Agarose Gel Membrane Research Articles

Solar energy-based electromembrane extraction using agarose gel for the determination of nonsteroidal anti-inflammatory drugs from biological samples: An environmentally friendly strategy

This paper presents the application of solar energy as a renewable resource in gel electromembrane extraction (G-EME). The extraction driving force (electrical field) generated from solar energy is stored through photovoltaic panels and significantly contributes to reducing the emission of greenhouse gasses. Moreover, the replacement of the polypropylene membrane and organic extracting solvents with biodegradable agarose membrane and the aqueous extracting solutions makes the presented approach compatible with the principles of green chemistry. Naproxen (NAP) and ibuprofen (IBF) were extracted from urine samples to the aqueous acceptor phase containing an anode electrode situated on the other side of the agarose gel membrane. Acceptable linearity was obtained in the concentration ranges of 6–100 and 9–100 μg. L − 1 with detection limits of 2 and 3 μg. L − 1 for NAP and IBF, respectively.

In-tube gel electro-membrane combined with microfluidic paper-based device: A green and miniaturized extraction mode for the chromium speciation

• In-tube gel electro-membrane was for the first time introduced as a scaled-down version of G-EME. • Extractions in transparent tube enable visual monitoring during the extraction processes. • With this new set-up, the DP/AP volume ratio can be easily increased to achieve a high enrichment factor. • Gel membrane and microfluidic paper-based analytical devices (µPADs) were a homemade fabrication. • The proposed workflow shows the ease of operation and is highly environmentally friendly. For the first time, in-tube gel electro-membrane microextraction (IT-G-EME) system followed by microfluidic paper-based analytical devices (µPADs) as a low-cost reading platform was fabricated for the speciation of trivalent chromium (Cr(III)) and hexavalent chromium (Cr(VI)) as model cationic and anionic compounds. In this new miniaturized extraction mode, a transparent narrow-bore polymeric tube was used as housing of the aqueous acceptor phase (AP, 30 µL), while an agarose gel membrane was placed as a micro plug (2.5 mm) at the beginning of the tube. A circular shape vial (1.5 mL, pH 5.5) containing chromium species as donor phase (DP) was connected between two tubes which previously filled with gel membrane and aqueous AP. By applying electric potential, the positively charged Cr(III) and negatively charged Cr(VI) in the DP migrated selectively toward cathodic and anodic tubes, respectively. After extraction, each AP was analyzed by µPAD, which had already been modified with diphenylcarbazide (DPC). Under the optimized extraction conditions, a good limit of detection (LOD) equal to 7.0 µg L –1 was achieved for both analytes, while the extraction recoveries for Cr(VI) and Cr(III) were 72% and 84%. In addition, the developed approach was used for the quantification of chromium species in water samples.

Evaluation of complexing agents in the gel electro-membrane extraction: An efficient approach for the quantification of zinc (II) ions in water samples

In this work, gel electro-membrane extraction (G-EME) combined with flame atomic absorption spectrometry (FAAS) was used for the determination of zinc ions (Zn2+) in water samples. For the first time, the effect of the presence of three types of complexing agents such as phenanthroline (Phen), crown ethers (12C4, 15C5, 18C6), and ethylene-diamine-tetra-acetic acid (EDTA) on the extraction efficiency of zinc ions was studied. In addition, the electroendosmosis (EEO) flow as an unwanted actuator was monitored in the presence and absence of complexing agents. By applying 50 V electrical potential across the membrane, the positive charged Zn2+ ions were migrated from a donor phase (pH 5.0) through the agarose gel membrane (pH 5.0, containing a complexing agent) into the acceptor phase (pH 3.0). The obtained results showed that the highest extraction recoveries were obtained when crown ethers, especially 1% (w/v) 18C6 was added to the gel membrane. In addition, EEO flow was decreased in the presence of all complexing agents (except EDTA), probably due to the increase in electrical resistance. Using the optimum conditions, the limit of detection (LOD), the limit of quantification (LOQ), and extraction recovery% (ER%) were 5.0 μg L−1, 15.0 μg L−1, and 92.5%, respectively. In the end, the applicability of the developed approach was successfully evaluated to determine Zn2+ in tap, mineral, and river water samples.

