FRET theoretical predictions concerning freely diffusive dyes inside spherical container: how to choose the best pair?

FRET has been massively used to see if biomolecules were bounded or not by labelling both biomolecules by one dye of a FRET pair. This should give a digital answer to the question (fluorescence of the acceptor: high FRET efficency: molecules associated, fluorescence of the donor: low FRET efficency: molecules dissociated). This has been used, inter alia, at the single-molecule scale in containers, such as liposomes. One perspective of the field is to reduce the container's size to study the effect of confinement on binding. The problem is that if the two dyes are encapsulated inside a small liposome, they could have a significant probability to be close one from the other one (and thus to undergo a high FRET efficiency event without binding). This is why we suggest here a theoretical model which gives mean FRET efficiency as a function of liposome radius (the model applies to any spherical container) and Förster radius to help the experimentalist to choose their experimental set-up. Besides, the influence of side effect on mean FRET efficiency has been studied as well. We show here that if this "background FRET" is most of the time non-quantitative, it can remain significant and which makes data analysis trickier. We could show as well that if this background FRET obviously increases when liposome radius decreases, this variation was lower than the one which could be expected because of side effect. We show as well the FRET efficiency function distribution which let the experimentalist know the probability to get one FRET efficiency value.

A Physiologically Responsive Nanocomposite Hydrogel for Treatment of Head and Neck Squamous Cell Carcinoma via Proteolysis-Targeting Chimeras Enhanced Immunotherapy.

Although immunotherapy has revolutionized oncotherapy, only ≈15% of head and neck squamous cell carcinoma (HNSCC) patients benefit from the current therapies. An immunosuppressive tumor microenvironment (TME) and dysregulation of the polycomb ring finger oncogene BMI1 are potential reasons for the failure. Herein, to promote immunotherapeutic efficacy against HNSCC, an injectable nanocomposite hydrogel is developed with a polymer framework (PLGA-PEG-PLGA) that is loaded with both imiquimod encapsulated CaCO3 nanoparticles (RC) and cancer cell membrane (CCM)-coated mesoporous silica nanoparticles containing a peptide-based proteolysis-targeting chimeras (PROTAC) for BMI1 and paclitaxel (PepM@PacC). Upon injection, this nanocomposite hydrogel undergoes in situ gelation, after which it degrades in the TME over time, releasing RC and PepM@PacC nanoparticles to respectively perform immunotherapy and chemotherapy. Specifically, the RC particles selectively manipulate tumor-associated macrophages and dendritic cells to activate a T-cell immune response, while CCM-mediated homologous targeting and endocytosis delivers the PepM@PacC particles into cancer cells, where endogenous glutathione promotes disulfide bond cleavage to release the PROTAC peptide for BMI1 degradation and frees the paclitaxel from the particle pores to elicit apoptosis meanwhile enhance immunotherapy. Thus, the nanocomposite hydrogel, which is designed to exploit multiple known vulnerabilities of HNSCC, succeeds in suppressing both growth and metastasis of HNSCC.

Evaluation of Dimercaptosuccinic Acid-Coated Iron Nanoparticles Immunotargeted to Amyloid Beta as MRI Contrast Agents for the Diagnosis of Alzheimer's Disease.

Nanoparticle-based magnetic contrast agents have opened the potential for magnetic resonance imaging (MRI) to be used for early non-invasive diagnosis of Alzheimer's disease (AD). Accumulation of amyloid pathology in the brain has shown association with cognitive decline and tauopathy; hence, it is an effective biomarker for the early detection of AD. The aim of this study was to develop a biocompatible magnetic nanoparticle targeted to amyloid beta (Aß) plaques to increase the sensitivity of T2-weighted MRI for imaging of amyloid pathology in AD. We presented novel iron core-iron oxide nanoparticles stabilized with a dimercaptosuccinic acid coating and functionalized with an anti-Aß antibody. Nanoparticle biocompatibility and cellular internalization were evaluated in vitro in U-251 glioblastoma cells using cellular assays, proteomics, and transmission electron microscopy. Iron nanoparticles demonstrated no significant in vitro cytotoxicity, and electron microscopy results showed their movement through the endocytic cycle within the cell over a 24 h period. In addition, immunostaining and bio-layer interferometry confirmed the targeted nanoparticle's binding affinity to amyloid species. The iron nanoparticles demonstrated favourable MRI contrast enhancement; however, the addition of the antibody resulted in a reduction in the relaxivity of the particles. The present work shows promising preliminary results in the development of a targeted non-invasive method of early AD diagnosis using contrast-enhanced MRI.

