Sequential enzymatic reactions and stability of biomolecules immobilized onto phospholipid polymer nanoparticles

Polymer nanoparticles for sequential enzymatic reactions were prepared by combining a phospholipid polymer shell with a polystyrene core. The active ester groups for the bioconjugation and phospholipid polar groups were incorporated into the phospholipid polymer backbone using a novel active ester monomer and 2-methacryloyloxyethyl phosphorylcholine. For the sequential enzymatic reactions, acetylcholinesterase, choline oxidase, and horseradish peroxidase-labeled IgG were immobilized onto the nanoparticles. As substrates, acetylcholine chloride, choline chloride, and tetramethylbenzidine were added to the nanoparticle suspension, the acetylcholine chloride was converted to choline chloride, the choline chloride was oxidized by choline oxidase, and hydrogen peroxide was then formed as an enzymatic degradation product. The hydrogen peroxide was used for the next enzymatic reaction (oxidized by peroxidase) with tetramethylbenzidine. The sequential enzymatic reactions on the nanoparticles via degradation products (hydrogen peroxide) were significantly higher than that of the enzyme mixture. This result indicated that the diffusion pathway of the enzymatic products and the localization of the immobilized enzyme were important for these reactions. These nanoparticles were capable of facilitating sequential enzymatic reactions.     

Polymer nanoparticles covered with phosphorylcholine groups and immobilized with antibody for high-affinity separation of proteins

Novel polymer nanoparticles were prepared for the selective capture of a specific protein from a mixture with high effectiveness. The nanoparticle surface was covered with hydrophilic phosphorylcholine groups and active ester groups for easy immobilization of antibodies. Phospholipid polymers (PMBN) composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate, were synthesized for the surface modification of poly( l-lactic acid) nanoparticles. Surface analysis of the nanoparticles using laser-Doppler electrophoresis and X-ray photoelectron spectroscopy revealed that the surface of nanoparticles was covered with PMBN. Protein adsorption was evaluated with regard to the nonspecific adsorption on the nanoparticles that was effectively suppressed by the phosphorylcholine groups. The immobilization of antibodies on nanoparticles was carried out under physiological conditions to ensure specific binding of antigens. The antibody immobilized on the nanoparticles exhibited high activity and strong affinity for the antigen similar to that exhibited by an antibody in a solution. The selective binding of a specific protein as an antigen from a protein mixture was relatively high compared to that observed with conventional antibody-immobilized polymer nanoparticles. In conclusion, nanoparticles having both phosphorylcholine and active ester groups for antibody immobilization have strong potential for use in highly selective separation based on the biological affinities between biomolecules.     

Single molecule FRET for the study on structural dynamics of biomolecules

Single molecule fluorescence resonance energy transfer (FRET) is the technique that has been developed by combining FRET measurement and single molecule fluorescence imaging. This technique allows us to measure the dynamic changes of the interaction and structures of biomolecules. In this study, the validity of the method was tested using fluorescence dyes on double stranded DNA molecules as a rigid spacer. FRET signals from double stranded DNA molecules were stable and their average FRET values provided the distance between the donor and acceptor in agreement with B-DNA type helix model. Next, the single molecule FRET method was applied to the studies on the dynamic structure of Ras, a signaling protein. The data showed that Ras has multiple conformational states and undergoes transition between them. This study on the dynamic conformation of Ras provided a clue for understanding the molecular mechanism of cell signaling switches.     

Artificial cell membrane-covered nanoparticles embedding quantum dots as stable and highly sensitive fluorescence bioimaging probes

To obtain a stable and highly sensitive bioimaging fluorescence probe, polymer nanoparticles with embedded quantum dots were covered with an artificial cell membrane. These nanoparticles were designed by assembling phospholipid polar groups as a platform, and oligopeptide was immobilized as a bioaffinity moiety on the surface of the nanoparticles. The polymer nanoparticles showed resistance to cellular uptake from HeLa cells owing to the nature of the phosphorylcholine groups. When arginine octapeptide was immobilized at the surface of the nanoparticles, they were able to penetrate the membrane of HeLa cells effectively. Cytotoxicity of the nanoparticles was not observed even after immobilization of oligopeptide. Thus, we obtained stable fluorescent polymer nanoparticles covered with an artificial cell membrane, which are useful as an excellent bioimaging probe and as a novel evaluation tool for oligopeptide functions in the target cells.     

