Protein-based Material Research Articles

Selective chemical modification of soy protein for a tough and applicable plant protein-based material.

Soy protein, one of the most abundant plant proteins, has gained considerable attention and popularity in recent years due to its biocompatibility, biodegradability, and renewability. However, the poor mechanical properties and high moisture sensitivity of soy protein-based materials have limited their wider application in practical use. In the present study we have overcome these shortcomings by the use of an aqueous reagent, tetrakis(hydroxylmethyl) phosphonium chloride (THPC), to modify the amino groups of the lysine (Lys) and arginine (Arg) residues present in soy protein. Solid state 13C cross polarization/magic-angle spinning nuclear magnetic resonance (CP/MAS NMR) and 31P NMR spectroscopy have confirmed the success of the chemical reaction by replacement of the hydroxymethyl arm of THPC, both qualitatively and quantitatively. Fourier-transform infrared (FTIR) spectroscopy, rheological observations, transmission electron microscopy (TEM) and atomic force microscopy (AFM) have been used to confirm the changes in the coherent tertiary structure of soy protein after modification, increasing the interconnection between the molecular chains. Finally, we have been able to produce a modified soy protein film, which provides a good combination of tensile strength and extensibility under either dry (10 ± 2 MPa and 25 ± 3%) or wet (5 ± 1 MPa and 200 ± 20%) conditions. In addition the modified soy protein film has unexpectedly been found to exhibit antimicrobial properties, and this adds to the merits of the final product. We believe that the method described further broadens the application of natural soy protein-based materials, for example in antimicrobial packaging films.

Introducing a combinatorial DNA-toolbox platform constituting defined protein-based biohybrid-materials

The access to defined protein-based material systems is a major challenge in bionanotechnology and regenerative medicine. Exact control over sequence composition and modification is an important requirement for the intentional design of structure and function. Herein structural- and matrix proteins provide a great potential, but their large repetitive sequences pose a major challenge in their assembly. Here we introduce an integrative “one-vector-toolbox-platform” (OVTP) approach which is fast, efficient and reliable. The OVTP allows for the assembly, multimerization, intentional arrangement and direct translation of defined molecular DNA-tecton libraries, in combination with the selective functionalization of the yielded protein-tecton libraries. The diversity of the generated tectons ranges from elastine-, resilin, silk- to epitope sequence elements. OVTP comprises the expandability of modular biohybrid-materials via the assembly of defined multi-block domain genes and genetically encoded unnatural amino acids (UAA) for site-selective chemical modification. Thus, allowing for the modular combination of the protein-tecton library components and their functional expansion with chemical libraries via UAA functional groups with bioorthogonal reactivity. OVTP enables access to multitudes of defined protein-based biohybrid-materials for self-assembled superstructures such as nanoreactors and nanobiomaterials, e.g. for approaches in biotechnology and individualized regenerative medicine.

Study of materials and techniques in painted ceiling panels from a palace in Cremona (Italy, 15thcentury)

Abstract Background This study concerns the analysis of four painted wooden panels from the second half of the 15th century which embellish the ceiling of a palace situated in Cremona (Northern Italy). This type of painting was generally used to decorate the top of the walls and the ceilings of the rooms in aristocratic palaces and they usually portray the owner’s ancestors, family members or important visitors. The paintings are of historical importance since they enable us to study the clothing and hair styles which were in fashionable in Italy during the Renaissance. These panels were heavily damaged and required a lot of restoration including the cleaning of the paint layers. This enabled us to carry out a thorough examination of the materials and techniques used for creating these paintings. Results Various micro-samples were taken in order to distinguish the materials and, in particular: (i) to analyze the ground layer and study its function; (ii) to typify the organic binders and the pigments of the polychrome films. The stratigraphic sequence of micro-samples was analyzed using optical microscopy, the elemental composition was determined by means of the SEM-EDX technique while the organic binders were analyzed by means of μFT-IR spectroscopy. The main component of ground layer proved to be clay. The organic binder was probably composed of a protein-based material such as animal glue. This type of ground layer is similar to the traditional 15th century Flemish formula and seems to confirm the strong influence of that artistic technique in Northern Italy. Cross-section investigation showed a primer layer over the ground layer made of white lead pigment. In fact, a white primer was used to cover the ground layer of yellowish clay common in Central Europe. The importance of this research is also confirmed by the study of paint films, for which pigments such as azurite or cinnabar were used. Red lake pigment for painting the subjects’ complexions was emphasized. Conclusions The analyses provide new detailed information concerning the materials used for painting the ceiling panels, the layer sequence of the paints and Northern Italian 15th century painting techniques.

