Hydrophilic Microspheres Containing αα-tert Butoxy-ωω-vinylbenzyl-polyglycidol for Immunodiagnostics: Synthesis,Properties and Biomedical Applications

Summary: Synthesis, characteristics and medical diagnostic application of core-shell poly(styrene/α-tert-butoxy-ω-(vinylbenzyl)polyglycidol) (P(S/PGL)) microspheres are described. The particles were prepared by soap-free emulsion polymerization of styrene and α-tert-butoxy-ω-(vinylbenzyl)polyglycidol macromonomer (PGL) initiated with potassium persulfate. The polymerization in water yielded microspheres with diameters in the range from 220 to 650 nm, depending on concentration of the macromonomer. Polydispersity was usually below 1.06. The fraction of PGL in interfacial layer of microspheres ranged from 0 to 42 mol.% (estimated by XPS). Microspheres suspended in NaCl solutions manifested fully reversible swelling-deswelling properties of their interfacial layer with characteristic transition temperature (T_t) slightly dependent on NaCl concentration. The adsorption of human serum albumin on the surface of P(S/PGL) microspheres was highly reduced when the PGL surface fraction exceeded 40 mol.%. P(S/PGL) microspheres with immobilized antigens Helicobacter pylori were used for detection of antibodies against. The test was based on monitoring differences in electrophoretic mobility of P(S/PGL) microspheres covalently bound H. pylori antigens and antibodies against H. pylori in blood serum.     

Properties of poly(styrene/alpha-tert-butoxy-omega-vinylbenzyl-polyglycidol) microspheres suspended in water. Effect of sodium chloride and temperature on particle diameters and electrophoretic mobility

Hydrodynamic and electrophoretic properties of core-shell poly(styrene/alpha- tert-butoxy-omega-vinylbenzyl-polyglycidol) (P(S/PGL)) microspheres suspended in water are described. The microspheres were obtained by surfactant-free emulsion copolymerization of styrene and alpha- tert-butoxy-omega-vinylbenzyl-polyglycidol macromonomer ( M n = 2800, M w/ M n = 1.05). The process yielded microspheres with number average diameter D n = 270 nm and with low diameter dispersity index D w/ D n = 1.01. Shells of P(S/PGL) microspheres were enriched in polyglycidol. Molar fraction of polyglycidol monomeric units in the shells (determined by X-ray photoelectron spectroscopy) was equal to 0.34, which is much higher than the average molar fraction of polyglycidol monomeric units in whole particles of 0.048. Influences of NaCl concentration and temperature on P(S/PGL) microsphere diameters and on their electrophoretic mobility were investigated. It was found that hydrodynamic diameter of P(S/PGL) microspheres, determined by photon correlation spectroscopy, decreased significantly when temperature did exceed a certain value (transition temperature, T t). It has been found that the decrease is more pronounced for higher concentrations of NaCl in the medium. For microspheres suspended in 10 (-1) M NaCl, the hydrodynamic diameter decreased by 8% whereas for the same particles in pure water the diameter decreased by 5.2%. The process of shrinkage was fully reversible. Values of T t for P(S/PGL) microspheres were lower for higher concentrations of NaCl. Adjustment of salt concentration allowed controlling T t in a range from 44.4 to 49.9 degrees C. 13C NMR relaxation time measurements (T 1) for carbon atoms in polyglycidol macromonomer revealed that T 1 did increase with increasing temperature (in temperature range from 25 to 75 degrees C) indicating higher motion of chains at higher temperature. Addition of NaCl did not induce a substantial change of T 1 in the mentioned temperature range. The swelling-deswelling properties of P(S/PGL) microspheres' interfacial layer affected adsorption of P(S/PGL) particles on modified with (3-aminopropyl)triethoxysilane mica. It was shown that the deposition of P(S/PGL) microspheres at 25 degrees C on mica led to formation of two-dimensional crystal-shape assemblies, whereas at 60 degrees C (far above T t = 49.8 degrees C in H2O) the microspheres were randomly adsorbed without formation of colloidal crystal assemblies.     