Enrichment of psychotropic drugs using rhamnolipid bioaggregates after electromembrane extraction based on an agarose gel using a rotating electrode as a green and organic solvent-free strategy

We here present an efficient approach for the tandem extraction of psychotropic drugs using biodegradable materials. In this regard, gel electromembrane extraction (G-EME) was combined with the emulsification-based microextraction (ME) technique by rhamnolipid bioaggregates as a green extraction approach. The tandem extraction technique consists of two stages: (i) extraction of psychotropic drugs from human urine samples to the acceptor phase situated on the other side of the agarose gel membrane, and (ii) transfer of analytes from the acceptor phase into a colloidal phase of rhamnolipid biosurfactants. The colloidal phase was formed by adding rhamnolipid biosurfactants to the extracted phase of the first step. The colloidal phase was finally injected into a liquid chromatographic system for quantitative analysis. G-EME mechanism is based on electrokinetic migration of charged species toward oppositely charged electrode located in the acceptor solution under the influence of the electric field. After extraction, the analytes were trapped in an emulsion phase floating on the surface of the solution and at the end were injected into the liquid chromatographic system. The method provided good linearity in the ranges of 5-100 and 10-100 μg. L−1 for methamphetamine and amphetamine, respectively with (r2 > 0.992). Also, the detection limits (LODs) were 1 and 5 μg. L−1 for methamphetamine and amphetamine, respectively. The mean extraction recoveries by G-EME-ME for real samples at three spiked concentrations were in the range 95.9-101.1% and complete analytical workflow within only 18 min.

Two-phase agarose gel-electromembrane extraction: Effect of organic solvent as an acceptor phase in electroendosmosis flow phenomenon

In this study, a new mode of gel electromembrane extraction (G-EME) namely as "Two-phase G-EME", is suggested for the sensitive quantification of five basic drugs (desipramine, clomipramine, trimipramine, citalopram and clozapine) in biological samples. Compared to classical G-EME which is based on aqueous-gel-aqueous layout, herein, the aqueous acceptor phase (AP) was replaced with organic solvent. Briefly, negative electrode was immersed into the organic AP (with low conductivity) and positive electrode into the aqueous donor phase (DP). Based on our results, this simple adjustment significantly reduced electroendosmosis (EEO) flow phenomenon which is considered as the main issue in G-EME. In the workflow, target analytes were extracted from the 7.0 mL sample, across the fabricated agarose gel membrane, to the 100 μL of the AP under the optimized extraction conditions (organic solvent type: acetonitrile; pH of gel membrane: 5.0, pH of sample solution: 4.0, voltage: 45 V and extraction time: 22 min). Then, the organic AP with analytes was analyzed by gas chromatography (GC) instrument with flame-ionization detector (FID). The methodology offered limits of detection (LODs) and recoveries in the range of 1.0–1.5 ng mL−1 and 48.5–89.0 %, respectively. Finally, we note that two-phase G-EME assembly was able to extract analytes-of-interest in the convenient and safe manner from the hazardous and difficult-to-process biological specimens such as human serum and urine.