Complementary analytical methods in identifying gilding and painting techniques of ancient clay-based polychromic sculptures

The gilding and painting techniques for the clay-based polychromic sculptures in Jizo Hall of Chongqing Temple, Shanxi Province of China, are analyzed by complementary techniques of microscopy, environmental scanning electron microscopy (ESEM), energy dispersive X-ray mapping (EDX), micro-Raman spectroscopy (μ-RS), and Fourier transform infrared spectroscopy (FTIR) with a reasonable sequence. 7 gilded samples and 6 pigmented samples are collected from Jizo Hall. For the 7 gilded polychromic samples, drying oil-based, Chinese lacquer-based and glue-based “Jin Jiao” techniques are identified respectively according to their different adhesive layers used for jointing gold leaf to the preparation layer. But the presence of a copper foil top surface, as well as the inner layers with crocoites (PbCrO4) and modern red pigment in one alleged gilded polychromic sample indicate a later restoration work. Among them, two gold layers have been identified in 4 samples due to the regilded work in history. For 6 pigmented samples, painting layer is generally applied on a white preparation layer made of kaolinite or the mixture of kaolinite and gypsum. Atacamite, cinnabar, minium, yellow ochre and gypsum are identified as the main pigments, and the protein-based material is confirmed as the binding medium for red pigments. This research not only reveals the Chinese traditional gilding and painting techniques employed in polychromic sculptures of Jizo Hall, but also demonstrates the advantages and limitations of each analytical technique for identifying polychromic samples, and the necessity of complementary analytical techniques approach.

A Safe and Efficient Method to Retrieve Mesenchymal Stem Cells from Three-Dimensional Fibrin Gels

Mesenchymal stem cells (MSCs) display multipotent characteristics that make them ideal for potential therapeutic applications. MSCs are typically cultured as monolayers on tissue culture plastic, but there is increasing evidence suggesting that they may lose their multipotency over time in vitro and eventually cease to retain any resemblance to in vivo resident MSCs. Three-dimensional (3D) culture systems that more closely recapitulate the physiological environment of MSCs and other cell types are increasingly explored for their capacity to support and maintain the cell phenotypes. In much of our own work, we have utilized fibrin, a natural protein-based material that serves as the provisional extracellular matrix during wound healing. Fibrin has proven to be useful in numerous tissue engineering applications and has been used clinically as a hemostatic material. Its rapid self-assembly driven by thrombin-mediated alteration of fibrinogen makes fibrin an attractive 3D substrate, in which cells can adhere, spread, proliferate, and undergo complex morphogenetic programs. However, there is a significant need for simple cost-effective methods to safely retrieve cells encapsulated within fibrin hydrogels to perform additional analyses or use the cells for therapy. Here, we present a safe and efficient protocol for the isolation of MSCs from 3D fibrin gels. The key ingredient of our successful extraction method is nattokinase, a serine protease of the subtilisin family that has a strong fibrinolytic activity. Our data show that MSCs recovered from 3D fibrin gels using nattokinase are not only viable but also retain their proliferative and multilineage potentials. Demonstrated for MSCs, this method can be readily adapted to retrieve any other cell type from 3D fibrin gel constructs for various applications, including expansion, bioassays, and in vivo implantation.

Poly(ɛ-caprolactone) modified with fusion protein containing self-assembled hydrophobin and functional peptide for selective capture of human blood outgrowth endothelial cells

Human blood outgrowth endothelial cells (HBOECs)-specific binding peptide, TPSLEQRTVYAK (TPS), was proposed to be applied on autologous cell therapy for treating cardiovascular diseases. Hydrophobins, as a family of self-assembly proteins originated from fungi, have demonstrated unique characteristics to modulate surface properties of other materials coated with these amphiphilic proteins in previous studies. In this report, a fusion protein which was composed of class I hydrophobin HGFI originated from Grifola frondosa and functional peptide TPS was expressed by Pichia pastoris expression system. Then, we purified this fusion protein by ultrafiltration and reverse-phase high performance liquid chromatography. Water contact angle, X-ray photoelectron spectroscopy measurements indicated that the surface properties of hydrophobin were greatly preserved by this fusion protein while comparing with wild HGFI. Cell binding assay showed that this fusion protein demonstrated specific binding property to HBOECs while coating on biodegradable poly(ɛ-caprolactone) (PCL) grafts in the presence of fetal bovine serum, whereas HGFI-coated PCL non-selectively enhanced all types of cells attachments. Methylthiazol tetrazolium assay was employed to verify the cytocompatibility of this fusion protein-based material. This work presented a new perspective to apply hydrophobin in tissue engineering and regenerative medicine and provided an alternative approach to study endothelial progenitor cells.

