Analytica Chimica Acta (v.470, #1)
Editorial board (iii).
calendar of forthcoming meetings (N1-N2).
Foreword by Ulrich J Krull (1).
Low density lipoprotein interaction with amino acid-modified self assembled monolayers on surface plasmon resonance surfaces by Katharina Gaus; Elizabeth A.H Hall (3-17).
Using a 1-mercaptundeconoic acid assembled on Au, surface plasmon resonance (SPR) was used to investigate specificity of low density lipoprotein (LDL) interaction with the surface. The 1-mercaptundeconoic acid was dispersed in a mixed 1-mercaptoundecanol: 1-mercapto-octyl-hexa(ethylene glycol) (80:20) self assembled monolayer (SAM) showing an LDL adsorption dependent on 1-mercaptundeconoic acid. The 1-mercaptundeconoic acid SAM was modified by coupling amino acids via the N-terminal. Glycine modification gave a surface that resisted LDL adsorption. Amino acids with –COOH side chains favoured LDL adsorption, whereas lysine encourages oxidised LDL (oxLDL) adsorption. Comparison with LDL and macrophage scavenger receptors indicated some useful amino acid combinations that were shown to have a high affinity for native but not oxLDL (aspartate–glutamate) or vice versa (lysine–lysine). Sequences with affinity for oxidised but not native LDL were either net positively charged or contained thiol-groups: a correlation with known oxLDL scavengers did not show complete homology here, but indicated some similarities. The ‘best’ (GlyCystineSerAspGlu and GlyLysLys–OH) surfaces were selected from the library for determination of native and oxLDL, respectively and detection limits of 1 μg ml−1 estimated in both cases. The ratio of the adsorption on these surfaces was shown to give a robust estimate of the degree of LDL oxidation as related to the relative electrophoretic mobility (REM).
Keywords: Low density lipoprotein; Self assembled monolayer; Surface plasmon resonance;
Screening of antagonists based on induced dissociation of a calmodulin–melittin interaction entrapped in a sol–gel derived matrix by Kulwinder K Flora; Tracey Keeling-Tucker; Christopher W Hogue; John D Brennan (19-28).
Sol–gel derived materials offer a unique advantage for the development of sensing and screening platforms in that they allow for the entrapment of multiple species within a confined space. In this work, we show that it is possible to entrap an intact protein–peptide interaction, consisting of bovine calmodulin (bCaM) and melittin, into a sol–gel derived silicate material. Fluorescence emission data demonstrate that the entrapped complex behaves similarly to the complex in solution, and can undergo reversible dissociation upon introduction of the denaturant guanidine hydrochloride. Screening of antagonists of the bCaM–melittin complex was accomplished based on induced dissociation of the entrapped complex, which was followed by measuring the loss of sensitization of Tb(III) luminescence originating from energy transfer from the Trp of melittin to Tb(III) bound in the loops of bCaM. This study shows that entrapped protein–peptide complexes can be used as targets for drug screening or for fluorescence-based biosensing.
Keywords: Protein complex; Calmodulin; Sol–gel; Terbium; Fluorescence;
Electron paramagnetic resonance spin label titration: a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties by D.Allan Butterfield; Joshua Colvin; Jiangling Liu; Jianquan Wang; Leonidas Bachas; Dibakar Bhattacharrya (29-36).
The immobilization of biological molecules onto polymeric membranes to produce biofunctional membranes is used for selective catalysis, separation, analysis, and artificial organs. Normally, random immobilization of enzymes onto polymeric membranes leads to dramatic reduction in activity due to chemical reactions involved in enzyme immobilization, multiple-point binding, etc., and the extent of activity reduction is a function of membrane hydrophilicity (e.g. activity in cellulosic membrane⪢polysulfone membrane). We have used molecular biology to effect site-specific immobilization of enzymes in a manner that orients the active site away from the polymeric membrane surface, thus resulting in higher enzyme activity that approaches that in solution and in increased stability of the enzyme relative to the enzyme in solution. A prediction of this site-specific method of enzyme immobilization, which in this study with subtilisin and organophosphorus hydrolase consists of a fusion tag genetically added to these enzymes and subsequent immobilization via the anti-tag antibody and membrane-bound protein A, is that the active site conformation will more closely resemble that of the enzyme in solution than is the case for random immobilization. This hypothesis was confirmed using a new electron paramagnetic resonance (EPR) spin label active site titration method that determines the amount of spin label bound to the active site of the immobilized enzyme. This value nearly perfectly matched the enzyme activity, and the results suggested: (a) a spectroscopic method for measuring activity and thus the extent of active enzyme immobilization in membrane, which may have advantages in cases where optical methods can not be used due to light scattering interference; (b) higher spin label incorporation (and hence activity) in enzymes that had been site-specifically immobilized versus random immobilization; (c) higher spin label incorporation in enzymes immobilized onto hydrophilic bacterial cellulose membranes versus hydrophobic modified poly(ether)sulfone membranes. These results are discussed with reference to analysis and utilization of biofunctional membranes.
