Aptamer decorated PDA@magnetic silica microparticles for bacteria purification.

One significant constraint in the advancement of biosensors is the signal-to-noise ratio, which is adversely affected by the presence of interfering factors such as blood in the sample matrix. In the present investigation, a specific aptamer binding was chosen for its affinity, while exhibiting no binding affinity towards non-target bacterial cells. This selective binding property was leveraged to facilitate the production of magnetic microparticles decorated with aptamers. A novel assay was developed to effectively isolate S. pneumoniae from PBS or directly from blood samples using an aptamer with an affinity constant of 72.8 nM. The capture experiments demonstrated efficiencies up to 87% and 66% are achievable for isolating spiked S. pneumoniae in 1 mL PBS and blood samples, respectively.

Biosensor for ATP detection via aptamer-modified PDA@POSS nanoparticles synthesized in a microfluidic reactor.

This study introduces aptamer-functionalized polyhedral oligomeric silsesquioxane (POSS) nanoparticles for adenosine triphosphate (ATP) detection where the POSS nanoparticles were synthesized in a one-step, continuous flow microfluidic reactor utilizing thermal polymerization. A microemulsion containing POSS monomers was generated in the microfluidic reactor which was designed to prevent clogging by using a continuous oil flow around the emulsion during thermal polymerization. Surfaces of POSS nanoparticles were biomimetically modified by polydopamine. The aptamer sequence for ATP was successfully attached to POSS nanoparticles. The aptamer-modified POSS nanoparticles were tested for affinity-based biosensor applications using ATP as a model molecule. The nanoparticles were able to capture ATP molecules successfully with an affinity constant of 46.5 [Formula: see text]M. Based on this result, it was shown, for the first time, that microfluidic synthesis of POSS nanoparticles can be utilized in designing aptamer-functionalized nanosystems for biosensor applications. The integration of POSS in biosensing technologies not only exemplifies the versatility and efficacy of these nanoparticles but also marks a significant contribution to the field of biorecognition and sample preparation.

Antioxidant Activity and Hemocompatibility Study of Quercetin Loaded Plga Nanoparticles.

Quercetin (QU) is an important flavonoid compound presenting lots of biological activities, but its application has been limited due to its low aqueous solubility and instability. In this study, conducted to improve these properties of the quercetin, quercetin-encapsulated PLGA nanoparticles were prepared, characterized, and evaluated for antioxidant and hemolytic activity. Nanoparticles were produced by single emulsion solvent evaporation method. Four different process parameters initial QU amount, PVA concentration, PVA volume, and initial PLGA amount were investigated to obtain the nanoparticles which have minimum particle size and maximum entrapment efficiency. Synthesized nanoparticles were evaluated for particle size, entrapment efficiency, and reaction yield. Additionally, antioxidant properties and in-vitro hemolytic activity of quercetin loaded nanoparticles with different particle size were also evaluated for the first time in the literature. The antioxidant activity results showed that nanoparticles have different antioxidant activity, depending on the amount of quercetin release from nanoparticles at different particle sizes. The hemolytic activity results show that all nanoparticles exhibited favorable compatibility to red blood cells and no significant hemolytic effect was observed.

Rapid and label-free detection of Brucella melitensis in milk and milk products using an aptasensor.

In this work, a novel quartz crystal microbalance (QCM) aptasensor is designed for the diagnosis of Brucella melitensis bacteria, which affects the Mediterranean fever (brucellosis) from the zoonotic diseases that are very common in the Middle East Countries. The method is based on the selection of B. melitensis bacterium from solutions using B. melitensis specific binding aptamer (Apt) attached magnetic nanoparticles. The surface of the magnetic nanoparticles (i.e.,Fe3O4) was modified by 3-aminopropyltriethoxysilane (APTES) and then grafted with a hydrophilic macromonomer poly(ethyleneglycol)-methacrylate (PEG-MA) as a first block polymer and glycidylmethacrylate (GMA) as a second block functional polymer via atom transfer radical polymerization (ATRP) method [Fe3O4 @SiO2 @p(PEG-MA-GMA)], then, the specific binding aptamer was immobilized. The aptamer immobilized magnetic nanoparticles were used for the pre-concentration of the target bacterium, and the same aptamer sequence was also immobilized on the QCM chip and used for the quantitative detection of B. melitensis using QCM aptasensor. The detection limits of the QCM aptasensor were in the range 1.02-1.07 CFU mL-1, with recoveries up to 79%. The synthesized [Fe3O4 @SiO2 @p(PEGMA-GMA)] nanoparticles showed a good permanence and high isolation recoveries for the pull down of the target bacterium from food samples, after recycling eight times. The method was successfully applied to target bacterium determinations in milk and milk product samples.

