Simple method for detection of extremely diluted anti beta-casein antibodies from glass bead based receptors

The current work describes the elaboration of a simple, sensitive and reliable β-casein modified glass beads, for the detection and quantification of its specific antibody anti β-casein. This is an elementary receptor without electronic part, developed by grafting glass bead surface with the antigenic β-casein via 3-aminopropyltriethoxy silane and then glutaraldehyde as cross-linker. The whole is realized by a classical process, called in two steps and in mild conditions where chemical protocol is optimized for β-casein use. The detection and quantification of the specific reaction antibody-receptor is carried out by the technique of the second antibody labeled with horse radish peroxidase (HRP). Our receptor can detect the β-casein antibody present in the serum at dilutions up to a factor 10⁷ in strong ionic strength medium. The same antibody of the same serum and in the same conditions can be detected by ELISA test at dilutions up to a factor 10⁵. The whole test, after our receptor realization, takes about 5 h.     

Different Strategies of Covalent Attachment of Oligonucleotide Probe onto Glass Beads and the Hybridization Properties

The glass bead is a new biochip support material for immobilization biomolecules, due to its independence and convenient rearrangement. In order to optimize the immobilization efficiency of oligonucleotides onto glass beads and obtain the highest hybridization efficiency, three commonly used coupling strategies have been studied for covalently attaching oligonucleotides onto large glass beads. Glass beads with 250 μm diameter were amino-silaned with 2% 3-aminopropyltrimethoxysilane (APTMS) and then reacted separately with glutaraldehyde, succinic anhydride and 1,4-phenylene diisothiocyanate (PDITC) to derive CHO beads, COOH beads and isothiocyanate-modified beads (NCS-Beads) accordingly. Afterwards, amino-terminal oligonucleotides were covalently attached onto the surface of beads achieved by three strategies mentioned above. The immobilization efficiency were studied to compare the three strategies, which turned out 2.55 × 10¹³ probes/cm² for CHO-Beads, 3.21 × 10¹³ probes/cm² for COOH beads and 6.68 × 10¹³ probes/cm² for NCS beads. It meant that the immobilization efficiency based on NCS beads was most acceptable. And the method, developed by attaching amino-terminal oligonucleotides onto these cyanate active beads, could be regarded as an efficient one for immobilizing oligonucleotides onto a solid surface. Moreover, in this paper, the hybridization properties of NCS bead-based oligonucleotides have been studied by employing Cy5-tagged complementary oligonucleotides. It was found that the high probe density NCS beads led to low hybridization efficiency possibly due to the existence of steric crowding. In addition, the equilibrium binding constant K _A was determined by employing Langmuir isotherm model, which was 7.0 × 10⁶ M⁻¹ for NCS beads with the density of 6.7 × 10¹³ probes/cm². Furthermore, it only took 60 min to reach hybridization equilibrium. These large microspheres (>100 μm) can be employed in the mesofluidic systems for automated heterogeneous assays.     

Development of hydrogen peroxide biosensor based on in situ covalent immobilization of horseradish peroxidase by one-pot polysaccharide-incorporated sol-gel process

A simple and reliable one-pot approach was established for the development of a novel hydrogen peroxide (H₂O₂) biosensor based on in situ covalent immobilization of horseradish peroxidase (HRP) into biocompatible material through polysaccharide-incorporated sol-gel process. Siloxane with epoxide ring and trimethoxy anchor groups was applied as the bifunctional cross-linker and the inorganic resource for organic-inorganic hybridization. The reactivity between amine groups and epoxy groups allowed the covalent incorporation of HRP and the functional biopolymer, chitosan (CS) into the inorganic polysiloxane network. Some experimental variables, such as mass ratio of siloxane to CS, pH of measuring solution and applied potential for detection were optimized. HRP covalently immobilized in the hybrid matrix possessed high electrocatalytic activity to H₂O₂ and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H₂O₂ ranged from 2.0 × 10⁻⁷ to 4.6 × 10⁻⁵ mol l⁻¹ with a detection limit of 8.1 × 10⁻⁸ mol l⁻¹. The apparent Michaelis-Menten constant was determined to be 45.18 μmol l⁻¹. Performance of the biosensor was also evaluated with respect to possible interferences. The fabricated biosensor exhibited high reproducibility and storage stability. The ease of the one-pot covalent immobilization and the biocompatible hybrid matrix serve as a versatile platform for enzyme immobilization and biosensor fabricating.     