Electromembrane extraction based on agarose gel for the extraction of phenolic acids from fruit juices

Extraction of polar acidic compounds is a challenging task in electromembrane extraction. In this study, gel-electromembrane extraction was employed for the extraction of phenolic acids as the polar acidic compounds from fruit juices. For this aim, the extraction of phenolic acids from the juice samples (4 mL, pH = 6.0) was carried out across the agarose gel membrane (concentration of agarose; 3% (w/v), pH of gel; 10.0, and thickness of membrane: 3 mm) into the acceptor solution (100 μL, pH = 12.0). Also, this extraction process was conducted by applying the optimum potential (25 V) for 15 min to the extraction system. Under the optimized condition, acceptable linearity (R2 ≥ 0.993) over a concentration range of 10.0–2500 ng mL−1 was achieved. The limits of detection were between 3.0 and 15.2 ng mL−1, while the corresponding repeatabilities ranged from 5.3 to 11.4% (n = 4). The recoveries achieved for the extraction of target compounds were ranged from 26.8 to 74.4%. The proposed method was used for the extraction of phenolic acids from orange, apple and kiwi juices, and the obtained relative recoveries in the range of 78.0–104.2% and RSDs in the range of 6.3 to 11.3% indicated successful extraction of phenolic acids.

Gel electro-membrane extraction of propranolol and atenolol from blood serum samples: Effect of graphene-based nanomaterials on extraction efficiency of gel membrane

In this work, gel electro-membrane extraction (G-EME) method is suggested for extraction and determination of propranolol and atenolol in complex biological samples. An in-house membrane based on agarose was used as green and biodegradable gel membrane. Essential chemical parameters that influence on extraction efficiency were tested, optimized and evaluated via a central composite design (CCD) and response surface methodology (RSM). Optimal conditions for extraction of drugs from the 7.0 mL sample were as follows: 3% (w/v) agarose with 0.1% (v/v) acetic acid functioning as membrane, voltage: 50 V; pH of the donor phase (DP): 8.1; pH of the AP: 3.3; extraction time: 35.9 min. Under these conditions, the acceptable normalized extraction recoveries were obtained such as 71.9 ± 5.4% that were in good agreement with the predicted values (i.e., 73.1 ± 0.9%). Limits of detection (LODs) for propranolol and atenolol were 5.0 ng mL˗1 and 7.5 ng mL˗1, respectively. Moreover, for the first time, the effect of presence of four graphene-based nanomaterials such as graphene (G), graphene oxide (GO), three-dimensional nitrogen doped graphene oxide (3D-ND-GO) and high nitrogen doped graphene oxide (HND-GO) in agarose gel membrane on extraction efficiency, was investigated. The results showed that in presence of these nanomaterials, the normalized recovery depressed significantly due to increasing of electric current and electroendosmosis (EEO) phenomenon. Eventually, the proposed method was applied to quantify basic drugs in real plasma samples with relative recoveries in the range of 85.7–97.5%, indicating good reliability of the assay.

Combination of gel-electromembrane extraction with switchable hydrophilicity solvent-based homogeneous liquid-liquid microextraction followed by gas chromatography for the extraction and determination of antidepressants in human serum, breast milk and wastewater

The current study presents for the first time a combination of the gel electromembrane extraction (GEL-EME) and switchable hydrophilicity solvent-based homogeneous liquid-liquid microextraction (SHS-HLLME) methods which can be used as an efficient hyphenated extraction procedure. This coupled method, which was followed by GC-FID, was applied for quantification of antidepressants (desipramine, clozapine, and citalopram) in biological and wastewater samples. The effective parameters of both GEL-EME and SHS-HLLME procedures were optimized. Using an agarose gel membrane, analytes were extracted from 7.0 mL of the sample solution to 500 µL of the aqueous acceptor solution. The maximum extraction of analytes of interest was obtained under the optimized conditions (pH of acceptor solution, 5.0; pH of gel membrane, 5.0; pH of sample solution, 7.0, voltage value, 30 V; and extraction time, 30 min). Then, the acceptor solution was transferred to the extraction cell and the SHS-HLLME procedure was conducted again under the optimized conditions. Dipropylamine (50 µL) was selected as an extraction solvent. The introduced technique exhibited good linearities with coefficients of determinatin (R2) higher than 0.983 and an acceptable linear range of 5.0–1000 ng/mL. Accordingly, the limit of detection was ≤ 1.0 ng/mL (S/N = 3) for all analytes, and the high enrichment factors were obtained in the range of 178.7–194.8. Moreover, the corresponding repeatability was from 4.0 to 8.7% (n = 3). The proposed method was successfully utilized to determine trace levels of the drugs in human serum, wastewater, and breast milk samples.