Keywords: Electron paramagnetic resonance; Site-specific immobilization; Enzymes; Biofunctional membranes;
Optimization and development of a high-performance liquid chromatography-based one-site immunometric assay with chemiluminescence detection by Matthew R Oates; William Clarke; Alden Zimlich; David S Hage (37-50).
Various practical and theoretical considerations were examined in the creation and optimization of a high-performance liquid chromatography (HPLC)-based one-site immunometric assay. This method used an HPLC analyte analog column and post-column chemiluminescence detection. The specific analyte chosen as the model for this study was l-thyroxine (also known as T4). In this technique, a sample containing thyroxine was first combined with an excess of anti-T4 antibody Fab fragments that had earlier been conjugated with chemiluminescent acridinium ester labels. After incubation, the mixture was injected onto a column that contained immobilized T4. The amount of thyroxine in the original sample was then determined by measuring the labeled Fab fragments that appeared in the non-retained fraction, or the decrease in excess Fab fragments that were bound to and later eluted from the column. Items considered in creating this assay included the preparation of acridinium ester-labeled Fab fragments, the detection of these fragments with a post-column reactor, and the creation of a suitable immobilized analog column for capturing excess labeled Fab fragments. The final method could measure T4 in standards at clinically-relevant concentrations and provided a response within 1.5 min of sample injection, following a 20–45 min incubation with the labeled Fab fragments. Possible applications of this method include its use in clinical chemistry and the screening of proteomic or combinatorial libraries.
Keywords: Chromatographic immunoassay; Chemiluminescence; One-site immunometric assay; Post-column detection; Thyroxine;
Immobilization of oligonucleotides onto silica nanoparticles for DNA hybridization studies by Lisa R Hilliard; Xiaojun Zhao; Weihong Tan (51-56).
This paper describes the development of oligonucleotide-functionalized nanoparticles. We used disulfide-coupling chemistry for the immobilization of oligonucleotides onto silica nanoparticles and subsequently demonstrated the properties of the resulting DNA nanoparticles. Factors influencing the immobilization and hybridization processes were examined and optimized. The oligonucleotide-modified silica nanoparticles provide an efficient substrate for hybridization and can be used in the development of DNA biosensors and biochips.
Keywords: Covalent immobilization; Oligonucleotides; Silica nanoparticles; Disulfide-coupling; Thiol/disulfide exchange reaction;
Tethered thiazole orange intercalating dye for development of fibre-optic nucleic acid biosensors by Xiaofeng Wang; Ulrich J Krull (57-70).
Single-stranded DNA (ssDNA) oligonucleotide in solution, or that is immobilized onto a surface to create a biosensor, can be used as a selective probe to bind to a complementary single-stranded sequence. Fluorescence enhancement of thiazole orange (TO) occurs when the dye intercalates into double-stranded DNA (dsDNA). TO dye has been covalently attached to probe oligonucleotides (homopolymer and mixed base 10mer and 20mer) through the 5′ terminal phosphate group using polyethylene glycol linker. The tethered TO dye was able to intercalate when dsDNA formed in solution, and also at fused silica surfaces using immobilized ssDNA. The results indicated the potential for development of a self-contained biosensor where the fluorescent label was available as part of the immobilized oligonucleotide probe chemistry. The approach was shown to be able to operate in a reversible manner for multiple cycles of detection of targeted DNA sequences.
Keywords: Fluorescence; Intercalation; Thiazole orange; DNA; Biosensors; Hybridization;
Covalent binding of genetically engineered microorganisms to porous glass beads by Lisa C Shriver-Lake; Wm.Bryce Gammeter; Sookie S Bang; Mehran Pazirandeh (71-78).
Several covalent immobilization methods, which have been routinely used with proteins and antibodies, were studied for their ability to immobilize genetically engineered Escherichia coli cells to glass beads. The cells used in this study expressed a metal binding peptide that binds cadmium (Cd) and mercury (Hg). The initial work focused on a method employing 2.5% aminopropyltrimethoxy silane and 2.5% glutaraldehyde for covalent immobilization of cells onto porous glass beads. Scanning electron microscopy (SEM) demonstrated cell attachment (average of 3.0×108 cells per bead) to the irregular surface. Columns containing cells immobilized with the 2.5% aminosilane and 2.5% glutaraldehyde removed more than 90% of the Cd from solutions with 50 ppb and 1 ppm levels. Following removal of the bound Cd with HCl elution and regeneration to pH 6.0, the columns were shown to effectively bind additional cadmium. Various concentrations of aminosilane and glutaraldehyde were tested for improved cell density.Glutaraldehyde is a universal and convenient cross-linker, but there are some concerns with its effects on the cells and proteins, therefore, two additional covalent techniques were examined. One method employed the aminopropyltrimethoxy silane and carbodiimide, and the other used mercaptopropyltrimethoxy silane and the heterobifunctional cross-linker GMBS. Some comparisons of these two immobilization methods to the method employing glutaraldehyde are described.