Preparation of a new Cd(II)-imprinted polymer and its application to determination of cadmium(II) via flow-injection-flame atomic absorption spectrometry

A new cadmium(II)-imprinted polymer based on cadmium(II) 2,2′-{ethane-1,2-diylbis[nitrilo(E)methylylidene]} diphenolate-4-vinylpyridine complex was obtained via suspension polymerization. The beads were used as a minicolumn packing for flow-injection-flame atomic absorption spectrometry (FI-FAAS) determination of cadmium(II) in water samples. Sorption effectiveness was optimal within pH range of 6.6-7.7. Nitric acid, 0.5% (v/v) was used as eluent. Fast cadmium(II) sorption by the proposed material enabled to apply sample flow rates up to 10 mL min⁻¹ without loss in sorption effectiveness. Enrichment factor (EF), concentration efficiency (CE) and limit of detection (LOD, 3σ) found for 120-s sorption time were 117, 39.1 min⁻¹ and 0.11 μg L⁻¹, respectively. Sorbent stability was proved for at least 100 preconcentration cycles (RSD = 2.9%). When compared to non-imprinted polymer the new Cd(II)-imprinted polymer exhibited improved selectivity towards cadmium(II) against other heavy metal ions, especially Cu(II) and Pb(II), as well as light metal ions. Accuracy of the method was tested for ground water and waste water certified reference materials and fortified water. The method was applied to Cd(II) determination in natural water samples.     

Protein microarrays enhanced performance using nanobeads arraying and polymer coating

A nanosize material composed of 330 nm glass beads coated with a copolymer of N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS) and [3-(methacryloyl-oxy)propyl]trimethoxysilane (MAPS) was developed to improve the protein immobilization on biochips. The developed material, bearing rabbit-IgG proteins, was arrayed as 150 μm spots trapped at the surface of a poly(dimethylsiloxane) elastomer (PDMS), and compared to copoly(DMA-NAS-MAPS)-coated glass slides and latex beads based biochips. Evidences were made through scanning electron microscopy that the newly developed material based microarray exhibited surface irregularities at the submicron level leading to high specific area.The combination of such large immobilization area with the highly efficient protein immobilization of the copoly(DMA-NAS-MAPS) polymer, enabled the achievement of microarrays exhibiting good performances both in pure media and complex samples (human sera). Indeed, high specific/non-specific signal ratio was found using this optimized immobilization procedure.Chemiluminescent detection of anti-rabbit-IgG was obtained through peroxidase labeled antibodies in the 5 μg/l to 10 mg/l range. Application of the developed system to real samples was achieved for the detection of rheumatoid factor (RF) through a capture assay. Interesting results were obtained, with a RF detection over the 5.3-485 IU/ml range and without measurable matrix effect or non-specific signal.     

Enzyme immobilization procedures on screen-printed electrodes used for the detection of anticholinesterase pesticides: Comparative study

A comparison between several acetylcholinesterase (AChE) immobilization procedures on the 7,7,8,8-tetracyanoquinodimethane (TCNQ)-modified graphite working electrodes is presented. The immobilization methods employed crosslinking with glutaraldehyde in presence of BSA protein and photopolymerization with poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ). The main variations were related to the enzyme charge in each electrode and the enzyme conditioning and storage conditions after immobilization. Initially, the enzyme-substrate reaction was carried out and the following parameters were chrono-amperometrically and -coulometrically monitored: current intensities, time to stabilize the current response, and the mass transfer represented by the Coulomb charge. The screen-printed biosensors that presented best characteristics were then used to perform the inhibition assays and to verify the sensitivity against the following NMC insecticides: aldicarb, carbaryl, carbofuran, and methomyl.In general, diffusion of electrons into the sensitive layer, mass transfer, and time to stabilize the current were adequate in all cases. The Cottrell law was followed before the 1 min of enzyme-substrate reaction. Adequate reproducibility within electrochemical measurements was also observed, with relative standard deviations varying from 6.5 to 18.6%.AChE immobilization with glutaraldehyde allow to obtain robust and reproducible biosensors, but they need a much higher enzyme content (80 mUA per electrode) to achieve current values comparable to that constructed by immobilizing the AChE through photopolymerization with PVA-SbQ (0.7 to 1 mUA per electrode). The limits of detection were determined with a minimum 10% inhibition, and varied from 10⁻⁹ to 8×10⁻⁹ M (0.2 to 1.5 ppb) by employing the enzyme immobilization through photopolymerization with PVA-SbQ. In practice, this kind of immobilization procedure is much simpler and produces good results: fast response, adequate reproducibility, large pesticides working ranges, and excellent sensitivities to N-methylcarbamates (NMCs) which in general do not present enzyme inhibition power as elevated as for the organophosphate pesticides.     