Conceptual Models for Synthetic Bipedal Motors

Biomolecular nano-motors have provided the inspiration for the design and construction of artificial nanoscale motors and machines based on several types of molecule including DNA. However, no synthetic nano-motors have yet been constructed from building blocks of protein-based material even though biomotors themselves are proteins. The HFSP motor group (1) are in the process of developing a bottom-up approach to the understanding of bio-motors by designing and constructing synthetic protein motors and numerically simulating their kinetic properties. One such concept is the “tripedal tumbleweed” motor (1) and another is the “Inchworm”. In this context we present the results of numerical simulations for a bipedal motor with two connected peptide legs and a simple model for the “inchworm” motor in terms of harmonic bonds. These motors walk on a one-dimensional track of periodically arranged binding sites. The sequence of binding sites on the track of each motor is AB-AB-AB..... and the motors are powered by a temporally periodic sequence of composite washes which modulate the ligand concentrations and the leg angles. The washes cause the motors to undergo directed motion by a hand-over-hand mechanism and an inchworm mechanism, respectively, on a track with asymmetric spacing between the AB and the BA binding sites. We will show simulation results for two-dimensional motor action which includes stepping diagrams, stall forces processivity and first passage times for a range of parameters. These motor has the following properties observed for biological molecular motors: binding, power stroke and diffusional search. Extensions of the model will also be discussed. 1. E. Bromley et al. HFSP Journal Vol. 3 pp.204-212, (2009).

Bottom-Up Multiscale Simulations of Silk Fiber Mechanics

Silk is an intriguing protein-based material that combines elasticity and strength to an extent not yet reached by any synthetic material today. Silk fibers are composed of highly-ordered beta-sheet crystals and an amorphous peptide matrix that both contribute to its outstanding mechanical properties. However, how the way of organization of these two components affects silk fiber mechanics has remained unclear. To quantify the mechanics of silk fibers and its components, we here in a bottom-up approach extract elastic and rupture parameters of silk composite units from all-atom molecular simulations and a novel force distribution analysis serve as input for finite element analysis [1,2]. By doing so, we can derive macroscopic fiber mechanics from the nano-structure, in quantitative agreement with experiments. One of our most striking predictions is that a serial arrangement of silk crystals in the fiber, as commonly observed in form of lamellae for other block copolymers, outperforms a random distribution, in sharp contrast to the current view of silk fiber organization [3]. We also show why the typical beta-strand length of eight residues in silk crystals is mechanically optimal [2]. Finally, a smaller cross-sectional area of silk crystals (∼1nm2) in fibers provides a better reinforcement of the amorphous phase than larger ones. [3] We expect our straightforward multiscale approach to serve as a guideline for the design of silk-like synthetic materials. 1. S Xiao, W Stacklies, M Cetinkaya, B Markert, and F Graeter. Biophys J, 96(10):3997–4005, May 2009. 2. S Xiao, W Stacklies, C Debes, and F Graeter. Soft Matter, 2010. 3. M Cetinkaya, S Xiao, B Markert, W Stacklies, and F Graeter. in review, 2010.

Rapid Nanoimprinting of Silk Fibroin Films for Biophotonic Applications

With soft micro- and nanopatterned materials becoming increasingly useful for various, optical, mechanical, electronic, microfluidic, and optofluidic devices, the extension of this paradigm to a pure protein-based material substrate provides entirely new options for such devices. Silk fibroin combines the properties of an ideal nanoimprint resist with superior optical quality and biocompatibility making it a new technology platform that seamlessly combines nanophotonics, biopolymeric, and biocompatible materials.

