutically acceptable carrier for chondroitinase.

Comparison of antibody immobilization strategies in detection of Vibrio cholerae by surface plasmon resonance.

Surface plasmon resonance immunosensor for the detection of bacterial cells was first reported in 1998 with high detection limit as much as 107 cfu/ml. Since then, many efforts have been made aiming to lower the detection limit and improve the sensitivity of detection. The aim of this study was to compare the effect of four most frequently used immobilization strategies, including direct physical adsorption (physisorption), covalent immobilization via self-assembled monolayer (SAM) formation, bioaffinity immobilization using protein G-mediated immobilization and using mixed SAM of alkane thiols on signal strength of detection of Vibrio cholerae using these modified surfaces. The most widely used strategy, covalent binding of antibodies to sensor chip via SAM formation, gave the highest immobilization density and mixed SAM of 20/80 (v/v) of 11-mercaptoundecanoic acid (11-MUA)/9-mercapto-1-nonanol resulted in the least surface coverage in antibody immobilization step. To optimize surface density in covalent immobilization, four different concentrations (12.5, 25, 50, and 100 µg/ml) of anti-OmpW were immobilized on 11-MUA modified gold chips and maximum interaction response was achieved at 25 µg/ml. The interaction response signals for detection of V. cholerae using immobilized anti-OmpW were in this order: Oriented immobilization using protein G/antibody complex > mixed SAM of 11-MUA and 9-mercapto-1-nonanol > homogenous 11-MUA SAM > direct physical adsorption. In order to evaluate interaction studies in real sample condition, waste water samples that were artificially spiked with V. cholerae were tested and the authors concluded that for real samples, it is better to setup experiment with low surface coverage such as mixed SAM to overcome nonspecific adsorption.

Development of an immunosensor using oriented immobilized anti-OmpW for sensitive detection of Vibrio cholerae by surface plasmon resonance.

The first SPR sensor for detection of bacteria was reported in 1998 with high detection limit as much as 10(7)cfu/mL. Since then, a lot of effort has been made to lower detection limit and increase sensitivity of detection mainly by using of different assay formats, immobilization strategies, suitable antibodies, minimizing non-specific adsorption and improving the quality of SPR devices. The aim of this paper is to introduce the potential of an antibody against recombinant outer membrane protein (anti-OmpW) in sensitive detection of Vibrio cholerae by developing an immunosensor based on SPR and compare the sensitivity of this method with former report for detection of V. cholerae published in 2006. Recombinant OmpW antigen (a bacterial outer-membrane protein) of V. cholerae was expressed and purified and raising of polyclonal rabbit anti-OmpW was done. Protein G was covalently immobilized on 11-MUA SAM via amine coupling and bioaffinity-based oriented immobilization of anti-OmpW was done on protein G layer. The results showed high affinity interaction between OmpW and anti-OmpW (KD=2.4×10(-9)M) and the detection limit of fabricated immunosensor was 43 cells/mL. The apparent reasons for achieving this low LOD are discussed.

Misoprostol Abortion: Ultrasonography versus Beta-hCG Testing for Verification of Effectiveness.

BACKGROUND AND OBJECTIVE: Miscarriage is a common complication of early pregnancy with medical and psychological consequences. Dilation and Curettage are considered as two standard caring ways for early pregnancy failure. Alternatively misoprostol has been used as a single agent for termination of early pregnancy. Aim of the present study was to compare the usefulness of serum ß-hCG measurement and ultrasound examination to predict complete abortion after medical induction. METHODS: There were one hundred and thirty three patients experiencing missed abortion or blighted ovum. Ultrasound examination and serum ß-hCG test were performed before treatment and during follow-up in all these patients. RESULTS: Treatment was successful without any need for surgical intervention in 92.4% of the cases. Both methods could verify the complete abortion among all the patients at the end of the study (4(th) week). Kappa agreement coefficient for the two methods of diagnosis was 0.327 (P < 0.5). CONCLUSION: Based on our results, ß- hCG is as effective as ultrasound in confirming a successful medically induced abortion in early pregnancy, but it should be used as supplements to clinical assessments.