Development of sensitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) for monitoring bisphenol-A in canned foods and beverages.

Enzyme-linked immunosorbent assays (ELISAs) are investigated in this work for the detection of bisphenol-A (BPA), a plastic monomer and a critical contaminant in food and environment. A series of polyclonal antibodies generated in vivo using BPA-butyrate-protein conjugate and BPA-valerate-protein conjugate were evaluated on direct and indirect competitive assay formats with five competing haptens (BPA-butyrate, BPA-valerate, BPA-crotonate, BPA-acetate, and BPA-2-valerate). Two indirect ELISAs and one direct ELISA exhibiting high sensitivity and specificity for BPA were developed. The 50 % inhibition of antibody binding (IC(50)) values were 0.78 ± 0.01-1.20 ± 0.26 µg L(-1), and the limits of detection as measured by the IC(20) values were 0.10 ± 0.03-0.20 ± 0.04 µg L(-1). The assays were highly specific to BPA, only displaying low cross-reactivity (3-8 % for the indirect assays and 26 % for the direct assay) for 4-cumylphenol (4-CP), at pH 7.2. The degree of cross-reaction of 4-CP was influenced by the antibody/hapten conjugate combination, assay conditions, and the assay format. The assays were optimized for the analysis of BPA in canned vegetables, bottled water and carbonated drinks. The limits of quantification for these three evaluated sample types, based on the spike and recovery data, were 0.5, 2.5, and 100 µg L(-1), respectively.

Using an electrical potential to reversibly switch surfaces between two states for dynamically controlling cell adhesion.

Smart surfaces presenting both antifouling molecules with a charged functional group at their distal end, and molecules that are terminated by RGD peptides for cell adhesion, were fabricated and characterized (see picture). By applying potentials of +300 or -300 mV, the surfaces could be dynamically switched to make the peptide accessible or inaccessible to cells.

Nanorepairers Rescue Inflammation-Induced Mitochondrial Dysfunction in Mesenchymal Stem Cells.

Mitochondrial dysfunction in tissue-specific mesenchymal stem cells (MSCs) plays a critical role in cell fate and the morbidity of chronic inflammation-associated bone diseases, such as periodontitis and osteoarthritis. However, there is still no effective method to cure chronic inflammation-associated bone diseases by physiologically restoring the function of mitochondria and MSCs. Herein, it is first found that chronic inflammation leads to excess Ca2+ transfer from the endoplasmic reticulum to mitochondria, which causes mitochondrial calcium overload and further damage to mitochondria. Furthermore, damaged mitochondria continuously accumulate in MSCs due to the inhibition of mitophagy by activating the Wnt/ß-catenin pathway under chronic inflammatory conditions, impairing the differentiation of MSCs. Based on the mechanistic discovery, intracellular microenvironment (esterase and low pH)-responsive nanoparticles are fabricated to capture Ca2+ around mitochondria in MSCs to regulate MSC mitochondrial calcium flux against mitochondrial dysfunction. Furthermore, the same nanoparticles are able to deliver siRNA to MSCs to inhibit the Wnt/ß-catenin pathway and regulate mitophagy of the originally dysfunctional mitochondria. These precision-engineered nanoparticles, referred to as "nanorepairers," physiologically restore the function of mitochondria and MSCs, resulting in effective therapy for periodontitis and osteoarthritis. The concept can potentially be expanded to the treatment of other diseases via mitochondrial quality control intervention.

Cellobiose dehydrogenase aryl diazonium modified single walled carbon nanotubes: enhanced direct electron transfer through a positively charged surface.