Inside gel electromembrane extraction: A novel green methodology for the extraction of morphine and codeine from human biological fluids

In this work, a new mode of gel-electromembrane extraction (G-EME), called “inside” gel-EME (IG-EME) is proposed for the extraction of morphine and codeine as model basic drugs from complex biological samples. Here, an aqueous media that was captured inside the agarose gel membrane, acted as both gel membrane and the acceptor phase (AP) at the same time. In this regard, the membrane served as the separation filter (membrane) and supported liquid acceptor phase (SLAP) as well. With this new development, unwanted changes of the AP volume during the extraction, which is a common issue in the G-EME (due to electroendosmosis (EEO) phenomenon), was addressed properly. Briefly, the setup involved insertion of negative electrode inside the gel membrane and positive electrode into the donor phase (DP). Following that, the IG-EME was easily performed using optimal conditions (pH of the DP: 6.0; membrane composition (agarose concentration: 1% (w/v) in aqueous media with pH 3.0, and 15 mm thickness); voltage: 25 V; and extraction time: 30 min). After extraction, the agarose gel was withdrawn and centrifuged for 5 min with 12000 rpm, to disrupt its framework to release the “trapped aqueous AP” apart from the gel structure. The separated AP was finally injected into the HPLC-UV for the analysis. The limits of detection (LODs) and recoveries in this proposed method were obtained 1.5 ng mL−1 and 67.7 %–73.8 %, respectively. The system feasibility was examined by the quantification of model drugs in the real plasma and urine samples.

Gel electromembrane extraction: Study of various gel types and compositions toward diminishing the electroendosmosis flow

For the first time, the effect of electroendosmosis (EEO) flow phenomenon was investigated in detail, on the gel electromembrane extraction (G-EME). When electric field is used as an actuator of ion transport in G-EME, the EEO flow phenomenon can occur within the agarose gel membrane interface and is generally considered the dominant issue. In this regard, various agarose-based gel membrane types with low-, medium- and high-EEO were fabricated and tested. To further diminish the EEO effect, the different gel membrane additives such as dextrin, chitosan and xylan with different concentrations i.e., 5%, 10% and 15% were also examined. The positively charged Cr(III) and negatively charged Cr(VI) were chosen as model analytes. The Cr(III) and Cr(VI) were simultaneously extracted from an aqueous sample (pH 3.0) via the cathodic and anodic gel membranes (pH 3.0 for both membranes), into the cathodic and anodic aqueous acceptors (200 µL each, pH 2.0), respectively. After extraction at the optimal conditions (i.e., voltage: 32 V, extraction time: 22 min), the both compounds were quantified by a cheap and easy-to-perform reader platform termed as microfluidic paper-based analytical device (μPAD). The results showed that high extraction recoveries such as 87% for Cr(III) and 75% for Cr(VI), were acquired when the low-EEO agarose gel membrane with 5% (w/v) dextrin was used. On the other hand, using the high-EEO agarose gel membrane with no additive lead to the high EEO flow and the increase of cathodic acceptor volume (equal to 350 µL) while the recovery depressed to about 50% due to the dilution effect. Simultaneously, the volume of anodic acceptor became almost vacant (~50 µL). Ultimately, by means of such simple gel membrane adjusting (i.e., low-EEO agarose with 5% dextrin), the both target compounds were quantitatively extracted at a single step achieving limits of detection (LODs) of 0.5 and 0.7 ng mL−1 for Cr(III) and Cr(VI), respectively.