Enhanced antitumor activity of carbendazim on HeLa cervical cancer cells by aptamer mediated controlled release.

Carbendazim, is a broad-spectrum fungicide and also a promising experimental antitumor drug as reproduction and developmental toxicant, which is currently under phase II preclinical trials. In this study, an approach based on controlled and targeted release with aptamers and mesoporous silica nanoparticles was investigated to improve the antitumor activity of carbendazim. To this end, we synthesized aptamer conjugated silica nanoparticles for testing cytotoxicity properties in vitro with human cervical adenocarcinoma (HeLa) cultured cells. Nucleolin (AS1411) binding aptamers were used to entrap carbendazim molecules inside nanopores of MCM-41 type silica nanoparticles to obtain a stimuli-dependent release system. The effect of carbendazim loaded aptamer silica complex was tested and compared to free carbendazim treatment on HeLa cells, demonstrating 3.3 fold increase of toxicity on targeted cells with our delivery system. In addition, cytotoxicity of the complex was determined to be mostly due to increased apoptosis and to a less extend necrosis related pathways.

Fast and Sensitive Detection of Salmonella in Milk Samples Using Aptamer-Functionalized Magnetic Silica Solid Phase and MCM-41-Aptamer Gate System.

General detection methods for S. enterica include PCR analysis, immunologic methods, solid culturing techniques, and various microscopic studies. Milk and other food samples demonstrate an especially difficult challenge for direct detection, resulting from high biological contents. In this report, we aimed for fast detection of pathogen cells through an efficient magnetic capture and subsequent quick detection based on aptamer affinity. The Fe3O4@SiO2@pGMA and MCM-41 particles were prepared separately and used for preconcentration and detection, respectively. Aptamer oligonucleotide sequences against S. enterica were fixed on both amine-functionalized MCM-41 and Fe3O4@SiO2@pGMA particles via glutaraldehyde coupling. The captured Salmonella cells were determined by a fluorescent homogeneous assay in the samples by aptamer-gated MCM-41 silica particles. Our method achieved a sensitive assay to detect Salmonella cells in milk samples as low as 103 CFU/ml without any culturing. Hence, the proposed sensing strategy might be an efficient platform for pathogen detection in a food matrix.

Improvement stability and performance of invertase via immobilization on to silanized and polymer brush grafted magnetic nanoparticles.

In this study, magnetic nanoparticles (Fe3O4) were modified sequentially with silica (Fe3O4@SiO2), glycidyl methacrylate (GMA) by surface initiated atom transfer radical polymerization (SI-ATRP) and hexamethylene diamine (as a spacer arm). The p(GMA) grafted and SA modified form (i.e., Fe3O4@SiO2@pGMA-SA-3) was used for covalent immobilization of invertase (EC 3.2.1.26). The amount of immobilized enzyme on Fe3O4@SiO2@p(GMA) and Fe3O4@SiO2@p(GMA)-SA-3 was 36.1±0.9 and 33.4±1.3mg/g, respectively. The Km and Vmax values of immobilized invertase were found to be 39.4mmol/L and 349.5mmol/L min, and not significantly changed compared with free form (34.3mmol/L and 387.2mmol/Lmin), respectively, revealed that the applied protocol did not have any detrimental effect on the retained activity of immobilized invertase.

Staphylococcus aureus detection in blood samples by silica nanoparticle-oligonucleotides conjugates.