Keywords: Cell immobilization; Heavy metal; Remediation; Bacteria;
Microbial biosensor for p-nitrophenol using Moraxella sp. by Priti Mulchandani; Yu Lei; Wilfred Chen; Joseph Wang; Ashok Mulchandani (79-86).
A novel microbial biosensor/electrode for highly selective, sensitive and rapid quantitative determination of p-nitrophenol (PNP) was developed using PNP-degrading organisms immobilized in a membrane on a dissolved oxygen electrode. Moraxella sp. specifically oxidizes PNP while consuming oxygen. A change in oxygen concentration was determined by a Clark oxygen electrode and correlated to the PNP concentration. The sensor signal and response time were optimized with respect to the buffer pH, temperature, time of cell growth and weight of cells immobilized. The best sensitivity and response time were obtained using a sensor constructed with 0.3 mg of cells and operating in pH 7.5, 20 mM phosphate buffer. Using these conditions, the biosensor was used to measure as low as 14 ppb (0.1 μM) of PNP extremely selectively without interference from structurally similar compounds, such as phenol, nitrophenols and chlorophenols. The biosensor had very good storage and multiple use stability when stored in the operating buffer at 4 °C.
Keywords: p-Nitrophenol; Microbial biosensor; Moraxella sp.; Respiration;
Surface modification of polymer-based microfluidic devices by Steven A Soper; Alyssa C Henry; Bikas Vaidya; Michelle Galloway; Musundi Wabuyele; Robin L McCarley (87-99).
We report the chemical modification of poly(methyl methacrylate) (PMMA), and poly(carbonate) (PC) surfaces for applications in microfluidic systems. For PMMA, a reaction of the surface methyl ester groups with a monoanion of α,ω-diaminoalkanes (aminolysis reaction) to yield amine-terminated PMMA surfaces will be described. Furthermore, it was found that the amine functionalities were tethered to the PMMA backbone through an alkane bridge to amide bonds formed during the aminolysis of the surface ester functionalities. The electro-osmotic flow (EOF) in aminated-PMMA microchannels was reversed when compared to that in unmodified channels. Finally, the availability of the surface amine groups was further demonstrated by their reaction with n-octadecane–1-isocyanate to form PMMA surfaces terminated with well ordered and highly crystalline octadecane chains, appropriate for performing reverse-phase separations. Examples of reverse-phase separations of ion-paired double-stranded DNAs in electric fields (capillary electrochromatography (CEC)) will be demonstrated using a PMMA-based fluidic chip. For PC, sulfonation of the surface with SO3 will be described; this sulfonation makes the surface very hydrophilic. EOF studies of the sulfonated-PC surfaces indicated changes in the pH-dependent profile when compared to unmodified PC.
Keywords: Surface modification; Polymer microdevices; MEMS; DNA electrophoresis;
Integrated chemical sensor array platform based on a light emitting diode, xerogel-derived sensor elements, and high-speed pin printing by Eun Jeong Cho; Frank V Bright (101-110).
We report a new, solid-state, integrated optical array sensor platform. By using pin printing technology in concert with sol–gel-processing methods, we form discrete xerogel-based microsensor elements that are on the order of 100 μm in diameter and 1 μm thick directly on the face of a light emitting diode (LED). The LED serves as the light source to excite chemically responsive luminophores sequestered within the doped xerogel microsensors and the analyte-dependent emission from within the doped xerogel is detected with a charge coupled device (CCD). We overcome the problem of background illumination from the LED reaching the CCD and the associated biasing that results by coating the LED first with a thin layer of blue paint. The thin paint layer serves as an optical filter, knocking out the LEDs red-edge spectral tail. The problem of the spatially-dependent fluence across the LED face is solved entirely by performing ratiometric measurements. We illustrate the performance of the new sensor scheme by forming an array of 100 discrete O2-responsive sensing elements on the face of a single LED. The combination of pin printing with an integrated sensor and light source platform results in a rapid method of forming (∼1 s per sensor element) reusable sensor arrays. The entire sensor array can be calibrated using just one sensor element. Array-to-array reproducibly is <8%. Arrays can be formed using single or multiple pins with indistinguishable analytical performance.
Keywords: Solid-state sensor array platform; Light emitting diode; Pin printing;