Evaluation of dispersive liquid–liquid microextraction by coupling with green-based agarose gel-electromembrane extraction: An efficient method to the tandem extraction of basic drugs from biological fluids

Nowadays, developing new methods for the effective extraction/separation of drugs (present at trace levels) from complicated matrices (as biological fluids) is certainly a great challenge for many operators. In this regard, green-based agarose gel electromembrane extraction (G-EME) was for the first time combined with dispersive liquid–liquid microextraction (DLLME) toward G-EME/DLLME methodology (i.e., tandem extraction approach). Two basic drugs such as trimipramine (TRI) and clomipramine (CLO) extracted from the urine samples, were used as model compounds. Regarding method workflow, analytes were extracted from the 5 mL sample, through a synthesized agarose gel membrane, to the 700 µL aqueous acceptor phase under the optimized conditions (pH of acceptor phase: 2.0; pH of gel membrane: 2.0; pH of donor phase: 4.0, voltage value: 30 V, and extraction time: 25 min). In the next step, acceptor solution was poured to a conic vial and mixed with 100 µL alkaline solution (NaOH, 1 M). Afterwards, DLLME procedure took place again at optimal conditions, i.e., extraction solvent was carbon tetrachloride (10 µL), and dispersive solvent was acetone (100 µL). Ultimately, gas chromatography (GC) was applied for the detection and quantification of drugs. Such G-EME/DLLME configuration has brought two main advantages. Firstly, interferences such as proteins and other large biological molecules were eliminated from biological fluids via G-EME. Further, high enrichment factors (EFs of 260–370 refer to extraction recoveries of 52–74%) were obtained using DLLME with acceptable detection limits (1.0–3.0 ng mL−1). Finally, the suggested approach was successfully utilized to determine drugs at trace levels in urine samples.

Three-Electrode on-Chip Sensors for Voltammetric Detection of Trace Metals in Natural Waters

This paper reports on the fabrication and electrochemical characterization of newly designed three-electrode on-chip devices for in situ voltammetric detection of trace metals in marine environment. Sensitivity values higher than those previously reported in literature were obtained for cadmium (Cd(II)), lead (Pb(II)), copper (Cu(II)), zinc (Zn(II)) [1] and arsenite (As(III)) [2]. The key novelty of these devices is the on-chip integration of a reference electrode (RE) towards the miniaturization of the voltammetric in situ profiling system [3].Compared to the microdisc arrays reported by Belmont et al. [1], [4], our sensors also differ in number of iridium (Ir) microdiscs (190 vs. 100), center to center distance of the microdiscs, as well as deposition/patterning techniques used during the fabrication. The sensors were fabricated on silicon wafers passivated with silicon nitride (Si3N4). Ir deposited by sputtering was patterned by Ion Beam Etching, followed by a Si3N4layer deposited by Plasma-Enhanced Chemical Vapor Deposition and subsequently patterned by Deep Reactive-Ion Etching to define the microelectrodes and bonding pads. Ir electrodes were used as either counter electrode (CE) without further modification or as a seed layer for the fabrication of the working and reference electrode (WE, RE). The WE was obtained by plating Ir microdisc array with mercury or gold nanoparticles for direct quantification of respectively cadmium, lead, copper , zinc [5] and arsenite [2], the RE was realized by plating the Ir with silver (Ag) by galvanostatic electrodeposition and chloridizing Ag to silver chloride (AgCl). A schematic view of the sensor is shown in Figure 1 as well as a picture of the microfabricated device. The electrode surface topography and cleanness were analyzed by Scanning Electron Microscopy (SEM) showing an excellent quality of the Si3N4 layer patterning in terms of geometry definition and absence of fabrication residue (as seen in Figure 2). The material properties and the electrochemical behavior of the Ir-based electrodes were characterized by cyclic voltammetry: the integrity of the thin-film Ir layer was assessed in degassed 1 M H2SO4(Figure 3a) and the absence of shielding effect was demonstrated in 1 mM potassium ferricyanide at different scan rates from 1 mV/s to 50 mV/s, as shown by the S-shaped current response in Figure 3b. The measured diffusion controlled steady-state current (118.9±2.24 nA) was found to be in good agreement with the theoretical value calculated for a recessed geometry (119.5 nA). Afterwards SU-8 was patterned by photolithography around the WE and RE to serve as containment ring for an antifouling agarose gel membrane. The pseudo-reference Ag/AgCl RE was tested in seawater showing a potential of about 37 mV against an Ag/AgCl/3 M KCl RE stable over more than 2 months as shown in Figure 4, and in synthetic solutions with different chloride concentrations giving a potential slope of 50.1 mV/decade at 25 °C. The use of an Ionic Liquid (IL) poly(vinyl chloride) (PVC) membrane [6] is currently under investigation in order to develop an all-solid-state RE towards a versatile use in brackish water as well. The first Square Wave Anodic Stripping Voltammetry (SWASV) measurements were carried out in synthetic solutions of Pb(II), Cu(II) and Cd(II) or As(III) against an external RE. Figure 5 shows the SWASV measurements obtained for different concentrations of Pb(II), Cd(II) and Cu(II) in 0.1 M NaNO3 background electrolyte for 600 s deposition time. Registered sensitivity values were 2.73 nAnM-1 min-1, 1.23 nAnM-1 min-1, 1.29 nAnM-1 min-1 and 0.6 nAnM-1 min-1for Pb(II), Cu(II), Cd(II) and As(III), respectively. SWASV measurements against on-chip RE are currently under investigation.