A fast, specific and sensitive homogeneous assay for Staphylococcus aureus detection was developed by measuring the activity of secreted nuclease from the bacteria via a modified DNA oligonucleotide. As biosensor format, an effective system, Nanokeepers as previously reported, were used for triggered release of confined fluorophores, and hence specific detection of S. aureus on nuclease activity was obtained. The interference from blood components for fluorescent quantification was eliminated by a pre-purification by aptamer-functionalized silica magnetic nanoparticles. The reported assay system was exclusively formed by nucleic acid oligos and magnetic or mesoporous silica nanoparticles, that can be used on blood samples in a stepwise manner. The assay was successfully used as a sensing platform for the specific detection of S. aureus cells as low as 682 CFU in whole blood.

Small molecule detection by lateral flow strips via aptamer-gated silica nanoprobes.

A fast, sensitive and ratiometric biosensor strategy for small molecule detection was developed through nanopore actuation. The new platform engineers together, a highly selective molecular recognition element, aptamers, and a novel signal amplification mechanism, gated nanopores. As a proof of concept, aptamer gated silica nanoparticles have been successfully used as a sensing platform for the detection of ATP concentrations at a wide linear range from 100 µM up to 2 mM.

A facile and efficient method of enzyme immobilization on silica particles via Michael acceptor film coatings: immobilized catalase in a plug flow reactor.

A novel method was developed for facile immobilization of enzymes on silica surfaces. Herein, we describe a single-step strategy for generating of reactive double bonds capable of Michael addition on the surfaces of silica particles. This method was based on reactive thin film generation on the surfaces by heating of impregnated self-curable polymer, alpha-morpholine substituted poly(vinyl methyl ketone) p(VMK). The generated double bonds were demonstrated to be an efficient way for rapid incorporation of enzymes via Michael addition. Catalase was used as model enzyme in order to test the effect of immobilization methodology by the reactive film surface through Michael addition reaction. Finally, a plug flow type immobilized enzyme reactor was employed to estimate decomposition rate of hydrogen peroxide. The highly stable enzyme reactor could operate continuously for 120 h at 30 °C with only a loss of about 36 % of its initial activity.

Fibrous polymer grafted magnetic chitosan beads with strong poly(cation-exchange) groups for single step purification of lysozyme.

Lysozyme is an important polypetide used in medical and food applications. We report a novel magnetic strong cation exchange beads for efficient purification of lysozyme from chicken egg white. Magnetic chitosan (MCHT) beads were synthesized via phase inversion method, and then grafted with poly(glycidyl methacrylate) (p(GMA)) via the surface-initiated atom transfer radical polymerization (SI-ATRP). Epoxy groups of the grafted polymer, were modified into strong cation-exchange groups (i.e., sulfonate groups) in the presence of sodium sulfite. The MCTH and MCTH-g-p(GMA)-SO3H beads were characterized by ATR-FTIR, SEM, and VSM. The sulphonate groups content of the modified MCTH-g-p(GMA)-4 beads was found to be 0.53mmolg(-1) of beads by the potentiometric titration method. The MCTH-g-p(GMA)-SO3H beads were first used as an ion-exchange support for adsorption of lysozyme from aqueous solution. The influence of different experimental parameters such as pH, contact time, and temperature on the adsorption process was evaluated. The maximum adsorption capacity was found to be 208.7mgg(-1) beads. Adsorption of lysozyme on the MCTH-g-p(GMA)-SO3H beads fitted to Langmuir isotherm model and followed the pseudo second-order kinetic. More than 93% of the adsorbed lysozyme was desorbed using Na2CO3 solution (pH 11.0). The purity of the lysozyme was checked by HPLC and SDS gel electrophoresis. In addition, the MCTH-g-p(GMA)-SO3H beads prepared in this work showed promising potential for separation of various anionic molecules.

Development of a paper-type tyrosinase biosensor for detection of phenolic compounds.

A low-cost, portable, and disposable paper-type tyrosinase biosensor was developed for determination of phenolic compounds, using a paper-strip absorption method. Tyrosinase and a chromophore (3-methyl-2-benzothiazolinone hydrazone) were immobilized on paper strips to manufacture the biosensor, which was tested on a nontoxic substrate (l-dopamine). The biosensor was responsive to phenolic compounds such as 4-chlorophenol, catechol, m-cresol, and p-cresol. The sensor showed stability for 70 days. The developed biosensor can be used for remote on-site qualitative monitoring of phenolic compounds in wastewater samples.