Polymer Nano- and Microparticle Based Systems for Medical Diagnostics

Summary: Synthesis, properties and medical diagnostic applications of hydrophilic nano- and microspheres with carboxyl, aldehyde and hydroxyl groups on their surface are described. The particles were obtained by emulsion copolymerization of styrene, acrolein, methyl methacrylate, methacrylic acid, and 2-hydroxyethyl methacrylate carried on in water media and initiated with potassium persulfate. Stabilization of particles' suspensions was provided by addition of sodium dodecyl sulfate to polymerizing mixture or by formation of surfactants in situ in copolymerization involving acrolein or α-tert-butoxy-ω-vinylbenzyl-polyglycidol macromonomer (PGL). Relations between interfacial properties of these particles and their ability for covalent immobilization of proteins, with eliminated or at least reduced nonspecific adsorption of these species were investigated. The particles with covalently attached proteins (antigens or antibodies) were used for preparation of diagnostic tests based on visual or turbidimetric observation of particles' aggregation or by monitoring changes in their electrophoretic mobility accompanying specific antigen (or antibody) binding. The later test was directed toward determination of antibodies against Helicobacter pylori. Principle of a new type of diagnostic test based on photonic crystals of microspheres are described.     

Online immunoaffinity assay-CE using magnetic nanobeads for the determination of anti-Helicobacter pylori IgG in human serum

About two-thirds of the world's population is infected with Helicobacter pylori (H. pylori). This Gram-negative bacterium is the most important etiological agent of chronic active type B gastritis and peptic ulcer diseases. Conventional methods such as gastric biopsy, ELISA and culture, require a long time for the determination of H. pylori infections. Moreover, the antibodies in human serum sample are capable to react immunologically with the purified H. pylori antigens immobilized on different kinds of support like magnetic nanobeads. In this study, we have developed an online immunoaffinity assay-CE to determine the concentration of anti-H. pylori IgG using magnetic nanobeads as a support of the immunological affinity ligands and an LIF as a detector. The separation was performed in 0.1 M glycine-HCl, pH 2, as the background electrolyte. The linear calibration curve to predict the concentration of H. pylori-specific immunoglobulin G antibodies in serum was produced within the range of 0.12-100 U/mL. The linear regression equation was i = 492.86+96.03 × C(anti-H. pylori), with the linear regression coefficient r(2) = 0.999. The LOD calculated by fluorescence detection procedure was of 0.06 U/mL. The whole assay was done in no more than 35 min and it was entirely automatized. The development of immunoaffinity assay-CE in this study demonstrates that there is a large possibility to introduce nanotechnology in several fields with significant advantages over the classic methodologies. Our proposition comprises the diagnosis and screening field.     

Immuno-column for on-line quantification of human serum IgG antibodies to Helicobacter pylori in human serum samples

This study report an human serum IgG antibodies to Helicobacter pylori quantitation procedure based on the multiple use of an immobilized H. pylori antigen on an immuno-column incorporated into an a flow-injection (FI) analytical system. The immuno-adsorbent column was prepared by packing 3-aminopropyl-modified controlled-pore glass (APCPG) covalently linking H. pylori antigens in a 3-cm of Teflon tubing (0.5 i.d.). Antibodies in the serum sample are allowed to react immunologically with the immobilized H. pylori antigen, and the bound antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. p-Aminophenyl phosphate (pAPP) was converted to p-aminophenol (pAP) by AP and an electroactive product was quantified on glassy carbon electrode (GCE) modified with multiwall carbon nanotubes (MWCNT) (GCE-CNTs) at 0.30 V. The total assay time was 25 min. The calculated detection limits for amperometric detection and the ELISA procedure are 0.62 and 1.8 UmL(-1), respectively. Reproducibility assays were made using repetitive standards of H. pylori-specific antibody and the intra- and inter-assay coefficients of variation were below 5%. The immuno-affinity method showed higher sensitivity and lower time-consumed, demonstrate its potential usefulness for early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori.     

Multiplex suspension array for human anti-carbohydrate antibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.     

Studies of the surface layer structure and properties of poly(styrene/α-t-butoxy-ω-polyglycidol) microspheres by carbon nuclear magnetic resonance,X-ray photoelectron spectroscopy,and the adsorption of human serum albumin and γ-globulins

