Quartz crystal microbalance-based aptasensor integrated with magnetic pre-concentration system for detection of Listeria monocytogenes in food samples.

A fast and accurate identification of Listeria monocytogenes. A new quartz crystal microbalance (QCM) aptasensor was designed for the specific and rapid detection of L. monocytogenes. Before detection of the target bacterium from samples in the QCM aptasensor, a magnetic pre-enrichment system was used to eliminate any contaminant in the samples. The prepared magnetic system was characterized using ATR-FTIR, SEM, VSM, BET, and analytical methods. The saturation magnetization values of the Fe3O4, Fe3O4@PDA, and Fe3O4@PDA@DAPEG particles were 57.2, 40.8, and 36.4 emu/g, respectively. The same aptamer was also immobilized on the QCM crystal integrated into QCM flow cell and utilized to quantitatively detect L. monocytogenes cells from the samples. It was found that a specific aptamer-magnetic pre-concentration system efficiently captured L. monocytogenes cells in a short time (approximately 10 min). The Fe3O4@PDA@DA-PEG-Apt particles provided selective isolation of L. monocytogenes from the bacteria-spiked media up to 91.8%. The immobilized aptamer content of the magnetic particles was 5834 µg/g using 500 ng Apt/mL. The QCM aptasensor showed a very high range of analytical performance to the target bacterium from 1.0 × 102 and 1.0 × 107 CFU/mL. The limit of detection (LOD) and limit of quantitation (LOQ) were 148 and 448 CFU/mL, respectively, from the feeding of the QCM aptasensor flow cell with the eluent of the magnetic pre-concentration system. The reproducibility of the aptasensor was more than 95%. The aptasensor was very specific to L. monocytogenes compared to the other Listeria species (i.e., L. ivanovii, L. innocua, and L. seeligeri) or other tested bacteria such as Staphylococcus aureus, Escherichia coli, and Bacillus subtilis. The QCM aptasensor was regenerated with NaOH solution, and the system was reused many times.

Tramates trogii biomass in carboxymethylcellulose-lignin composite beads for adsorption and biodegradation of bisphenol A.

Tramates trogii biomass was immobilized in carboxymethyl cellulose-lignin composite beads via cross-linking with Fe(III) ions (i.e., Fe(III)-CMC@Lig(1-4)@FB). The composite beads formulations were used for the adsorption and degradation of bisphenol A (BPA) using the free fungal biomass as a control system. The maximum adsorption capacity of the free fungal biomass and Fe(III)-CMC@Lig-3@FB for BPA was found to be 57.8 and 95.6, mg/g, respectively. The degradation rates of BPA were found to be 87.8 and 89.6% for the free fungal biomass and Fe(III)CMC@Lig-3@FB for 72 h in a batch reactor, respectively. Adsorption of BPA on the free fungal biomass and Fe(III)CMC@Lig-3@FB fungal preparations described by the Langmuir and Temkin isotherm models, and the pseudo-second-order kinetic model. The values of Gibbs free energy of adsorption (ΔG°) were - 20.7 and - 25.8 kJ/mol at 298 K for BPA on the free fungal biomass and Fe(III)-CMC@Lig-3@FB beads, respectively. Moreover, the toxicities of the BPA and degradation products were evaluated with three different test organisms: (i) a freshwater micro-crustacean (Daphnia magna), (ii) a freshwater algae (Chlamydomonas reinhardti), and (iii) a Turkish winter wheat seed (Triticum aestivum L.). After treatment with the Fe(III)CMC@Lig-3@FB formulation, the degradation products had not any significant toxic effect compared to pure BPA. This work shows that the prepared composite bioactive system had a high potential for degradation of BPA from an aqueous medium without producing toxic end-products. Thus, it could be a good candidate for environmentally safe biological methods.

Biodegradation of Cibacron Blue 3GA by insolubilized laccase and identification of enzymatic byproduct using MALDI-ToF-MS: Toxicity assessment studies by Daphnia magna and Chlorella vulgaris.

The presented paper describes a detailed study on the use of immobilized laccase for effective degradation of Cibacron Blue 3GA dye. The amount of laccase loading on the cyclic carbonate groups containing poly(hydroxyethyl methacrylate-co-vinylene carbonate), p(HEMA-co-VC), microbeads was 27.8 mg g-1, and the retained immobilized enzyme activity was 73% compared to free enzyme. The toxicity of the dye and its byproducts were studied using Daphnia magna as test organism. The micro-algal growth inhibition was also studied using a green micro algae "Chlorella vulgaris". MALDI-ToF-MS was used to verify dye degradation byproducts. After 60 min of incubation period, Cibacron Blue 3GA (CB3GA) and its byproducts disappeared from the medium. After 60-min enzymatic treatment, the non-toxic nature of medium was confirmed by toxicity studies. On the other hand, the initial byproducts of the dye seemed to be more toxic than the later formed dye products. It should be noted that the information obtained from this study can be beneficial for understanding the initial degradation byproducts toxicities of the enzymatically treated dyes to provide information about environmental protection.