One of the challenges in the field of biosensors and biofuel cells is to establish a highly efficient electron transfer rate between the active site of redox enzymes and electrodes to fully access the catalytic potential of the biocatalyst and achieve high current densities. We report on very efficient direct electron transfer (DET) between cellobiose dehydrogenase (CDH) from Phanerochaete sordida (PsCDH) and surface modified single walled carbon nanotubes (SWCNT). Sonicated SWCNTs were adsorbed on the top of glassy carbon electrodes and modified with aryl diazonium salts generated in situ from p-aminobenzoic acid and p-phenylenediamine, thus featuring at acidic pH (3.5 and 4.5) negative or positive surface charges. After adsorption of PsCDH, both electrode types showed excellent long-term stability and very efficient DET. The modified electrode presenting p-aminophenyl groups produced a DET current density of 500 µA cm(-2) at 200 mV vs normal hydrogen reference electrode (NHE) in a 5 mM lactose solution buffered at pH 3.5. This is the highest reported DET value so far using a CDH modified electrode and comes close to electrodes using mediated electron transfer. Moreover, the onset of the electrocatalytic current for lactose oxidation started at 70 mV vs NHE, a potential which is 50 mV lower compared to when unmodified SWCNTs were used. This effect potentially reduces the interference by oxidizable matrix components in biosensors and increases the open circuit potential in biofuel cells. The stability of the electrode was greatly increased compared with unmodified but cross-linked SWCNTs electrodes and lost only 15% of the initial current after 50 h of constant potential scanning.

Modular immune-homeostatic microparticles promote immune tolerance in mouse autoimmune models.

The therapeutic goal for autoimmune diseases is disease antigen-specific immune tolerance without nonspecific immune suppression. However, it is a challenge to induce antigen-specific immune tolerance in a dysregulated immune system. In this study, we developed immune-homeostatic microparticles (IHMs) that treat multiple mouse models of autoimmunity via induction of apoptosis in activated T cells and reestablishment of regulatory T cells. Specifically, in an experimental model of colitis, IHMs rapidly released monocyte chemotactic protein-1 after intravenous administration, which recruited activated T cells and then induced their apoptosis by conjugated Fas ligand on the IHM surface. This triggered professional macrophages to ingest apoptotic T cells and produce high quantities of transforming growth factor-ß, which drove regulatory T cell differentiation. Furthermore, the modular design of IHMs allowed IHMs to be engineered with the autoantigen peptides that can reduce disease in an experimental autoimmune encephalomyelitis mouse model and a nonobese diabetic mouse model. This was accomplished by sustained release of the autoantigens after induction of T cell apoptosis and transforming growth factor-ß production by macrophages, which promoted to establish an immune tolerant environment. Thus, IHMs may be an efficient therapeutic strategy for autoimmune diseases through induction of apoptosis and reestablishment of tolerant immune responses.

How Nanoparticles Transform Single Molecule Measurements into Quantitative Sensors.

Single molecule measurements are revolutionizing the understanding of the stochastics of behavior of single molecules. There is a common theme referred to as a near-field approach, in how many single molecule measurements are being performed in assays. The term near field is used because the measurement volume is typically very small such that a single molecule, or a single molecule binding pair, within that volume is of an appreciable concentration. The next development in detection will be performing many single molecule measurements at one time such that single molecule measurements can be used as the basis for quantitative analysis. There have already been some notable developments in this direction. Again, all have a common theme in that nanoparticles are used to create many near-field volumes that can be measured simultaneously. Herein, the coupled developments in nanoparticles and measurement strategies that allow nanoparticles to be the backbone of the next generation of sensing technologies are discussed.

High F-Content Perfluoropolyether-Based Nanoparticles for Targeted Detection of Breast Cancer by 19F Magnetic Resonance and Optical Imaging.

Two important challenges in the field of 19F magnetic resonance imaging (MRI) are the maintenance of high fluorine content without compromising imaging performance, and effective targeting of small particles to diseased tissue. To address these challenges, we have developed a series of perfluoropolyether (PFPE)-based hyperbranched (HBPFPE) nanoparticles with attached peptide aptamer as targeting ligands for specific in vivo detection of breast cancer with high 19F MRI sensitivity. A detailed comparison of the HBPFPE nanoparticles (NPs) with the previously reported trifluoroethyl acrylate (TFEA)-based polymers demonstrates that the mobility of fluorinated segments of the HBPFPE nanoparticles is significantly enhanced (19F T2 > 80 ms vs 31 ms), resulting in superior MR imaging sensitivity. Selective targeting was confirmed by auto- and pair correlation analysis of fluorescence microscopy data, in vitro immunofluorescence, in vivo 19F MRI, ex vivo fluorescence and 19F NMR. The results highlight the high efficiency of aptamers for targeting and the excellent sensitivity of the PFPE moieties for 19F MRI. Of relevance to in vivo applications, the PFPE-based polymers exhibit much faster clearance from the body than the previously introduced perfluorocarbon emulsions ( t1/2 ∼ 20 h vs up to months). Moreover, the aptamer-conjugated NPs show significantly higher tumor-penetration, demonstrating the potential of these imaging agents for therapeutic applications. This report of the synthesis of polymeric aptamer-conjugated PFPE-based 19F MRI CAs with high fluorine content (∼10 wt %) demonstrates that these NPs are exciting candidates for detecting diseases with high imaging sensitivity.