Microencapsulated Aliivibrio fischeri in alginate microspheres for monitoring heavy metal toxicity in environmental waters.

In this article a luminescence fiber optic biosensor for the microdetection of heavy metal toxicity in waters based on the marine bacterium Aliivibrio fischeri (A. fischeri) encapsulated in alginate microspheres is described. Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(II) were selected as sample toxic heavy metal ions for evaluation of the performance of this toxicity microbiosensor. The loss of bioluminescence response from immobilized A. fischeri bacterial cells corresponds to changes in the toxicity levels. The inhibition of the luminescent biosensor response collected at excitation and emission wavelengths of 287 ± 2 nm and 487 ± 2 nm, respectively, was found to be reproducible and repeatable within the relative standard deviation (RSD) range of 2.4–5.7% (n = 8). The toxicity biosensor based on alginate micropsheres exhibited a lower limit of detection (LOD) for Cu(II) (6.40 μg/L), Cd(II) (1.56 μg/L), Pb(II) (47 μg/L), Ag(I) (18 μg/L) than Zn(II) (320 μg/L), Cr(VI) (1,000 μg/L), Co(II) (1700 μg/L), Ni(II) (2800 μg/L), and Fe(III) (3100 μg/L). Such LOD values are lower when compared with other previous reported whole cell toxicity biosensors using agar gel, agarose gel and cellulose membrane biomatrices used for the immobilization of bacterial cells. The A. fischeri bacteria microencapsulated in alginate biopolymer could maintain their metabolic activity for a prolonged period of up to six weeks without any noticeable changes in the bioluminescence response. The bioluminescent biosensor could also be used for the determination of antagonistic toxicity levels for toxicant mixtures. A comparison of the results obtained by atomic absorption spectroscopy (AAS) and using the proposed luminescent A. fischeri-based biosensor suggests that the optical toxicity biosensor can be used for quantitative microdetermination of heavy metal toxicity in environmental water samples.