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.

Aminopyridine modified Spirulina platensis biomass for chromium(VI) adsorption in aqueous solution.

Chemical modification of Spirulina platensis biomass was realized by sequential treatment of algal surface with epichlorohydrin and aminopyridine. Adsorptive properties of Cr(VI) ions on native and aminopyridine modified algal biomass were investigated by varying pH, contact time, ionic strength, initial Cr(VI) concentration, and temperature. FTIR and analytical analysis indicated that carboxyl and amino groups were the major functional groups for Cr(VI) ions adsorption. The optimum adsorption was observed at pH 3.0 for native and modified algal biomasses. The adsorption capacity was found to be 79.6 and 158.7 mg g(-1), for native and modified algal biomasses, respectively. For continuous system studies, the experiments were conducted to study the effect of important design parameters such as flow rate and initial concentration of metal ions, and the maximum sorption capacity was observed at a flow rate of 50 mL h(-1), and Cr(VI) ions concentration 200 mg L(-1) with modified biomass. Experimental data fitted a pseudo-second-order equation. The regeneration performance was observed to be 89.6% and 94.3% for native and modified algal biomass, respectively.

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.

P(HPMA/EGDMA) beads grafted with fibrous chains by SI-ATRP method: agmatine functionalized affinity beads for selective separation of serum albumin.

In this paper, novel core-shell polymeric affinity beads based on fibrous grafting and functionalization with a salt resistance affinity ligand were developed to separate and deplete serum albumin (SA) from human serum. Poly(hydroxypropyl methacrylate/ethyleneglycole dimethacrylate), p(HPMA/EGDMA), beads were prepared via suspension polymerization, and were grafted with poly(glycidyl methacrylate) (p(GMA)) via surface-initiated atom transfer radical polymerization (SI-ATRP) method. The grafted p(GMA) fibrous chains on the beads were modified with an affinity ligand (i.e., agmatine). The binding capacity of the affinity beads to SA was determined using aqueous solution of SA in a batch system. Batch adsorption studies showed that the amount of adsorbed SA was found to be 156.7 mg/g at 25 °C. The maximum adsorption capacity for affinity beads was observed at around pH 5.5. Adsorption of SA onto affinity beads significantly increased with increasing temperature, and reached a value 177.8 mg/g beads at 35 °C. The equilibrium data were found to be well described by Langmuir model, while the kinetic data were well fitted to the pseudo-second-order kinetic. The degree of the purity of SA was determined by using HPLC. Before and after adsorption, the peak areas of SA were used in the calculation of separated SA.

Activity and stability of urease entrapped in thermosensitive poly(N-isopropylacrylamide-co-poly(ethyleneglycol)-methacrylate) hydrogel.

Urease was entrapped in thermally responsive poly(N-isopropylacrylamide-co-poly(ethyleneglycol)-methacrylate), p[NIPAM-p(PEG)-MA], copolymer hydrogels. The copolymer membrane shows temperature-responsive properties similar to conventional p(NIPAM) hydrogels, which reversibly swell below and de-swell above the lower critical solution temperature of p(NIPAM) hydrogel at around 32 °C. The retained activities of the entrapped urease (in p[NIPAM-p(PEG)-MA]-4 hydrogels) were between 83 and 53% compared to that of the same quantity of free enzyme. Due to the thermo-responsive character of the hydrogel matrix, the maximum activity was achieved at around 25 °C with the immobilized urease. Optimum pH was the same for both free and entrapped enzyme. Operational, thermal and storage stabilities of the enzyme were found to increase with entrapment of urease in the thermoresponsive hydrogel matrixes. As for reusability, the immobilized urease retained 89% of its activity after ten repeated uses.

Immobilization of glucoamylase onto polyaniline-grafted magnetic hydrogel via adsorption and adsorption/cross-linking.

Glucoamylase (GA) was immobilized onto polyaniline (PANI)-grafted magnetic poly(2-hydroxyethylmethacrylate-co-glycidylmethacrylate) hydrogel (m-p(HEMA-GMA)-PANI) with two different methods (i.e., adsorption and adsorption/cross-linking). The immobilized enzyme preparations were used for the hydrolysis of "starch" dextrin. The amount of enzyme loading on the ferrogel was affected by the medium pH and the initial concentration of enzyme. The maximum loading capacity of the enzyme on the ferrogel was found to be 36.7 mg/g from 2.0 mg/mL enzyme solution at pH 4.0. The adsorbed GA demonstrated higher activity (59%) compared to adsorbed/cross-linked GA (43%). Finally, the immobilized GA preparations exhibited greater stability against heat at 55 °C and pH 4.5 compared to free enzyme (50 °C and pH 5.5), suggesting that the ferrogel was suitable support for immobilization of glucoamylase.