A novel fluorescence sensor based on covalent immobilization of 3-amino-9-ethylcarbazole by using silver nanoparticles as bridges and carriers

A novel technique of covalent immobilization of indicator dyes in the preparation of fluorescence sensors is developed. Silver nanoparticles are used as bridges and carriers for anchoring indicator dyes. 3-amino-9-ethylcarbazole (AEC) was employed as an example of indicator dyes with terminal amino groups and covalently immobilized onto the outmost surface of a quartz glass slide. First, the glass slide was functionalized by (3-mercaptopropyl) trimethoxysilane (MPS) to form a thiol-terminated self-assembled monolayer, where silver nanoparticles were strongly bound to the surface through covalent bonding. Then, 16-mercaptohexadecanoic acid (MHDA) was self-assembled to bring carboxylic groups onto the surface of silver nanoparticles. A further activation by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) converted the carboxylic groups into succinimide esters. Finally, the active succinimide esters on the surface of silver nanoparticles were reacted with AEC. Thus, AEC was covalently bound to the glass slide and an AEC-immobilized sensor was obtained. The sensor exhibited very satisfactory reproducibility and reversibility, rapid response and no dye-leaching. Rutin can quench the fluorescence intensity of the sensor and be measured by using the sensor. The linear response of the sensor to rutin covers the range from 2.0 × 10⁻⁶ to 1.5 × 10⁻⁴ mol L⁻¹ with a detection limit of 8.0 × 10⁻⁷ mol L⁻¹. The proposed technique may be feasible to the covalent immobilization of other dyes with primary amino groups.     

A new flow injection preconcentration method based on multiwalled carbon nanotubes for the ETA-AAS determination of Cd in urine

A new flow injection (FIA) procedure for the preconcentration of cadmium in urine using multiwalled carbon nanotubes (MWCNT) as sorbent and posterior electrothermal atomization atomic absorption spectrometry (ETA-AAS) Cd determination has been developed. Cadmium was retained in a column filled with previously oxidized MWCNTs and it was quantitatively eluted with a nitric acid solution. The parameters influencing the adsorption-elution process such as pH of the sample solution, amount of sorbent and flow rates of sample as well as eluent solutions have been studied. Cd concentration in the eluent was measured by ETA-AAS under the optimized conditions obtained. The results indicated the elimination of urine matrix effect as a consequence of the preconcentration process performed. Total recovery of cadmium from urine at pH 7.2 using a column with 45 mg of MWCNTs as sorbent and employing a HNO₃ 0.5 mol L⁻¹ solution for elution was attained. The detection limit obtained was 0.010 μg L⁻¹ and the preconcentration factor achieved was 3.4. The method showed adequate precision (RSD: 3.4-9.8%) and accuracy (mean recovery: 97.4-100%). The developed method was applied for the determination of cadmium in real urine samples from healthy people (in the range of 0.14-2.94 μg L⁻¹) with satisfactory results.     

Determination of cadmium by an improved double chamber electrothermal vaporization inductively coupled plasma atomic emission spectrometry

An improved double chamber electrothermal vaporization (ETV) system was designed. A new inner chamber and its bottom plate made of quartz glass were attached with carrier support gas inlet port for the determination of cadmium by inductively coupled plasma atomic emission spectrometry (ICP-AES). The use of the inner chamber in combination with the plate played important roles to transport the metal vapor efficiently into argon ICP. Ten-μl sample aliquots were dried at 100 °C and subsequently heated at 1000 °C on the tungsten boat furnace. The evolved vapor was swept into the ICP source through PTFE tubing and the inner chamber by a 0.8 l/min H₂ (7%)-Ar carrier gas. The performance parameters of ETV-ICP-AES such as temperature program and gas flow rate were evaluated using cadmium standard solution. Under the optimized experimental conditions, the best attainable detection limit at Cd II 214.438 nm line was 0.2 ng/ml with linear dynamic ranges of 50 to 10,000 ng/ml for cadmium. The instrumental precision expressed as the relative standard deviation (RSD) from ten replicate measurements of 10,000 ng/ml for cadmium by ETV-ICP-AES was 0.85%. The present method has been successfully applied to the determination of cadmium in zinc-base materials.     