The composition and properties of the surface layers of poly(styrene/α-t-butoxy-ω-polyglycidol) [poly(styrene/VB-polyGL)] microspheres synthesized by the radical copolymerization of styrene and α-t-butoxy-ω-vinylbenzyl-polyglycidol (VB-polyGL) macromonomers [number-average molecular weight (M_n) = 950 or 2700] were investigated with X-ray photoelectron spectroscopy, ¹³C NMR, and the adsorption of human serum albumin and γ-globulins. The number-average diameter of the synthesized microspheres was 220 nm. Their surface layers were rich in polyglycidol, with polyglycidol-to-polystyrene unit ratios of 0.443 (VB-polyGL with M_n = 950) and 0.427 (VB-polyGL with M_n = 2700). In suspensions of poly(styrene/VB-polyGL) particles in D₂O, the polymer chains in the polyglycidol-rich surface layers were highly mobile, allowing the registration of polyglycidol ¹³C NMR spectra with standard procedures for polymer solutions. In these spectra, the signals of the relatively immobile polystyrene segments were absent. The spin–lattice relaxation times (T₁) measured for polyglycidol in the microsphere surface layers and for VB-polyGL macromonomers in solution were very close, indicating similar degrees of motion in bound (in particle surface layers) and free (in solution) polyglycidol macromolecules. Studies of protein adsorption revealed that hydrophilic polyglycidol layers were protein-repellent. It was found that longer polyglycidol chains in particle surface layers were more mobile (higher T₁ values) and provided better protection against protein adsorption. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 615–623, 2004     

Cell separations using targeted monoclonal antibodies against overproduced surface proteins

Applied Biochemistry and Biotechnology - The capacity of polystyrene microspheres with immobilized antibodies against type 1 pili ofE. coli was measured. Using pure IgG-type monoclonal antibodies...     

Multiplexed immunoassay for the rapid detection of anti-tumor-associated antigens antibodies

TAAs (tumor-associated antigens) microarrays were designed to detect auto-antibodies directly in patient sera. Twelve different probes were chosen according to their described occurrence in cancer pathologies (Cyclin B1, Cyclin D1, Complement factor H, c-myc, IMP1, p53, p62, survivin, Her2/neu, Koc, NY-ESO-1 and PSA). Microarrays of these 12 proteins were immobilized within the nitrocellulose/cellulose acetate membrane of a 96-well filtering microtiter plate bottom. The captured auto-antibodies were detected using a staining approach based on alkaline phosphatase labeling. Thus, the presence of specific auto-antibodies in samples was visualized through the positive staining of the corresponding TAA spots. The TAA HiFi microarrays were shown to be able to capture specific purified anti-TAA antibodies. In real samples, 9 proteins from the 12 TAAs panel were shown to generate specific signal and 5 antigens (p53, NY-ESO-1, IMP1, cyclin B1 and c-myc) were shown to have interaction with more than 10% of the positive sera from cancer patients. This protein subpanel was proven to be able to detect 72.2% of the cancer patients tested (within a 34 panel of 18 patients and 16 healthy donors).     

Micelle to solvent stacking of organic cations in micellar electrokinetic chromatography with sodium dodecyl sulfate

The on-line sample concentration technique, micelle to solvent stacking (MSS), was studied for small organic cations (quaternary ammonium herbicides, β-blocker drugs, and tricyclic antidepressant drugs) in reversed migration micellar electrokinetic chromatography. Electrokinetic chromatography was carried out in fused silica capillaries with a background solution of sodium dodecyl sulfate (SDS) in a low pH phosphate buffer. MSS was performed using anionic SDS micelles in the sample solution for analyte transport and methanol or acetonitrile as organic solvent in the background solution for analyte effective electrophoretic mobility reversal. The solvent also allowed for the separation of the analyte test mixtures. A model for focusing and separation was developed and the mobility reversal that involved micelle collapse was experimentally verified. The effect of analyte retention factor was observed by changing the % organic solvent in the background solution or the concentration of SDS in the sample matrix. With an injection length of 31.9 cm (77% of effective capillary length) for the 7 test drugs, the LODs (S/N=3) of 5-14 ng/mL were 101-346-fold better when compared to typical injection. The linearity (R(2), range=0.025-0.8 μg/mL), intraday and interday repeatability (%RSD, n=10) were ≥0.988, <6.0% and <8.5%, respectively. In addition, analysis of spiked urine samples after 10-fold dilution with the sample matrix yielded LODs=0.02-0.10 μg/mL. These LODs are comparable to published electrophoretic methods that required off-line sample concentration. However, the practicality of the technique for more complex samples will rely on dedicated sample preparation schemes.     