The biocompatibility of Pluronic ®F127 fibrinogen-based hydrogels

Our research is focused on the design of hydrogel biomaterials that can be used for 3-D cell encapsulation and tissue engineering. In this study, our goal was to engineer a temperature-responsive biomaterial to possess bioactive properties using polymer and protein chemistry, and at the same time provide the biomaterial with susceptibility to cell-mediated remodeling. Toward this goal, we developed a biomimetic material that can harness the bioactive properties of fibrinogen and the unique structural properties of Pluronic ®F127. Pluronic ®F127 is a synthetic block copolymer that exhibits reverse thermal gelation (RTG) in response to small changes in ambient temperature. We conjugated fibrinogen to Pluronic ®F127 to create a biosynthetic precursor with tunable physicochemical properties based on the relationship between the two constituents. A hydrogel matrix was formed from the fibrinogen-F127 adducts by free-radical polymerization using light activation (photo-polymerization). These materials displayed a reversible temperature-induced physical sol–gel transition and an irreversible light-activated chemical cross-linking. The susceptibility of this hydrogel biomaterial to protease degradation and consequent cell-mediated remodeling was controlled by the Pluronic ®F127 constituent. The protein-based material also conveyed inductive signals to cells through bioactive sites on the fibrinogen backbone, as well as through structural properties such as the matrix modulus. We apply these materials as a tissue engineering hydrogel scaffold for 3-D in vitro culture of dermal fibroblasts in order to gain a better understanding of how the material bioactivity and matrix properties can independently affect cell morphology and remodeling.

Conceptual Model for a Synthetic Bipedal Stepping Motor

Biomolecular nanomotors have provided the inspiration for the design and construction of artificial nanoscale motors and machines based on several types of molecule including DNA. However, no synthetic nano-motors have yet been constructed from building blocks of protein-based material even though biomotors themselves are proteins. The HFSP smotor group (1) are in the process of developing a bottom-up approach to the understanding of biomotors by designing and constructing synthetic protein motors and numerically simulating their kinetic properties. One such concept is the “tripedal tumbleweed” motor, which is described in (1). In this context we present the results of numerical simulations for a bipedal motor with two connected peptide legs and with some of the properties of the tumbleweed motor. This motor walks on a one-dimensional track of periodically arranged binding sites. The two “feet” at the end of the legs represent different ligand-gated binding proteins which can only bind to their specific binding sites on the track when the related ligands are themselves bound to the binding proteins. The sequence of binding sites on the track is AB-AB-AB…. and the motor is powered by a temporally periodic sequence of composite washes which modulate the ligand concentrations and the leg angles. The washes cause the motor to undergo directed motion by a hand-over-hand mechanism on a track with asymmetric spacing between the AB and the BA binding sites. We will show simulation results for both two-dimensional and three-dimensional motor action of our bipedal motor which will include stepping diagrams, stall forces and first passage times for a range of parameters. This motor has the following properties observed for biomolecular motors: binding, power stroke and diffusional search. Extensions of the model will also be discussed. 1. E. Bromley et al. HFSP Journal (2009)

Bioactive Silk Protein Biomaterial Systems for Optical Devices

Silk-based biomaterial systems have been previously explored for a variety of medical and nonmedical materials needs. The unique biophysical features of silks provide options to generate highly tailored structures and morphologies with this unique family of fibrous proteins. To exploit these features, we have optimized the all aqueous processing of silk fibroin into novel surface nanopatterned protein materials. We have exploited control of this nanomorphology to optimize the optical features of these silk protein systems. We demonstrate control of surface morphology down to 125 nm, with fidelity over large length scales. This surface nanopatterning allows the silk protein to be formed into diffractive optics such as diffraction gratings, pattern generators, and lenses due to novel aqueous processing into optically clear materials via control of beta sheet crystallinity. Further, we incorporate biological components, such as hemoglobin and the enzyme peroxidase, during the process of forming the silk diffraction gratings. The ambient processing of the silk protein in water, in combination with these bioactive components, allows these entrained molecules to retain activity and provide added functions and selectivity to the optically active silk films. Thus, combinations of biochemical and optical readout is feasible and provides in a single, disposable/all degradable element with both spectral discrimination and biological function. These new surface nanopatterned, bioactive silk protein-based material systems offer a unique combination of features potentially useful for a range of biosensor needs, particularly when considered in concert with the remarkable mechanical properties of these proteins, their biocompatibility, and controllable biodegradation.