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.

Real-Time Biosensing Bacteria and Virus with Quartz Crystal Microbalance: Recent Advances, Opportunities, and Challenges.

Continuous monitoring of pathogens finds applications in environmental, medical, and food industry settings. Quartz crystal microbalance (QCM) is one of the promising methods for real-time detection of bacteria and viruses. QCM is a technology that utilizes piezoelectric principles to measure mass and is commonly used in detecting the mass of chemicals adhering to a surface. Due to its high sensitivity and rapid detection times, QCM biosensors have attracted considerable attention as a potential method for detecting infections early and tracking the course of diseases, making it a promising tool for global public health professionals in the fight against infectious diseases. This review first provides an overview of the QCM biosensing method, including its principle of operation, various recognition elements used in biosensor creation, and its limitations and then summarizes notable examples of QCM biosensors for pathogens, focusing on microfluidic magnetic separation techniques as a promising tool in the pretreatment of samples. The review explores the use of QCM sensors in detecting pathogens in various samples, such as food, wastewater, and biological samples. The review also discusses the use of magnetic nanoparticles for sample preparation in QCM biosensors and their integration into microfluidic devices for automated detection of pathogens and highlights the importance of accurate and sensitive detection methods for early diagnosis of infections and the need for point-of-care approaches to simplify and reduce the cost of operation.

Development of a sensitive method for selection of affinity ligand for trypsin using quartz crystal microbalance sensor.

In this work, a new methodology is developed for selection of affinity ligands towards the enzyme "trypsin" using quartz crystals microbalance (QCM) technique. To achieve this goal, the surface amination of gold plated QCM crystals was achieved in 13.56 MHz plasma polymerization system by using ethylenediamine. Three different ligands (i.e., 4-aminobenzamidine, 4-aminobenzoic acid, and phenylalanine) were immobilized on the aminated QCM crystals surface via glutaraldehyde coupling. All three ligand immobilized QCM crystals were characterized and compared under different experimental conditions. It was observed that the benzamidine ligand showed higher affinity to trypsin with a dissociation constant on the order of 1.76 × 10(-9) M, which is within the range of 10(-4)-10(-8) M for affinity ligands. Thus, its selectivity was suitable for purification of trypsin from biological fluids.

Cross-linking of horseradish peroxidase adsorbed on polycationic films: utilization for direct dye degradation.

Horseradish peroxidase (HRP) was immobilized on the polyaniline (PANI) grafted polyacrylonitrile (PAN) films. The maximum HRP immobilization capacity of the PAN-g-PANI-3 film was 221 µg/cm(2). The HRP-immobilized PAN-g-PANI-3 film retained 79 % of the activity of the same quantity free enzyme. The HRP-immobilized PAN-g-PANI-3 film was operated for the decolorization of two different benzidine-based dyes in the presence of hydrogen peroxide. The maximum decolorization grade was obtained at pH 6.0 for both dyes. The HRP-immobilized PAN-g-PANI-3 film was very effective for removal of Direct Blue-53 compared to Direct Black-38 from aqueous solutions. The immobilized HRP exhibited high resistance to proteolysis by trypsin compared to the free counterpart. Immobilized HRP preserved 83 % of its original activity even after 8 weeks of storage at 4 °C, while the free enzyme lost its initial activity after 3 weeks of storage period.

Immobilization of Candida rugosa lipase on magnetic chitosan beads and application in flavor esters synthesis.

In this work, magnetic chitosan (MCH) beads were synthesized by phase-inversion method, and grafted with polydopamine (PDA) and then used for direct immobilization of Candida rugosa lipase by Schiff base reaction. The amount of immobilized enzyme and the retained activity were found to be 47.3 mg/g and 72.8%, respectively, at pH 7.0, and at 25 °C. The apparent Km (9.7 mmol/L), and Vmax (384 U/mg) values of the immobilized lipase were significantly changed compared to the free lipase. The MCH@PDA-lipase was better thermal and storage stability at different temperatures than those of the free lipase. In hexane medium, the esterification reaction results showed that the maximum conversions of isoamylalcohol and isopentyl alcohol to isoamyl acetate and isopentyl acetate using the MCH@PDA-lipase were found to be 98.4 ± 1.3% and 73.7 ± 0.7%, respectively. These results showed that the MCH@PDA-lipase can be used as an operative immobilized enzyme system for many biotechnological applications.

Selective isolation and sensitive detection of lysozyme using aptamer based magnetic adsorbent and a new quartz crystal microbalance system.