Energy dispersive X-ray fluorescence determination of cadmium in uranium matrix using Cd Kα line excited by continuum

An energy dispersive X-ray fluorescence method for determination of cadmium (Cd) in uranium (U) matrix using continuum source of excitation was developed. Calibration and sample solutions of cadmium, with and without uranium were prepared by mixing different volumes of standard solutions of cadmium and uranyl nitrate, both prepared in suprapure nitric acid. The concentration of Cd in calibration solutions and samples was in the range of 6 to 90 µg/mL whereas the concentration of Cd with respect to U ranged from 90 to 700 µg/g of U. From the calibration solutions and samples containing uranium, the major matrix uranium was selectively extracted using 30% tri-n-butyl phosphate in dodecane. Fixed volumes (1.5 mL) of aqueous phases thus obtained were taken directly in specially designed in-house fabricated leak proof Perspex sample cells for the energy dispersive X-ray fluorescence measurements and calibration plots were made by plotting Cd Kα intensity against respective Cd concentration. For the calibration solutions not having uranium, the energy dispersive X-ray fluorescence spectra were measured without any extraction and Cd calibration plots were made accordingly. The results obtained showed a precision of 2% (1σ) and the results deviated from the expected values by < 4% on average.     

Preparation and application of triangular silver nanoplates/chitosan composite in surface plasmon resonance biosensing

This paper reports the utilization of triangular silver nanoplates (TSNPs) to enhance the sensitivity of surface plasmon resonance (SPR) biosensor. TSNPs modified with 3-mercaptopropinic acid (MPA) were simply mixed with chitosan and glutaraldehyde to form TSNPs/chitosan composite. The composite was deposited on Au film as immobilization substrate for SPR biosensor. The novel structures of TSNPs are preserved against etching by MPA and chitosan polymer. Moreover, chitosan cross-linked by glutaraldehyde enables antibody to be immobilized on fabricated substrate directly via Schiff alkali reaction. In the optimized conditions, the resulting biosensor based on TSNPs/chitosan composite shows a satisfactory response to bovine IgG in the concentration range of 0.075–40.00 μg mL⁻¹. While the biosensor based on chitosan without TSNPs shows a response in the concentration range of 0.6–40 μg mL⁻¹ and the biosensor based on Au film shows a response in the concentration range of 2.5–40 μg mL⁻¹. The experiment results show that the sensitivity of SPR biosensor based on TSNPs/chitosan composite was significantly enhanced and the immobilization procedure of antibody was simplified.     

Electrochemical immunosensor designs for the determination of Staphylococcus aureus using 3,3-dithiodipropionic acid di(N-succinimidyl ester)-modified gold electrodes

The preparation of novel Staphylococcus aureus (S. aureus) amperometric immunosensing designs based on the covalent immobilization of RbIgG at gold electrodes using the heterobifunctional cross-linker 3,3-dithiodipropionic acid di(N-succinimidyl ester) (DTSP), are reported. Two different competitive immunosensing configurations have been tested and compared. In the first one, protein A-bearing S. aureus cells and HRP-labelled antiRbIgG compete for immobilized RbIgG binding sites, while in the second case HRP-labelled protein A was used. In both cases, the evaluation of the developed immunosensors performance was accomplished through the monitoring at 0.00 V (vs. Ag/AgCl) of the catalytic current originated after addition of hydrogen peroxide, using tetrathiafulvalene as redox mediator entrapped at the modified electrode surface by cross-linking with glutaraldehyde. Optimization of variables concerning the composition of the immunosensors as well as the detection conditions was carried out in 0.1 M NaAc/0.1 M NaCl buffer of pH 5.6. The configuration that employed antiRbIgG-HRP resulted in better analytical characteristics, with a detection limit of 1.4 × 10⁴ cells mL⁻¹ for S. aureus cells submitted to wall lyses by ultrasonic treatment. This immunosensor design was also evaluated using gold screen-printed electrodes in order to reduce the analysis time and cost. In this case, a limit of detection of 3.7 × 10² cells mL⁻¹ and a dynamic range from 1.3 × 10³ to 7.6 × 10⁴ cells mL⁻¹ was obtained. A RSD value of 10.5% was found for the responses to 9.6 × 10³S. aureus cells mL⁻¹ obtained with seven different Au/SPEs-immunosensors. These disposable immunosensors were applied to the quantification of S. aureus in milk spiked at two concentration levels, 1.2 × 10³ and 4.8 × 10³ cells mL⁻¹, with good recoveries.     