Influence of gelling properties on protein imprinted agarose gel membrane

ABSTRACTA method based on molecular imprinting technique was presented for preparing protein‐imprinted agarose gel membrane (AGM) under moderate conditions, and the influencing factors such as molecular weights and modified chemical groups on the adsorption ability and selectivity of AGMs were investigated. The agaroses used for AGMs were prepared through ultrasonic degradation, oxidation degradation, gel‐melting method, and sulfation, respectively. Bovine serum albumin (BSA) and hemoglobin were selectively recognized on AGMs. Results showed that the molecular weight was the most crucial influencing factor for the protein recognition ability of AGMs. The lower and upper limit of molecular weight was 100 and 130 kDa, respectively, where the AGMs could maintain both good mechanical strength and high recognition ability, with K value around 4.0. The enhancement of ionic strength could make the imprinting effect disappeared even when the concentration of salt was as low as 2 mmol/L. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40323.

Synthesis of tellurium nanotubes via a green approach for detection and removal of mercury ions

In the present study, we develop a green approach for the preparation of tellurium nanotubes (Te NTs) by a simple fructose-mediated reduction of Te ions in an alkaline aqueous solution. The influence of reaction conditions, including the types of saccharides involved, reaction temperature, and fructose and NaOH concentrations on the size and morphology of Te nanostructures are investigated and the structural evolution with respect to different growth rates are illustrated. It is proposed that a low reduction reaction rate plays a significant role in the controlled growth mechanism of Te NTs. A simple gel-based membrane is also developed for the removal and sensing of mercury ions (Hg2+) in aqueous solutions. An agarose gel is used to trap as-prepared Te NTs, leading to the preparation of a nanocomposite film composed of Te NTs-modified agarose gel membrane (Te NTs–AGM) for removing Hg2+ in the solution via the strong hybridization and galvanic replacement reaction of Te–Hg. The Te NTs–AGM adsorbent allows effective removal of mercury species spiked in tap, stream, and sea waters with efficiencies greater than 97%. In addition, Te NTs allow for the rapid and simple detection of Hg2+ at concentrations as low as 10 nM. We validate the practicality of the use of Te NTs–AGM in this application through analyses of Hg2+ in tap water, stream water, and sea water samples. The low-cost, effective, and stable Te NTs–AGM nanocomposite shows great potential for detection and economical removal of Hg2+.

Detection and removal of mercury and lead ions by using gold nanoparticle-based gel membrane

This paper describes the use of two types of bovine serum albumin (BSA)-capped 14.2 nm diameter gold nanoparticles (Au NPs) for the separate detection of mercury (Hg2+) and lead (Pb2+) ions in highly saline media. The BSA-capped Au NPs were stable in solutions containing up to 500 mM NaCl. Introduction of BSA to a solution of rhodamine 6G (R6G) and 3-mercaptopropionic acid (MPA)-modified Au NPs (R6G/MPA–Au NPs) provided a R6G/MPA–Au NP@BSA probe for the sensing of Hg2+ ions. We also used BSA-capped Au NPs to detect Pb2+ ions through a mechanism based on Pb2+ ions accelerating the leaching rate of Au NPs in the presence of thiosulfate (S2O32−) and 2-mercaptoethanol (2-ME). The resulting deposition of Hg2+ ions onto the Au NPs induced the release of R6G from the surfaces of the Au NPs, causing increased fluorescence from the R6G/MPA–Au NP@BSA solution. The Pb2+ ions accelerated the dissolution of the 2-ME/S2O32−–Au NPs@BSA into solution, leading to dramatic decreases in the absorption. These two Au NP-based probes were highly sensitive (LOD ≈ nM) and selective (over 100-fold against other metal ions) toward Hg2+ or Pb2+ ions. We validated the practicality of these two probes through analyses of seawater and urine samples. We also developed a simple gel-based membrane for removal and sensing of Hg2+ or Pb2+ in aqueous solutions. The agarose gel was used to trap BSA-Au NPs, leading to the preparation of a nanocomposite film of Au NPs@BSA-decorated agarose gel membrane (Au NPs@BSA/AGM) for removing Hg2+ or Pb2+ in solution. In addition, R6G/MPA–Au NP@BSA-trapped agarose gel membrane (R6G/MPA–Au NP@BSA/AGM) and 2-ME/S2O32−–Au NPs@BSA/AGM allowed for the rapid and simple detection of Hg2+ and Pb2+, respectively.