Lectin-based electrophoretic analysis of the expression of the 35 kDa inter-alpha-trypsin inhibitor heavy chain H4 fragment in sera of patients with five different malignancies

A 35 kDa glycoprotein whose abundance was previously demonstrated to be enhanced in sera of patients with endometrial adenocarcinoma (n = 12), was isolated from pooled sera of three of the cancer patients using champedak galactose-binding lectin affinity chromatography in the present study. Subjecting it to 2-DE and MS/MS, the glycoprotein was identified as the O-glycosylated fragment of inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4). When compared to control sera (n = 17), expression of the 35 kDa ITIH4 cleavage fragment was demonstrated to be significantly enhanced in sera of patients with breast carcinoma (n = 10), epithelial ovarian carcinoma (n = 10), and germ cell ovarian carcinoma (n = 10) but not in patients with nasopharyngeal carcinoma (n = 13) and osteosarcoma (n = 7). The lectin-based electrophoretic bioanalytical method adopted in the present study may be used to assess the physiological relevance of ITIH4 fragmentation and its correlation with different malignancies, their stages and progression.     

Preparation of quantum dots encoded microspheres by electrospray for the detection of biomolecules

In this paper, a novel method based on the electrospray technique has been developed for preparation of quantum dot (QD)-encoded microspheres for the fist time. By electrospraying the mixture of polymer solution and quantum dots solution (single-color QDs or multi-color QDs), it is accessible to obtain a series of composite microspheres containing the functional nanoparticle. Poly(styrene-acrylate) was utilized as the electrospray polymer materials in order to obtain the microsphere modified with carboxyl group on the surface. Moreover, to test the performance of the QD-encoded microsphere in bioapplication, it is carried out that immunofluorescence analysis between antigens of mouse IgG immobilized on the functional microsphere and FITC labeled antibodies of goat-anti-mouse IgG in experiment. To the best of our knowledge, this is the first report of QD-encoded microspheres prepared by electrospray technology. This technology can carry out the one-pot preparation of different color QD-encoded microspheres with multiple intensities. This technology could be also suitable for encapsulating other optical nanocrystals and magnetic nanoparticles for obtaining multifunctional microspheres. All of the results in this paper show that the fluorescence beads made by electrospray technique can be well applied in multiplex analysis. These works provide a good foundation to accelerate application of preparing microspheres by electrospray technique in practice.     

Electrophoresis of soft particles at high electrolyte concentrations: an interpretation by the Henry theory

The existence of electrophoretic mobility at high electrolyte concentrations defines a remarkable peculiarity in the electrosurface characteristics of soft particles. According to Ohshima [H. Ohshima, Colloids Surf. 103 (1995) 249], this effect is caused by the electroosmotic flow within the soft particle shell. An explanation supporting Ohshima's conclusion can be derived from classic electrokinetic theories. Based on the Henry theory [D.C. Henry, Proc. R. Soc. London Ser. A 133 (1931) 106], we demonstrate that the electrophoretic mobility of soft particles does not disappear at decinormal concentration.     

Hydrophilic core-shell microspheres: a suitable support for controlled attachment of proteins and biomedical diagnostics

Functional hydrophilic microspheres (latex particles) have found various applications in life sciences and in medicine - particularly in latex diagnostic tests. This paper presents a comprehensive review of studies on latex particles with a hydrophilic interfacial layer composed of various hydrophilic polymers with reactive groups at the ends of macromolecules or at each monomeric unit along the chain. Typical examples of these hydrophilic polymers are poly(2-hydroxyethyl methyl methacrylate), poly(acrylic acid), poly(N,N-dimethylacrylamide), polysaccharides, poly(ethylene oxide) and polyglycidol. Hydrophilic microspheres with different morphologies (uniform or core-shell, see Figure) have been synthesized by emulsion and dispersion polymerizations. The chemical structure of polymers which constitute the interfacial layer of microspheres has been investigated using a variety of instrumental techniques (such as XPS, SSIMS and NMR) and analytical methods based on specific chemical reactions suitable for the determination of particular functional groups. Microspheres are exposed to contact with proteins in the majority of medical applications. This paper presents examples of studies on the attachment of these biomacromolecules to microspheres. The relation between the structure of the interfacial layer of microspheres and the ability of these particles for the covalent binding of proteins is discussed. Several examples of diagnostic tests, in which hydrophilic microspheres with adsorbed or covalently immobilized proteins were used as reagents, are presented. The paper also contains a short review of the application of magnetic hydrophilic particles for protein separation. Examples of hydrophilic latex particles used for hemoperfusion or heavy metal ion separation are presented. Hydrophilic microspheres with uniform or core-shell morphologies.     