Examination of fabrication conditions of acrylate-based hydrogel formulations for doxorubicin release and efficacy test for hepatocellular carcinoma cell.

The objective of the present study was to develop 2-hydroxypropyl methacrylate-co-polyethylene methacrylate [p(HPMA-co-PEG-MEMA)] hydrogels that are able to efficiently entrap doxorubicin for the application of loco-regional control of the cancer disease. Systemic chemotherapy provides low clinical benefit while localized chemotherapy might provide a therapeutic advantage. In this study, effects of hydrogel properties such as PEG chains length, cross-linking density, biocompatibility, drug loading efficiency, and drug release kinetics were evaluated in vitro for targeted and controlled drug delivery. In addition, the characterization of the hydrogel formulations was conducted with swelling experiments, permeability tests, Fourier transform infrared, SEM, and contact angle studies. In these drug-hydrogel systems, doxorubicin contains amine group that can be expected a strong Lewis acid-base interaction between drug and polar groups of PEG chains, thus the drug was released in a timely fashion with an electrostatic interaction mechanism. It was observed that doxorubicin release from the hydrogel formulations decreased when the density of cross-linking, and drug/polymer ratio were increased while an increase in the PEG chains length of the macro-monomer (i.e. PEG-MEMA) in the hydrogel system was associated with an increase in water content and doxorubicin release. The biocompatibility of the hydrogel formulations has been investigated using two measures: cytotoxicity test (using lactate dehydrogenase assay) and major serum proteins adsorption studies. Antitumor activity of the released doxorubicin was assessed using a human SNU398 human hepatocellular carcinoma cell line. It was observed that doxorubicin released from all of our hydrogel formulations which remained biologically active and had the capability to kill the tested cancer cells.

Pathogen detection by core-shell type aptamer-magnetic preconcentration coupled to real-time PCR.

The presence of pathogenic bacteria is a major health risk factor in food samples and the commercial food supply chain is susceptible to bacterial contamination. Thus, rapid and sensitive identification methods are in demand for the food industry. Quantitative polymerase chain reaction (PCR) is one of the reliable specific methods with reasonably fast assay times. However, many constituents in food samples interfere with PCR, resulting in false results and thus hindering the usability of the method. Therefore, we aimed to develop an aptamer-based magnetic separation system as a sample preparation method for subsequent identification and quantification of the contaminant bacteria by real-time PCR. To achieve this goal, magnetic beads were prepared via suspension polymerization and grafted with glycidylmethacrylate (GMA) brushes that were modified into high quantities of amino groups. The magnetic beads were decorated with two different aptamer sequences binding specifically to Escherichia coli or Salmonella typhimurium. The results showed that even 1.0% milk inhibited PCR, but our magnetic affinity system capture of bacteria from 100% milk samples allowed accurate determination of bacterial contamination at less than 2.0 h with limit of detection around 100 CFU/mL for both bacteria in spiked-milk samples.

Design of a core-shell type immuno-magnetic separation system and multiplex PCR for rapid detection of pathogens from food samples.

We report an immuno-magnetic separation system developed by the immobilization of pathogen-specific antibodies on the core-shell magnetic beads. The magnetic beads were grafted with glycidylmethacrylate (GMA) using surface-initiated atom transfer radical polymerization (SI-ATRP). For immuno-magnetic separation (IMS) of target bacterial cells from others, antibodies for Escherichia coli and Salmonella enterica serovar Typhimurium cells were immobilized on the magnetic beads via glutaraldehyde coupling reaction. Our IMS system successfully separated Salmonella cells when the concentrations of target (i.e., Salmonella) and interfering (i.e., E. coli) cells were at the same level. Polymerase chain reaction (PCR) assays amplifying the rfb/rfbE region of the E. coli genome and a 647-bp fragment of the invA region of Salmonella were performed as the specific selection to accurately confirm the presence of E. coli and Salmonella, respectively. IMS and multiplex PCR methods can be used for specific and quantitative detection of pathogens from food samples. Thus, this study developed a reliable and direct system for rapid detection of Salmonella and E. coli in food samples. In addition, IMS method could be easily adapted to detect other pathogens by selecting the pertinent antibody.

Adsorption and separation of immunoglobulins by novel affinity core-shell beads decorated with Protein L and l-histidine.