G-quadruplex-forming oligonucleotide conjugated to magnetic nanoparticles: synthesis, characterization, and enzymatic stability assays

In the present work, we report the conjugation of superparamagnetic nanoparticles to a fluorescently labeled oligodeoxyribonucleotide (ODN) able to fold into stable unimolecular guanine quadruple helix under proper ion conditions by means of its thrombin-binding aptamer (TBA) sequence. The novel modified ODN, which contained a fluorescent dU(Py) unit at 3'-end and a 12-amino-dodecyl spacer (C(12)-NH(2)) at 5' terminus, was characterized by ESI-MS and optical spectroscopy (UV, CD, fluorescence), and analyzed by RP-HPLC chromatography and electrophoresis. From CD and fluorescence experiments, we verified that dU(Py) and C(12)-NH(2) incorporation does not interfere with the conformational stability of the G-quadruplex. Subsequently, the conjugation of the pyrene-labeled ODN with the magnetite particles was performed, and the ODN-conjugated nanoparticles were studied through optical spectroscopy (UV, CD, fluorescence) and by enzymatic and chemical assays. We found that the nanoparticles enhanced the stability of the TBA ODN to enzymatic degradation. Finally, we evaluated the amount of the TBA-conjugated nanoparticles immobilized on a magnetic separator in view of the potential use of the nanosystem for the magnetic capture of thrombin from complex mixtures.     

A bioconjugate of Pseudomonas cepacia lipase with alginate with enhanced catalytic efficiency

A bioconjugate of Pseudomonas cepacia lipase with alginate was prepared by simple adsorption. Atomic force microscope (AFM) images showed that this bioconjugate resulted from adsorption rather than entrapment of the enzyme as enzyme molecules were visible on the gel surface. The soluble bioconjugate exhibited increased enzyme activity in terms of high effectiveness factor (effectiveness factor was 3 for the immobilized preparation) and greater Vmax/Km value (Vmax/Km increased 25 times upon immobilization). This constitutes one of the less frequently observed instances of lipase activation by lid opening as a result of binding to a predominantly hydrophilic molecule. The bioconjugate was also more stable at 55 degrees C as compared to the free enzyme and could be reused for oil hydrolysis up to 4 cycles without any loss in activity. Fluorescence emission spectroscopy showed that the immobilized enzyme had undergone definite conformational changes.     

Characterization of lysine-tagged Bacillus stearothermophilus leucine aminopeptidase II immobilized onto carboxylated gold nanoparticles

Bacillus stearothermophilus leucine aminopeptidase II tagged C-terminally with either tri- or nona-lysine (BsLAPII-Lys_3/9) was constructed and over-expressed in Escherichia coli M15 (pRep4). The recombinant enzymes were purified to homogeneity by nickel-chelate chromatography and their molecular masses were determined to be approximately 45 kDa by SDS/PAGE. Surface modification of colloidal gold with 16-mercaptohexadecanoic acid was employed to generate the carboxylated nanoparticles. BsLAPII-Lys₉ was efficiently immobilized onto the carboxylated gold nanoparticles (AuNP-COOH) and the obtained bioconjugate showed excellent biocatalytic activity in the immobilized form. Additionally, the bioconjugate material exhibited a significant enhancement in temperature stability and could be reused over 5 successive cycles.     

Development of a method to evaluate caspase-3 activity in a single cell using a nanoneedle and a fluorescent probe

A method to detect an enzymatic reaction in a single living cell using an atomic force microscope equipped with an ultra-thin needle (a nanoneedle) and a fluorescent probe molecule was developed. The nanoneedle enables the low-invasive delivery of molecules attached onto its surface directly into a single cell. We hypothesized that an enzymatic reaction in a cell could be profiled by monitoring a probe immobilized on a nanoneedle introduced into the cell. In this study, a new probe substrate (NHGcas546) for caspase-3 activity based on fluorescent resonance energy transfer (FRET) was constructed and fixed on a nanoneedle. The NHGcas546-modified nanoneedle was inserted into apoptotic cells, in which caspase-3 is activated after apoptosis induction, and a change in the emission spectrum of the immobilized probe could be observed on the surface of the nanoneedle. Thus, we have developed a successful practical method for detecting a biological phenomenon in a single cell. We call the method MOlecular MEter with Nanoneedle Technology (MOMENT).     