Effect of the Surface Structure of Poly(styrene-co-acrolein) Microspheres and Its Modification by Protein on Electrosurface Properties

The electrophoretic mobility of poly(styrene-co-acrolein) microspheres was studied as a function of storage time. It was shown that pH_IEP2.0 is retained but the abnormal dependence of electrophoretic mobility on NaCl concentration is replaced by classical dependence. When comparing chemisorption of bovine serum albumin (BSA) on the microsphere surface for various latex samples, the differences in the isotherm patterns was revealed; moreover, the prevalence of surface concentration of carboxyl groups over that of aldehyde groups resulted in a decrease in adsorption. After the modification of the microspheres by protein, the values of pH_IEPfall within the range of 3.5–5.0 and their dependence on the amount of surface-bound protein passes the minimum. The results obtained are discussed in terms of the different arrangement patterns of protein molecules on the microsphere surface and the changes of BSA macromolecule conformations under the effect of a dispersion medium and as a result of chemical interaction with the polymer surface.     

Dependence of Temperature-Sensitivity of Poly(N-isopropylacrylamide-co-acrylic acid) Hydrogel Microspheres upon Their Sizes

Monodisperse poly(N-isopropylacrylamide-co-acrylic acid) hydrogel microspheres were prepared by a membrane emulsification method using membranes of pore diameters of 0.33, 0.73, 1.15, and 1.70 μm. The hydrogels were synthesized by polymerization of 3.6 M N-isopropylacrylamide (N-IPAAm or NIPAM) and 0.4 M acrylic acid (AAc). Their surface properties were studied by measuring the electrophoretic mobility of the microspheres in electrolyte solutions at pH 7.4 at 25, 30, 33, 35, 40, and 45 degrees C. Poly(N-IPAAm-co-AAc) microspheres have shown negative mobility. More negative values of electrophoretic mobility were obtained with the smaller microspheres than the larger ones at each temperature. The surface charge density of the microspheres increased and their surfaces became harder above 35 degrees C, since the microspheres contained thermosensitive poly(N-IPAAm) moiety and LCST increased by the addition of AAc, while that of poly(N-IPAAm) was 33 degrees C. It has recently been found that the smaller microspheres exhibit the stronger dependence of both surface charge density and softness on the temperature. Copyright 2000 Academic Press.     

Stepwise hydration of ionized aromatics. Energies, structures of the hydrated benzene cation, and the mechanism of deprotonation reactions

The stepwise binding energies (DeltaHdegree(n-1,n)) of 1-8 water molecules to benzene(.+) [Bz(.+)(H2O)n] were determined by equilibrium measurements using an ion mobility cell. The stepwise hydration energies, DeltaHdegree(n-1,n), are nearly constant at 8.5 +/- 1 kcal mol-1 from n = 1-6. Calculations show that in the n = 1-4 clusters, the benzene(.+) ion retains over 90% of the charge, and it is extremely solvated, that is, hydrogen bonded to an (H2O)n cluster. The binding energies and entropies are larger in the n = 7 and 8 clusters, suggesting cyclic or cage-like water structures. The concentration of the n = 3 cluster is always small, suggesting that deprotonation depletes this ion, consistent with the thermochemistry since associative deprotonation Bz(.+)(H2O)(n-1) + H2O-->C6H5. + (H2O)nH+ is thermoneutral or exothermic for n > or = 4. Associative intracluster proton transfer Bz(.+)(H2O)(n+1) + H2O-->C6H5.(H2O)nH+ would also be exothermic for n > or = 4, but lack of H/D exchange with D2O shows that the proton remains on C6H6(.+) in the observed Bz(.+)(H2O)n clusters. This suggests a barrier to intracluster proton transfer, and as a result, the [Bz(.+)(H2O)n]* activated complexes either undergo dissociative proton transfer, resulting in deprotonation and generation of (H2O)nH+, or become stabilized. The rate constant for the deprotonation reaction shows a uniquely large negative temperature coefficient of K = cT(-67+/-4) (or activation energy of -34+/- 1 kcal mol-1), caused by a multibody mechanism in which five or more components need to be assembled for the reaction.     