Magnetic chitosan beads and quartz crystal microbalance chip were decorated with lysozyme specific aptamer for isolation and detection of lysozyme, respectively. The lysozyme specific aptamer was immobilized on poly (dopamine) coated magnetic chitosan beads and the chip via Schiff base reaction. The percentage of the removal efficiency and purity of the isolated lysozyme from egg white were 87.6% and 91.8%, respectively. Further, the sensor system was contacted with different concentrations of lysozyme and other test proteins. This sensor system provided a method for the label-free, concentration-dependent, and selective detection of lysozyme with an observed detection limit of 17.9 ± 0.6 ng/mL. The sensor system was very selective and not significantly responded to the other tested proteins such as ovalbumin, trypsin, cytochrome C, and glucose oxidase. The prepared new sensor system showed a good durability and a high sensitivity for determination of lysozyme from solutions and whole egg white.

Surface plasmon resonance aptasensor for Brucella detection in milk.

A Surface Plasmon Resonance (SPR) aptasensor was developed for the detection of Brucella melitensis (B. melitensis) in milk samples. Brucellosis is a bacterial zoonotic disease with global distribution caused mostly by contaminated milk or their products. Aptamers recognizing B. melitensis were selected following a whole bacteria-SELEX procedure. Two aptamers were chosen for high affinity and high specificity. The high affinity aptamer (B70 aptamer) was immobilized on the surface of magnetic silica core-shell nanoparticles for initial purification of the target bacteria cells from milk matrix. Another aptamer, highly specific for B. melitensis cells (B46 aptamer), was used to prepare SPR sensor chips for sensitive determination of Brucella in eluted samples from magnetic purification since direct injection of milk samples to SPR sensor chips is known for a high background unspecific signal. Thus, we integrated a quick and efficient magnetic isolation step for subsequent instant detection of B. melitensis contamination in one ml of milk sample by SPR with a LOD value as low as 27 ± 11 cells.

Reversible immobilization of uricase on conductive polyaniline brushes grafted on polyacrylonitrile film.

Polyacrylonitrile film (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence of potassium dichromate as an oxidizing agent. The conductive films were used for immobilization of uricase. The surface resistance of the conductive film in this work was found to be 0.97 kΩ/cm. The maximum amount of immobilized enzyme on conductive film containing 2.4% PANI was about 216 µg/cm(2). The optimum pH for free and immobilized enzymes was observed at 7.0 and 7.5, respectively. The K (m) values for free and immobilized uricase were found to be 94 and 138 µM, respectively. V (max) values were calculated as 1.87 and 1.63 U/mg protein for the free and immobilized enzymes, respectively. Immobilized uricase exhibited ~68% of its original activity even after 2 months of storage at 4 °C while the free enzyme lost its initial activity within 4 weeks.

Poly(styrene-divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: performance of reversibly immobilized lipase in ester synthesis.

Fibrous poly(styrene-b-glycidylmethacrylate) brushes were grafted on poly(styrene-divinylbenzene) (P(S-DVB)) beads using surface-initiated atom transfer radical polymerization. Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The ligand attached beads were used for reversible immobilization of lipase. The influences of pH, ionic strength, and initial lipase concentration on the immobilization capacities of the beads have been investigated. Lipase adsorption capacity of the beads was about 78.1 mg/g beads at pH 6.0. The Km value for immobilized lipase was about 2.1-fold higher than that of free enzyme. The thermal, and storage stability of the immobilized lipase also was increased compared to the native lipase. It was observed that the same support enzyme could be repeatedly used for immobilization of lipase after regeneration without significant loss in adsorption capacity or enzyme activity. A lipase from Mucor miehei immobilized on styrene-divinylbenzene copolymer was used to catalyze the direct esterification of butyl alcohol and butyric acid.

Strong and weak cation-exchange groups generated cryogels films for adsorption and purification of lysozyme from chicken egg white.

Here, the macroporous poly(hydroxylmethyl methacrylate/glycidyl methacrylate [p(HEMA-GMA)] cryogels with large porous surface were prepared, and then the epoxy groups of the p(HEMA-GMA) cryogels were systematically modified into strong and weak cationic groups. The effects of initial protein concentrations, adsorption time, pH, salt concentrations and temperatures on adsorption efficiency of cation exchange cryogels for lysozyme were determined. The maximum lysozyme adsorption capacities of strong and weak cation exchange cryogels were found to be 188.3 and 79.7 mg/g cryogel at 25 °C, respectively. The performance of the strong cationic cryogel was evaluated by purification of lysozyme from egg white. The activity of the isolated lysozyme was found to be 21,347 U/mg. The cationic cryogel maintained its expected high adsorption capacity and efficiency of the purification levels during repeated adsorption desorption processes. Finally, the purpose of this work is the design a cation exchange system for purification of lysozyme from egg-white.