TiO2 nanowire FET device: encapsulation of biomolecules by electro polymerized pyrrole propylic acid

Silane-based methods have become the standards for the conjugation of biomolecules, especially for the preparation of one-dimensional nanomaterial biosensors. However, the specific binding of those target molecules might raise problems with regard to the sensing and non-sensing regions, which may contaminate the sensing devices and decrease their sensitivity. This paper attempts to explore the encapsulation of biomolecules on a one-dimensional nanomaterial field effect transistor (FET) biosensor using polypyrrole propylic acid (PPa). Specifically, the encapsulation of biomolecules via the electropolymerization of pyrrole propylic acid (Pa), a self-made low-conductivity polymer, on TiO(2)-nanowire (NW)-based FETs is presented. The energy dispersive spectrum (EDS) was obtained and electrical analysis was conducted to investigate PPa entrapping anti-rabbit IgG (PPa/1°Ab) on a composite film. The specificity, selectivity and sensitivity of the sensor were analyzed in order to determine the immunoreaction of PPa/1°Ab immobilized NW biosensors. Our results show that PPa/1°Ab achieved high specificity immobilization on NWs under the EDS analysis. Furthermore, the TiO(2)-NW FET immunosensor developed in this work successfully achieved specificity, selectivity and sensitivity detection for the target protein rabbit IgG at the nano-gram level. The combination of PPa material and the electropolymerization method may provide an alternative method to immobilize biomolecules on a specific surface, such as NWs.     

Use of dextrans as long and hydrophilic spacer arms to improve the performance of immobilized proteins acting on macromolecules

New dextran-agarose supports, suitable for covalent immobilization of enzymes and proteins acting on macromolecular substrates, were prepared. The thick internal fibers of agarose gels were covered by a low-density layer of long, flexible, hydrophilic, and inert dextran molecules. Rennin and protein A were immobilized on these novel supports and the resulting derivatives exhibited a very high capacity for biological recognition of soluble macromolecular substrates. Caseinolytic activity of this immobilized enzyme was 15-fold higher than activity of directly immobilized rennin, through short spacer arms, on agarose gels. Similarly, the new derivatives of immobilized protein A were able to adsorb up to 2 molecules of immunoglobulin per each molecule of immobilized protein A. When the immobilized proteins were secluded away from the support surface by using these new long and hydrophilic spacer arms, they exhibit minimal steric hindrances that could be promoted by the proximity of the support surface.     

On the preparation, characterization, and enzymatic activity of fungal protease-gold colloid bioconjugates

We present herein details pertaining to the preparation of bioconjugates of colloidal gold with aspartic protease from the fungus Aspergillus saitoi (F-prot) and their characterization and enzymatic activity. Simple mixing of the colloidal gold and protein solutions under protein-friendly conditions (pH = 3) followed by centrifugation (to remove uncomplexed gold nanoparticles and protein molecules) results in the formation of the fungal protease-gold nanoparticle conjugates. The protein-gold nanoparticle bioconjugate was redispersed in buffer solution and indicated the formation of efficient bioconjugates with intact native protein structures. The bioconjugates in solution were characterized by UV-vis spectroscopy, fluorescence spectroscopy, and biocatalytic activity measurements while drop-dried bioconjugate films on Si (111) substrates were characterized by scanning electron microscopy (SEM), energy dispersive analysis of X-rays (EDAX), and X-ray diffraction (XRD) measurements. Microscopy images do show some aggregate formation, but the intactness of the native structure of the enzyme in the bioconjugate material was verified by fluorescence and biocatalytic activity measurements. The enzyme retains substantial biocatalytic activity in the bioconjugate material and was comparable to that of free enzyme in solution.     

High-performance affinity chromatography for characterization of human immunoglobulin G digestion with papain

Reactive continuous rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) were prepared within the confines of a stainless steel column. Then papain was immobilized on these monoliths either directly or linked by a spacer arm. In a further step, a protein A affinity column was used for the characterization of the digestion products of human immunoglobulin G (IgG) by papain. The results showed that papain immobilized on the monolithic rod through a spacer arm exhibits higher activity for the digestion of human IgG than that without a spacer arm. The apparent Michaelis-Menten kinetic constants of free and immobilized papain, K(m) and V(max), were determined. The digestion conditions of human IgG with free and immobilized papain were optimized. Comparison of the thermal stability of free and immobilized papain showed that the immobilized papain exhibited higher thermal stability than the free enzyme. The half-time of immobilized papain reaches about a week under optimum pH and temperature conditions.     