Two-dimensional electrophoretic and immunoblot analysis of cell surface proteins of spiral-shaped and coccoid forms of Helicobacter pylori

Cell surface proteins of the human gastric pathogen Helicobacter pylori extracted during different in vitro growth phases were analyzed by one- and two-dimensional gelelectrophoresis (1-DE and 2-DE) and by 2-DE immunoblot. Broth-cultured H. pylori cells were stained with an acridine-orange dye to monitor the morphological status of the organism. In 2-day-cultures, 96% of the bacterial cells were spiral-shaped and four days later a morphological switch to coccoid forms occurred. In 10-day cultures spiral-shaped forms were not found. By 1-DE, proteins with the molecular masses of 87 and 120 kDa were detected in the 2-day cultures that disappeared in cells of 12-day cultures. A protein corresponding in size to the heat shock protein (GroEl homolog, Hsp60) and a 62 kDa protein, the ureaseB-subunit, were identified in extracted proteins of 2-, 8-, and 12-day cultures. 2-DE revealed an increased number of silver-stained spots of 8-day cultures (in average 250 spots) compared with protein extracted from 2-day cells (in average 160 spots). 2-DE immunoblots performed with sera containing antibodies to major H. pylori proteins such as the A- and B-subunits of urease and the Hsp60 showed similar reactivity to surface proteins extracted from 2-, 8-, and 12-day cultures, suggesting that these proteins remain immunologically intact. Pooled sera from infected patients absorbed with spiral-shaped cells showed an almost total blocking of the antibody reactivity to extracted coccoid proteins in 2-DE immunoblot. Eighteen spots were still visible, but this reactivity probably represents a solid overexpression by the coccoid cells of Hsp60 and ureaseB proteins and is thus difficult to block.     

Measurement of the amplitude and phase of the electrophoretic and electroosmotic mobility based on fast single-particle tracking

The electrophoretic mobility of micron-scale particles is of crucial importance in applications related to pharmacy, electronic ink displays, printing, and food technology as well as in fundamental studies in these fields. Particle mobility measurements are often limited in accuracy because they are based on ensemble averages and because a correction for electroosmosis needs to be made based on a model. Single-particle approaches are better suited for examining polydisperse samples, but existing implementations either require multiple measurements to take the effect of electroosmosis into account or are limited in accuracy by short measurement times. In this work, accurate characterization of monodisperse and polydisperse samples is achieved by measuring the electrophoretic mobility on a particle-to-particle basis while suppressing electroosmosis. Electroosmosis can be suppressed by measuring in the middle of a microchannel while applying an AC voltage with a sufficiently high frequency. An accurate measurement of the electrophoretic mobility is obtained by analyzing the oscillating particle motion for per particle with a high-speed camera measuring at , synchronized to the applied electric field. Attention is paid to take into account the effect of the rolling shutter and the non-uniform sampling in order to obtain the accurate amplitude and phase of the electrophoretic mobility. The accuracy of method is experimentally verified and compared with a commercial apparatus for polystyrene microspheres in water. The method is further demonstrated on a range of particle materials and particle sizes and for a mixture of positively and negatively charged particles.     

Surface plasmon resonance biosensors for detection of pathogenic microorganisms: strategies to secure food and environmental safety

This review describes the exploitation of exclusively optical surface plasmon resonance (SPR) biosensors for the direct and indirect detection of pathogenic microorganisms in food chains and the environment. Direct detection is, in most cases, facilitated by the use of defined monoclonal or polyclonal antibodies raised against (a part of) the target pathogenic microorganisms. The antibodies were immobilized to a solid phase of the sensor to capture the microbe from the sample. Alternatively, antibodies were used in an inhibition-like assay involving incubation with the target organism prior to analysis of nonbound antibodies. The free immunoglobins were screened on a sensor surface coated with either purified antigens or with Fc or Fab binding antibodies. Discussed examples of these approaches are the determination of Escherichia coli O1 57:H7, Salmonella spp., and Listeria monocytogenes. Another direct detection strategy involved SPR analysis of polymerase chain reaction products of Shiga toxin-2 genes reporting the presence of E. coli O157:H7 in human stool. Metabolic products have been exploited as biomarkers for the presence of a microbial agent, such as enterotoxin B and a virulence factor for the occurrence of Staphylococcus aureus and Streptococcus suis, respectively. Indirect detection, on the other hand, is performed by analysis of a humoral immune response of the infected animal or human. By immobilization of specific antigenic structures, infections with Herpes simplex and human immunodeficiency viruses, Salmonella and Treponema pallidum bacteria, and Schistosoma spp. parasites were revealed using human, avian, and porcine sera and avian eggs. Bound antibodies were easily isotyped using an SPR biosensor to reveal the infection history of the individual. Discussed studies show the recent recognition of the suitability of this type of instrument for (rapid) detection of health-threatening microbes to food and environmental microbial safety.