Nucleic acid hybridization on an electrically reconfigurable network of gold-coated magnetic nanoparticles enables microRNA detection in blood.

There is intense interest in quantifying the levels of microRNA because of its importance as a blood-borne biomarker. The challenge has been to develop methods that can monitor microRNA expression both over broad concentration ranges and in ultralow amounts directly in a patient's blood. Here, we show that, through electric-field-induced reconfiguration of a network of gold-coated magnetic nanoparticles modified by probe DNA (DNA-Au@MNPs), it is possible to create a highly sensitive sensor for direct analysis of nucleic acids in samples as complex as whole blood. The sensor is the first to be able to detect concentrations of microRNA from 10 aM to 1 nM in unprocessed blood samples. It can distinguish small variations in microRNA concentrations in blood samples of mice with growing tumours. The ultrasensitive and direct detection of microRNA using an electrically reconfigurable DNA-Au@MNPs network makes the reported device a promising tool for cancer diagnostics.

Preparation and characterisation of an aligned carbon nanotube array on the silicon (100) surface.

Arrays of aligned carbon nanotubes formed by self-assembly on a Si (100) surface are described. The surface of a Si (100) wafer has been modified by reaction of hydride-terminated Si (100) with ethyl undecylenate to give ethyl undecanoate self-assembled monolayers (SAMs) which were linked by stable silicon-carbon covalent bonds. The ester terminus of the monolayer was converted to an alcohol whereupon shortened carbon nanotubes were covalently attached using carbodiimide coupling. The formation of the SAM and its subsequent modification with nanotubes has been followed using a series of techniques including X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), scanning electron microscopy (SEM), IR spectroscopy and cyclic voltammetry.

Carbon-Quantum-Dots-Loaded Mesoporous Silica Nanocarriers with pH-Switchable Zwitterionic Surface and Enzyme-Responsive Pore-Cap for Targeted Imaging and Drug Delivery to Tumor.

Mesoporous silica nanocarriers with pH-switchable antifouling zwitterionic surface, enzyme responsive drug release properties and blue fluorescence are reported. Prolonged circulation in the blood system with zero premature release as well as efficient cellular uptake and intracellular drug release in tumor tissue are achieved.

Effects of Surface Epitope Coverage on the Sensitivity of Displacement Assays that Employ Modified Nanoparticles: Using Bisphenol A as a Model Analyte.

With the ever-increasing use of nanoparticles in immunosensors, a fundamental study on the effect of epitope density is presented herein, with a small molecule epitope, on the performance of the displacement assay format in an enzyme-linked immunosorbent assay (ELISA). Thiolated bisphenol A (BPA) functionalized gold nanoparticles (cysBPAv-AuNPs) and specific anti-BPA antibodies are employed for this purpose. It is shown that the displacement of cysBPAv-AuNPs bound to the immobilized antibodies was influenced by both the avidity of bound cysBPAv-AuNPs and the concentration of free BPA to displace it. The importance of surface epitope density was that it changed the number of epitopes in close proximity to the antibody-binding site. This then influenced the avidity of cysBPAv-AuNPs bound to the immobilized antibody. Furthermore, the molar epitope concentration in an assay appears to affect the degree of antibody binding site saturation. Controlling surface epitope density of the functionalized nanoparticles and molar epitope concentration in an assay leads to a decrease of the concentration of free BPA required to displace the bound cysBPAv-AuNP, and hence better assay performance with regards to the D50 value and dynamic range in the displacement assay.

Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP).

The loop-mediated isothermal amplification (LAMP) technique has the potential to revolutionize molecular biology because it allows DNA amplification under isothermal conditions and is highly compatible with point-of-care analysis. To achieve efficient genetic analysis of samples, the method of real-time or endpoint determination selected to monitor the biochemical reaction is of great importance. In this paper we briefly review progress in the development of monitoring methods for LAMP.