The use of immobilized alcohol oxidase in the continuous flow determination of ethanol with an oxygen electrode

Immobilized alcohol oxidase was used in the determination of blood alcohol. The alcohol oxidase catalyzed the aerobic oxidation of ethanol and the oxygen concentration was monitored with an oxygen membrane electrode in a flow cell. The enzyme was immobilized either by covalent attachment via glutaraldehyde to the inside walls of nylon tubing, or by adsorption onto three separate controlled-pore glass support materials: TiO₂, SiO₂, or AL₂O₃. The supports were packed into 10 cm lengths of 3 mm i.d. glass tubing or 30 cm lengths of 5 mm i.d. nylon tubing. The five methods of immobilization were compared for stability and activity toward ethanol. Immobilization on silanized glass beads results in the highest activity and greatest stability of the reactor.     

Immobilization of Acidithiobacillus ferrooxidans with complex of PVA and sodium alginate

A new Acidithiobacillus ferrooxidans cell immobilization technique utilizing the complex of PVA solution and sodium alginate solution crosslinked by Ca(NO₃)₂ as entrapment medium is reported. The mixture of A. ferrooxidans suspension and the entrapment complex were extruded into a solution of Ca(NO₃)₂ (1-5%) to form beads, then the beads were frozen at −20 °C for 1-2 days and thawed at room temperature. The forming mechanism, characteristic of this immobilized beads and the factors affecting activity of immobilized cells were also discussed. A maximum oxidation rate of 4.6 g Fe²⁺/(L h) was achieved in batch cultures by these immobilized cells. Precipitation formed during culture process was analyzed. The forming mechanism of this precipitation and how this precipitation affects the whole system were also discussed. In addition, the immobilization technique is operated simply, and the gel beads have high stability even under non-sterile conditions. So its application on an industrial scale would be more practicable.     

Optimization of Enzyme Co-Immobilization with Sodium Alginate and Glutaraldehyde-Activated Chitosan Beads

In this study, two different materials—alginate and glutaraldehyde-activated chitosan beads—were used for the co-immobilization of α-amylase, protease, and pectinase. Firstly, optimization of multienzyme immobilization with Na alginate beads was carried out. Optimum Na alginate and CaCl₂ concentration were found to be 2.5% and 0.1 M, respectively, and optimal enzyme loading ratio was determined as 2:1:0.02 for pectinase, protease, and α-amylase, respectively. Next, the immobilization of multiple enzymes on glutaraldehyde-activated chitosan beads was optimized (3% chitosan concentration, 0.25% glutaraldehyde with 3 h of activation and 3 h of coupling time). While co-immobilization was successfully performed with both materials, the specific activities of enzymes were found to be higher for the enzymes co-immobilized with glutaraldehyde-activated chitosan beads. In this process, glutaraldehyde was acting as a spacer arm. SEM and FTIR were used for the characterization of activated chitosan beads. Moreover, pectinase and α-amylase enzymes immobilized with chitosan beads were also found to have higher activity than their free forms. Three different enzymes were co-immobilized with these two materials for the first time in this study.     

Clearance of host cell impurities from plasmid-containing lysates by boronate adsorption

The ability of boronate adsorption to clear Escherichia coli impurities directly from plasmid-containing lysates (∼pH 5.2) was evaluated. Results show that 3-aminophenyl boronate (PB) controlled pore glass (CPG) is able to adsorb not only those species that bear cis-diol groups (RNA, lipopolysaccharides-LPS), and are thus able to form covalent bonds with boronate, but also cis-diol-free proteins and genomic DNA (gDNA) fragments, while leaving most plasmid DNA in solution. Control runs performed with phenyl Sepharose and with PB-free CPG beads ruled out hydrophobic interactions with the phenyl ring and non-specific interactions with the glass matrix, respectively, as being responsible for RNA and gDNA adsorption. In batch mode, up to 97.6 ± 3.1% of RNA, 94.6 ± 0.8% of proteins and 96.7 ± 11.7% of gDNA were cleared after 30 min, with a plasmid yield of 64%. In fixed-bed mode, most of the plasmid was recovered in the flowthrough (96.2 ± 4.0%), even though the RNA (65.5 ± 2.8%), protein (84.4 ± 1.3%) and gDNA clearance (44.7 ± 14.1%) were not as effective. In both cases, the LPS content was removed to a residual value of less than 0.005 EU/ml. The method is fast and straightforward, circumvents the need for pre-treatment of the feed and may contribute to shorten plasmid purification processes, as the treated streams can proceed directly to the final polishing steps.     