A novel core shell beaded chromatographic materials was prepared by grafting of glycidyl methacrylate (GMA) on to the surface of poly(hydoxypropyl methacrylate/ethyleneglycol dimethacrylate), p(HPMA/EGDMA) beads via surface-initiated atom transfer radical polymerization (SI-ATRP). For grafting GMA, p(HPMA/EGDMA) beads were first modified with an ATRP initiator. A reaction with 2-bromo-2-methylpropionyl bromide of the hydroxyl groups of the beads led to ATRP initiator-covered surfaces. The grafted p(GMA) fibrous chains on the beads were decorated with two different ligands (i.e., Protein L and l-histidine) for separation of Immunoglobulin's (Igs) from aqueous solution in batch system. The maximum Igs adsorptions on the p(HPMA/EGDMA)-g-p(GMA)-Protein L and p(HPMA/EGDMA)-g-p(GMA)-l-histidine affinity beads were found to be 81.8 and 112.3mg/g at pH 7.5 and 5.5, respectively. The purity of Igs from human serum was analyzed by HPLC. The Protein L immobilized affinity beads provided purity about 98%. The novel core shell polymeric beads decorated with Protein L showed a good selectivity for Igs molecules from diluted human serum. Adsorption studies of Igs onto Protein L and l-histidine immobilized affinity beads were also carried out in a continuous system.

Melting temperature of surface-tethered DNA.

A method for the accurate determination of the melting temperature (T(m)) of surface-immobilized DNA duplexes that exploits the fluorescence-quenching properties of gold is reported. A thiolated single-stranded DNA probe is chemisorbed onto a gold surface and then hybridized to a fluorophore-labeled complementary sequence. On formation of the duplex, the fluorescence of the label is effectively quenched by the gold surface. As the temperature is increased and the duplex denatures, the fluorophore label moves away from the gold surface and the fluorescence signal is again observed. The increase in fluorescence is measured as the temperature is ramped, and using first-derivative plots, the T(m) is determined. To demonstrate the approach, the T(m) of the cystic fibrosis DF508 mutation was determined in three different phases: in solution, in suspension immobilized on gold nanoparticles, and immobilized on gold film-coated substrate. The technique was further applied to optimize conditions for differentiation between a surface-immobilized DF508 mutant probe and a mutant/wild-type target exploiting increasing stringency in varying salt and formamide concentrations. The approach has application in optimization of assay conditions for biosensors that use gold substrates as well as in melting curve analysis.

Label free optical sensor for Avidin based on single gold nanoparticles functionalized with aptamers.

Optical spectroscopy of a single gold nanoparticle, functionalized with an aptamer, is used to sense the specific binding of avidin. Herewith, the field of single noble metal nanoparticle biosensors is extended to the important field of aptamer based assays. The sensitivity of this initial, but not yet optimized apta-nano-sensor is in the range of 20 nM. Due to its nanoscopic size, this single nanoparticle based apta-sensor may be used in nanoscopic volumes such as in array type assays or even inside cells.

Real-time apta-PCR for 20 000-fold improvement in detection limit.

A real-time apta-PCR for the ultrasensitive detection of thrombin is reported, where the thrombin aptamer acts not only as a biomolecular recognition element, but also as a label for amplification via real-time PCR. Aptamers can be easily converted to a reporter agent for detection by real-time PCR, simply via flanking of the aptamer's recognition moiety with primer sequences. The reported technique has the advantage of the ultrasensitivity achievable with immuno-PCR, but without the complications of addition of a DNA label, and is a technique generically applicable to all aptamers. Here, we use a sandwich format, where two existing thrombin binding aptamers with distinct binding epitopes have been utilised to capture and detect thrombin in a streptavidin-coated microtiter plate. The amount of thrombin is calculated from real-time PCR analysis of eluted captured reporter aptamer. However, the technique can also be used for aptamer-antibody sandwiches, or simply with single aptamers. A greater than 20 000-fold increase in sensitivity is achieved, highlighting the potential of this approach for the detection of very low levels of target analytes. The use of the aptamer itself as the reporter molecule eliminates the necessity of laborious enzyme/DNA labelling, facilitating a significantly more straightforward assay with a vastly enhanced sensitivity.