Hydrogels for cardiac tissue regeneration

Our research is focused on the design of engineered biomaterials that can harness natural cellular and molecular healing pathways to enhance functional tissue regeneration. Two important considerations for tissue regeneration are induction and remodeling. Although the healing process that leads to functional regeneration relies on numerous biological events, it can often be catalyzed and sustained by a single inductive biological factor. Ideally, one can engineer a synthetic biomaterial to possess inductive healing properties using protein immobilization techniques and also to be susceptible to cell-mediated remodeling. Toward this goal, we developed a novel biomimetic material that can harness the inductive properties of the natural blood clot protein fibrinogen. Using synthetic polymer conjugation chemistry, we modify the fibrinogen molecule with poly(ethylene glycol) (PEG) to create a biosynthetic precursor with tunable physicochemical properties based on the molecular relationship between the two constituents [1]. A hydrogel matrix is formed from the biocompatible liquid precursor by non-toxic free-radical polymerization using light activation (photopolymerization). The susceptibility of this hydrogel biomaterial to protease degradation and consequent cell-mediated remodeling is precisely controlled by the amount and size of the PEG constituent in the polymer network [4]. The protein-based material also conveys inductive signals to cells through bioactive sites on the fibrinogen backbone. This biomimetic material has been tested in cell-based tissue engineering applications and in acellular in vivo tissue regeneration applications with bone [7], cartilage [2,9], and cardiac tissues [11,12]. In cardiac cell therapy for example, the inability to locally deliver and retain cell grafts in the damaged cardiac muscle has limited the effectiveness of this important treatment option. As cardiac stem cell research addresses the issue of cell sourcing, there is still a need for biomaterials that can effectively deliver the cell grafts into the infarct region and promote structural and functional integration with the native myocardium, without damaging the cells or the heart muscle [3]. Injectable hydrogel biomaterials based on hydrophilic, biocompatible polymers are an optimal delivery system for cardiac tissue engineering [6]; the high water content of these polymers creates a tissue-like environment, and in situ polymerization provides a means of injection and gelation of a cell suspension polymer mixture directly in the site of the infarct [8]. In the current investigation, we explore the use PEGylated fibrinogen polymer hydrogels for myocardial tissue engineering. The optimization of hydrogel composition and cell seeding density were assessed

Characterization of a Protein-based Adhesive Elastomer Secreted by the Australian Frog Notaden bennetti

When provoked, Notaden bennetti frogs secrete an exudate which rapidly forms a tacky elastic solid ("frog glue"). This protein-based material acts as a promiscuous pressure-sensitive adhesive that functions even in wet conditions. We conducted macroscopic tests in air to assess the tensile strength of moist glue (up to 78 +/- 8 kPa) and the shear strength of dry glue (1.7 +/- 0.3 MPa). We also performed nanomechanical measurements in water to determine the adhesion (1.9-7.2 nN or greater), resilience (43-56%), and elastic modulus (170-1035 kPa) of solid glue collected in different ways. Dry glue contains little carbohydrate and consists mainly of protein. The protein complement is rich in Gly (15.8 mol %), Pro (8.8 mol %), and Glu/Gln (14.1 mol %); it also contains some 4-hydroxyproline (4.6 mol %) but no 5-hydroxylysine or 3,4-dihydroxyphenylalanine (L-Dopa). Denaturing gel electrophoresis of the glue reveals a characteristic pattern of proteins spanning 13-400 kDa. The largest protein (Nb-1R, apparent molecular mass 350-500 kDa) is also the most abundant, and this protein appears to be the key structural component. The solid glue can be dissolved in dilute acids; raising the ionic strength causes the glue components to self-assemble spontaneously into a solid which resembles the starting material. We describe scattering studies on dissolved and solid glue and provide microscopy images of glue surfaces and sections, revealing a porous interior that is consistent with the high water content (85-90 wt %) of moist glue. In addition to compositional similarities with other biological adhesives and well-known elastomeric proteins, the circular dichroism spectrum of dissolved glue is almost identical to that for soluble elastin and electron and scanning probe microscopy images invite comparison with silk fibroins. Covalent cross-linking does not seem to be necessary for the glue to set.

Designing new materials from wheat protein.

We recently discovered that wheat gluten could be formed into a tough, plasticlike substance when thiol-terminated, star-branched molecules are incorporated directly into the protein structure. This discovery offers the exciting possibility of developing biodegradable high-performance engineering plastics and composites from renewable resources that are competitive with their synthetic counterparts. Wheat gluten powder is available at a cost of less than dollars 0.5/lb, so if processing costs can be controlled, an inexpensive alternative to synthetic polymers may be possible. In the present work, we demonstrate the ability to toughen an otherwise brittle protein-based material by increasing the yield stress and strain-to-failure, without compromising stiffness. Water absorption results suggest that the cross-link density of the polymer is increased by the presence of the thiol-terminated, star-branched additive in the protein. Size-exclusion high performance liquid chromatography data of molded tri-thiol-modified gluten are consistent with that of a polymer that has been further cross-linked when compared directly with unmodified gluten, handled under identical conditions. Remarkably, the mechanical properties of our gluten formulations stored in ambient conditions were found to improve with time.