Novel trifunctional carrier molecule for the fluorescent labeling of haptens

We developed a novel trifunctional carrier molecule for the synthesis of hapten-fluorophore conjugates as reporter molecules in immunoassays. This carrier eliminates some of the disadvantages associated with currently used fluorophore-labeling procedures including high nonspecific binding. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain molecule. This polypeptide provides a single site (terminal amino group) for covalent coupling of the hapten, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the attached fluorophores. The sites for fluorophore attachment are 4, 17, and 21 amino acids away from the hapten attachment site. This spatial separation minimizes quenching of the fluorescence signal due to interaction of the fluorophores with each other and with the attached hapten. In this study, 2,4-dinitrophenol (DNP) was selected as model hapten, fluorescein as label, and S-sulfonate groups as hydrophilic residues. The properties of the DNP-insulin A-chain-fluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein moiety via a small lysine spacer (DNP-Lys-Fl). The DNP-Ins-Fl conjugate exhibited a 3-fold lower nonspecific adsorption to immobilized non-immune IgG contributing to an approximately 3-fold more efficient displacement from the binding sites of an immobilized monoclonal anti-DNP antibody by the antigen DNP-lysine. Furthermore, at equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than DNP-Lys-Fl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrolytic and enzymatic degradation of nanoparticles based on amphiphilic poly(gamma-glutamic acid)-graft-L-phenylalanine copolymers

Amphiphilic graft copolymers consisting of poly(gamma-glutamic acid) (gamma-PGA) as the hydrophilic backbone and L-phenylalanine ethylester (L-PAE) as the hydrophobic side chain were synthesized by grafting L-PAE to gamma-PGA. The nanoparticles were prepared by a precipitation method, and about 200 nm-sized nanoparticles were obtained due to their amphiphilic properties. The hydrolytic and enzymatic degradation of these gamma-PGA nanoparticles was studied by gel permeation chromatography (GPC), scanning electron microscopy (SEM), dynamic light scattering (DLS) and (1)H NMR measurements. The hydrolysis ratio of gamma-PGA and these hydrophobic derivatives was found to decrease upon increasing the hydrophobicity of the gamma-PGA derivates. The pH had an effect on the hydrolytic degradation of the polymer. The hydrolysis of the polymer could be accelerated by alkaline conditions. The degradation of the gamma-PGA backbone by gamma-glutamyl transpeptidase (gamma-GTP) resulted in a dramatic change in nanoparticle morphology. With increasing time, the gamma-PGA nanoparticles began to decrease in size and finally disappeared completely. Moreover, the gamma-PGA nanoparticles were degraded by four different enzymes (Pronase E, protease, cathepsin B and lipase) with different degradation patterns. The enzymatic degradation of the nanoparticles occurred via the hydrolysis of gamma-PGA as the main chain and L-PAE as the side chain. In the case of the enzymatic degradation of gamma-PGA nanoparticles with Pronase E, the size of the nanoparticles increased during the initial degradation stage and decreased gradually when the degradation time was extended. Nanoparticles composed of biodegradable amphiphilic gamma-PGA with reactive function groups can undergo further modification and are expected to have a variety of potential pharmaceutical and biomedical applications, such as drug and vaccine carriers.     

Near infrared sensing based on fluorescence resonance energy transfer between Mn:CdTe quantum dots and Au nanorods

A novel sensing system based on the near infrared (NIR) fluorescence resonance energy transfer (FRET) between Mn:CdTe quantum dots (Qdots) and Au nanorods (AuNRs) was established for the detection of human IgG. The NIR-emitting Qdots linked with goat anti-human IgG (Mn:CdTe-Ab1) and AuNRs linked with rabbit anti-human IgG (AuNRs-Ab2) acted as fluorescence donors and acceptors, respectively. FRET occurred by human IgG with the specific antigen–antibody interaction. And human IgG was detected based on the modulation in FRET efficiency. The calibration graph was linear over the range of 0.05–2.5 μM of human IgG under optimal conditions. The proposed sensing system can decrease the interference of biomolecules in NIR region and increase FRET efficiency in optimizing the spectral overlap of AuNRs with Mn:CdTe Qdots. This method has great potential for multiplex assay with different donor–acceptor pairs.     