Flow injection determination of choline in milk hydrolysates by an immobilized enzyme reactor coupled to a selective hydrogen peroxide amperometric sensor

A choline oxidase (ChO) immobilized enzyme reactor (IMER) prepared by glutaraldehyde coupling of the enzyme on aminopropyl modified controlled pore glass beads is described. The ChO-IMER was coupled, in a flow injection configuration system, to an interference free hydrogen peroxide amperometric sensor based on a Pt surface modified by an overoxidized polypyrrole film. The resulting analytical device responds selectively to choline and displays a sensitivity of 46.9 ± 0.2 μC mM⁻¹ and a limit of detection, calculated at a signal-to-noise ratio equal to 3, of 7 μM. Sensitivity remains constant for about 20 days and then starts to slowly deteriorate and after 2 months a 70% of the initial sensitivity was still retained. The application to choline determination in milk hydrolysates is demonstrated. Short- and long-term drift observed in the analytical response can be corrected by a bracketing technique.     

Immobilization of β-Galactosidase onto Magnetic Beads

A study of the cross-linking of β-galactosidase on magnetic beads is reported here. The magnetic beads were prepared from artemisia seed gum, chitosan, and magnetic fluid in the presence of a cross-linking regent (i.e., glutaraldehyde). The reactive aldehyde groups of the magnetic beads allowed the reaction of the amino groups of the enzymes. The animated magnetic beads were used for the covalent immobilization of β-galactosidase. The effect of various preparation conditions on the activity of the immobilized β-galactosidase, such as immobilizing time, amount of enzyme, and the concentration of glutaraldehyde, were investigated. The influence of pH and temperature on the activity and the stability of the enzyme, both free and immobilized, have been studied. And o-nitrophenyl-β-d-galactopyranoside (ONPG) was chosen as a substrate. The β-galactosidase immobilized on the magnetic beads resulted in an increase in enzyme stability. Optimum operational temperature for immobilized enzyme was 10 °C higher than that of free enzyme and was significantly broader.     

Sequential injection-capillary immunoassay system for determination of sialoglycoconjugates

An automatic immunoassay system for an assay of sialoglycoconjugates was developed based on the sequential injection technique. A cost effective plain glass capillary tube was used as a solid surface for immobilization of biomolecules via a simple physical adsorption which is adequate to tolerate the force of solution flowing through the capillary during the multi-steps immunoassay process. Immunoassay could be performed with many improvements—rapidity per sample as compared to the conventional micro-plate format (40 min vs. 5-8 h); lower cost and simpler as compared to fused silica capillary with covalent immobilization; and without problem of back pressure as compared to flow injection-bead based immunoassay. Performance of the sequential injection-capillary immunoassay was demonstrated by assay of sialoglycoconjugates level in human serum to differentiate cancer patients from healthy people.     

Removal of heavy metal ions from water by using poly(ethyleneglycol dimethacrylate-co-acrylamide) beads

Poly(ethyleneglycol dimethacrylate-co-acrylamide) (poly(EDGMA-co-AAm)) copolymer beads have been prepared for use in the separation Pb(II), Hg(II), and Cd(II), metal ions in aqueous solution by a batch equilibration technique. Adsorption capacity were increased with pH for Pb(II), Cd(II) and Hg(II) and then reached almost plateau value around 6.0. The high initial rate of metal ions uptake (<10 min) suggests that the adsorption occurs mainly at the bead surface. The metal uptake results show that poly(EGDMA-co-AAm) can be used for the adsorption of the following metals in the indicated order: Pb(II) > Cd(II) > Hg(II) expressed on a molar basis. However, when the uptake was expressed in terms of the amount of metal removed from solution was as follows: Pb(II) > Hg(II) > Cd(II). The beads still showed preference toward Pb(II) when this metal was in a mixture with Hg(II) and Cd(II). A linearized form of the Freundlich and the Langmuir isotherm model fits the experimental equilibrium concentration data of Hg(II) and Cd(II) better than isotherm type model of Pb(II). The recovery of the metal ions after adsorption and the regeneration of the adsorbent can be carried out by treatment of the loaded beads with either 0.5 M NaCl, or 1 M HNO₃.