Soy matrix drug delivery systems obtained by melt-processing techniques.

The aim of this study was to develop new soy protein drug delivery matrix systems by melt-processing techniques, namely, extrusion and injection moulding. The soy matrix systems with an encapsulated drug (theophylline, TH) were previously compounded by extrusion performed at two different pH values, (i) pH 4 (SIpDtp) and (ii) pH 7 (SIDtp), and further injection-moulded into a desired shape. During the extrusion process the matrixes SIDtp were also cross-linked with glyoxal (0.6X-SIDtp) and reinforced with a bioactive filler, hydroxylapatite (SI-HADtp). The obtained mouldings were used to study the drug-release mechanisms from the plastic soy-TH matrixes. In an isotonic saline solution (ISS) buffered at pH 5.0 (200 mM acetate buffer), the resulting release kinetics could be described using the Fick's second law of diffusion. Because the diffusion coefficients were found to be constant and the boundary conditions to be stationary, these systems are drug-diffusion controlled. Conversely, the dominant phenomena in an isotonic saline solution buffered at pH 7.4 (200 mM Tris/HCl buffer) are more complex. In fact, because of the higher polymer solubility, the resulting matrix is time-variant. So, the drug release is affected by swelling, drug diffusion, and polymer dissolution, being faster when compared to ISS-200 mM acetate buffer, pH 5.0. The changes in the formulation composition affecting the correspondent release rates were also investigated. At pH 7.4, increasing the cross-linking degree of the polymer matrix (via reaction with glyoxal or heat treatment) or decreasing the net charge (extruding at pH near its isoelectric point) led to lower release rates. The incorporation of ceramic filler caused the opposite effect. Because of the low solubility of the matrix at pH 5.0, no significant variations were detected with variations in the selected formulations. These systems, based on a nonstandard protein-based material, seem to be very promising to be used as carriers for drug delivery.

An evaluation of the performance of blood gas analysis in the UK using tonometered haemolysate material

WEQAS is one of the largest and the most rapidly expanding national EQA Schemes in the UK with over 500 participants. Since April 1995, the Scheme has distributed a tonometered protein-based aqueous material for blood gas analytes (H+, pCO2, pO2), electrolytes, glucose and lactate. Current performance of the new generation of blood gas analysers show marked bias between analysers which a number of manufacturers have attributed to a “matrix” effect. This study was undertaken to investigate the “true” analytical performance of these analysers utilising a tonometered haemolysate blood product with oxygen saturation kinetics identical to that of fresh blood. One hundred laboratories that represented the major analyzers used in the UK were selected to participate in the study. Participants were asked to analyse the samples within 1 week of tonometry of the material. Performance data, expressed as interlaboratory variation, was compared with concentration matched data from the same group using the aqueous material. Significant improvement in instrument Bias was observed for the AVL Omni and iStat analysers for pO2. Surprisingly, there was also an improvement in bias for the electrolyte parameters on the IL synthesis. However, a significant positive bias was observed for the AVL Opti for Potassium and for the Nova analyzer for glucose using this material. Similar performance was observed for the co-oximetry, parametres for fresh and lyophilised material for the majority of analyzers.

Simultaneous multielement analysis of microliter quantities of serum for copper, iron, and zinc by graphite furnace atomic absorption spectrometry

A method is described for the simultaneous determination of Cu, Fe, and Zn in 25 ..mu..L of serum, using simultaneous, multielement atomic absorption spectrometry (SIMAAC) and graphite furnace atomization. Peak height measurements are more adversely affected than peak area measurements by both the organic and inorganic constituents in serum. Peak area measurements are significantly more precise and accurate than peak height measurements. Determinations are carried out at a 1:21 dilution of serum, in the concentration range with the optimum precision and minimum carryover contamination and memory effects for these elements, using SIMAAC. The serum specimens are diluted with Triton X-100 (10 mL/L), 10 mM nitric acid, and 30 mM ammonium nitrate. The use of peak area measurements and atomization from a L'vov platform permits calibration with acidified aqueous standards. A commercially available, protein-based reference material, Cation-Cal (American Dade) and a bovine serum pool are used as controls. The method is shown to have good correlation to comparative methods (single-element flame AAS after deproteinization) for the analysis of 37 serum samples.