Horseradish peroxidase-functionalized gold nanoparticle label for amplified immunoanalysis based on gold nanoparticles/carbon nanotubes hybrids modified biosensor

This paper describes the combination of electrochemical immunosensor using gold nanoparticles (GNPs)/carbon nanotubes (CNTs) hybrids platform with horseradish peroxidase (HRP)-functionalized gold nanoparticle label for the sensitive detection of human IgG (HIgG) as a model protein. The GNPs/CNTs nanohybrids covered on the glass carbon electrode (GCE) constructed an effective antibody immobilization matrix and made the immobilized biomolecules hold high stability and bioactivity. Enhanced sensitivity was obtained by using bioconjugates featuring HRP labels and secondary antibodies (Ab₂) linked to GNPs at high HRP/Ab₂ molar ratio. The approach provided a linear response range between 0.125 and 80 ng/mL with a detection limit of 40 pg/mL. The immunosensor showed good precision, acceptable stability and reproducibility and could be used for the detection of HIgG in real samples, which provided a potential alternative tool for the detection of protein in clinical laboratory.     

Protein A immobilized affinity cartridge for immunoglobulin purification

Recombinant Protein A was immobilized on a cellulose and acrylic composite matrix through Schiff base formation. Various factors that could affect the binding of immunoglobulin by the Protein A molecules immobilized on the solid matrix were studied to achieve optimum affinity purification. The spacer arm length and ligand concentration of Protein A were verified as factors crucial to optimized IgG purification. Liquid-phase environmental conditions such as pH and salt concentration also play important roles in adsorption capacity by affecting the molecular interaction between IgG and the immobilized Protein A. The rate of interaction between Protein A and IgG is rather fast, with minimal differences observed at 10-fold increases in the cartridge loading rate. This paper describes a cellulose/acrylic composite matrix for immobilizing Protein A, at an optimized ligand concentration, installed on a spacer arm of adequate length, to purify immunoglobulins from animal plasma. The fast-flow property of the cartridge made from such a matrix and its simplicity in operation provide effective means for purifying immunoglobulins on a relatively large scale.     

Multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using Au nanoparticle probes and quantum dots as labels

A novel microfluidic device with microbeads array was developed and sensitive genotyping of human papillomavirus was demonstrated using a multiple-enzyme labeled oligonucleotide-Au nanoparticle bioconjugate as the detection tool. This method utilizes microbeads as sensing platform that was functionalized with the capture probes and modified electron rich proteins, and uses the horseradish peroxidase (HRP)-functionalized gold nanoparticles as label with a secondary DNA probe. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. Through "sandwich" hybridization, the enzyme-functionalized Au nanoparticles labels were brought close to the surface of microbeads. The oxidation of biotin-tyramine by hydrogen peroxide resulted in the deposition of multiple biotin moieties onto the surface of beads. This deposition is markedly increased in the presence of immobilized electron rich proteins. Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal. Enhanced detection sensitivity was achieved where the large surface area of Au nanoparticle carriers increased the amount HRP bound per sandwiched hybridization. The on-chip genotyping method could discriminate as low as 1fmol/L (10zmol/chip, SNR>3) synthesized HPV oligonucleotides DNA. The chip-based signal enhancement of the amplified assay resulted in 1000 times higher sensitivity than that of off-chip test. In addition, this on-chip format could discriminate and genotype 10copies/μL HPV genomic DNA using the PCR products. These results demonstrated that this on-chip approach can achieve highly sensitive detection and genotyping of target DNA and can be further developed for detection of disease-related biomolecules at the lowest level at their earliest incidence.     

Occurrence of glucose polymer in undifferentiated PC12 cells

A large glucose polymer was found, following pronase digestion, in PC12 pheochromocytoma cells metabolically labeled with [1-3H]galactose. The polymer was excluded from a Bio-Gel A-0.5 m column and adsorbed by immobilized concanavalin A-Sepharose from which it was eluted with 10 mM alpha-methylmannoside. Glucose was found to be the sole component monosaccharide. Except for those capable of degrading glycogen, no exo- or endo-glycosidases cleaved the polymer. This is the first report on the occurrence of a glucose polymer in undifferentiated PC12 cells.