Diffusion of bacteriophages through artificial biofilm models

The simple two-chamber diffusion method was improved to study the diffusion properties of bacteriophage (phage) T4 through a model biofilm agarose gel membrane (AGM) embedded with dead host Escherichia coli K12 cells. The apparent diffusion coefficient (D(app) ) of phage T4 was calculated to be 2.4 × 10(-12) m(2) /s in 0.5% AGM, which was lower than the coefficient of 4.2 × 10(-12) m(2) /s in 0.5% AGM without host cells. The phage adsorption process by dead host cells slowed the apparent phage diffusion. The Langmuir adsorption equation was used to simulate phage adsorption under different multiplicity of infections (MOIs); the maximum adsorbed phage MOI was calculated to be 417 PFU/CFU, and the Langmuir adsorption constant K(L) was 6.9 × 10(-4) CFU/PFU. To evaluate the effects of phage proliferation on diffusion, a simple syringe-based biofilm model was developed. The phage was added into this homogenous biofilm model when the host cells were in an exponential growth phase, and the apparent diffusion coefficient was greatly enhanced. We concluded that D(app) of phages through biofilms could be distinctly affected by phage adsorption and proliferation, and that the idea of D(app) and these methods can be used to study diffusion properties through real biofilms.

Diffusion properties of bacteriophages through agarose gel membrane

A simple two-chamber diffusion method was developed to study the diffusion properties of bacteriophages (phages). The apparent diffusion coefficients (D(app)) of Myoviridae phage T4 and filamentous phage fNEL were investigated, and the diffusion of the phages was found to be much slower than the diffusion of three antibiotics, ciprofloxacin, penicillin G, and tetracycline. D(app) of T4 and fNEL in water through filter paper were calculated to be 2.8 x 10⁻¹¹ m²/s and 6.8 x 10⁻¹² m²/s, respectively, and D(app) of fNEL through agarose gel membrane, an artificial biofilm, was also calculated to be smaller than that of T4. In addition, D(app) of phages through agarose gel was dependent on agarose concentration due to the similar size of phage and agarose gel mesh. We concluded that D(app) of phages through an artificial biofilm is dependent on both phage morphology and biofilm density, and suggest the use of this method to study diffusion properties through real biofilms.

Small Agarose Microcapsules with Cell-Enclosing Hollow Core for Cell Therapy: Transplantation of Ifosfamide-Activating Cells to the Mice with Preestablished Subcutaneous Tumor

Cell transplantation after enclosing in microcapsules has been studied as an alternative approach for treatment of wide variety of diseases. In the present study, we examined the feasibility of using agarose microcapsules, having a cell-enclosing hollow core of 100-150 microm in diameter and agarose gel membrane of about 20 microm in thickness, as a device for the methodology. We enclosed cells that had been genetically engineered to express cytochrome P450 2B1, an enzyme that activates the anticancer prodrug ifosfamide. The enclosed cells were shown to express the enzymatic function in the microcapsules in that they suppressed the growth of tumor cells in medium containing ifosfamide. In addition, a more significant regression of preformed tumors was observed in the nude mice implanted with the cell-enclosing microcapsules compared with those implanted with empty capsules after administration of ifosfamide. Preformed tumors shrank by less than 40% in volume in 6 of the 10 recipients implanted with cell-enclosing microcapsules. In contrast, only 1 in 10 of the preformed tumors in the recipient implanted with empty microcapsules shrank by this amount. These results suggest that agarose microcapsules containing cytochrome P450 2B1 enzyme-expressing cells are feasible devices for improving the chemotherapy of tumors. Thus, agarose microcapsule having hollow cores are generally a good candidate as vehicles for cell-encapsulation approaches to cell therapy.

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.