Affinity separations using microfabricated microfluidic devices:<Emphasis Type="Italic">In situ</Emphasis> photopolymerization and use in protein separations

The use of microfabricated microfluidic devices offers significant advantages over current technologies including fast analysis time and small reagent requirements. In the context of proteomic research, the possibility of using affinity-based separations for prefractionation of samples using microfluidic devices has significant potential. We demonstrate the use of microscale devices to achieve affinity separations of proteins using a device fabricated from borosilicate glass wafers. Photolithography and wet etching are used to pattern individual glass wafers and the wafers are fusion bonded at 650°C to obtain enclosed channels. A polymer has been successfully polymerizedin situ and used either as a frit for packing beads or, when derivatized with Cibacron Blue 3GA, as a separation matrix. Both of these technologies are based onin situ UV photopolymerization of glycidyl methacrylate (GMA) and trimethylolpropane trimethacrylate (TRIM) in channels.     

Parallel sample preparation of proteins,from crude samples to crystals ready for MALDI-MS,in an integrated microfluidic system

A microfluidic structure is presented where selective capture of proteins in complex samples, followed by clean-up, enzymatic processing, and MALDI-MS sample preparation of peptides generated, can be performed. The structure uses an affinity column to capture the protein while all other components in the sample are disposed of. The protein of interest is then eluted from the affinity column and captured on a second column on which the enzymatic processing is performed. Salts and hydrophilic contaminants are then removed before the products from the enzymatic reaction are eluted together with a suitable MALDI matrix and the solvent evaporated in a designated MALDI target structure. All steps can be performed automatically in 54 parallel microstructures on a microfluidic compact disc. The process is demonstrated by the selective capture and tryptic digest of recombinant IgG molecules from samples containing other proteins: an excess of bovine serum albumin or spent cell culture media.     

Biospecific properties of lysine-containing polyelectrolyte coatings for blood contacting devices

Biospecific properties of thromboresistant bilayer and multilayer coatings based on polyelectrolyte complexes of modified copolymer of N-vinylpyrrolidone and maleic acid (VPMA) with chitosan, amphiphilic chitosan or albumin were investigated. VPMA contained an affinity ligand towards plasminogen, α-amino coupled lysine residues. Polyethylene and polystyrene surfaces were investigated before and after their covering by protective polyelectrolyte coatings. The specific adsorption of plasminogen (precursor of the fibrinolytic enzyme plasmin) from its solutions and from human plasma was investigated using these model systems. It was found that all coatings with the outer contact layer of the lysine-containing affinity polymer exhibited affinity towards plasminogen. However, multilayer polyelectrolyte coatings were more efficient than the bilayer coatings with a single layer application and the affinity polymer coatings without interlayer. The decrease in the degree of thrombogenicity of the materials modified by polyelectrolyte coatings has been demonstrated in vitro and ex vivo. Employment of the proposed modification of surfaces will improve hemocompatibility of medical devices.     

Immobilized hemin affinity chromatography as a probe for proteins having potentiality to bind with heme

After Sepharose 4B polymer beads were activated by using epichlorohydrin, hemin was binded with them to prepare an immobilized hemin affinity chromatography column. The coupling rate of this column was very high, more than 0.25mg hemin could be fixed by 1g of wet Sepharose 4B beads. The column equilibrated with deionized water and eluated with pH 3.0 NaAc-HAc buffer was applied to capture the proteins in human serum, earthworm body and Bacillus subtilis cells. Three polypeptides in human serum were captured, one of which was verified as serum albumin after comparison to the control. At least one polypeptide in earthworm body, two in Bacillus subtilis cells displayed the powerful binding specificity to hemin. Our experiments demonstrated that the immobilized hemin affinity chromatography was available as a probe for some proteins having potentiality to bind with heme.     

Practical applications of high-affinity, albumin-binding proteins from a group G streptococcal isolate

Binding proteins that have high affinities for mammalian plasma proteins that are expressed on the surface of bacteria have proven valuable for the purification and detection of several biologically important molecules from human and animal plasma or serum. In this study, we have isolated a high affinity albumin-binding molecule from a group G streptococcal isolate of bovine origin and have demonstrated that the isolated protein can be biotinylated without loss of binding activity and can be used as a tracer for quantification of human serum albumin (HSA). The binding protein can be immobilized and used as a selective capture reagent in a competitive ELISA format using a biotinylated HSA tracer. In this assay format, the sensitivity of detection for 50% inhibition of binding of HSA was less than 1 μg/ml. When attached to the bacterial surface, this binding protein can be used to deplete albumin from human plasma, as analyzed by surface-enhanced laser desorption ionization time of flight mass spectrometry.     

Capturing cancer cells using aptamer-immobilized square capillary channels

We report a simple square capillary-based cell affinity chromatography device that utilizes a coating of aptamers for selective capture of target cancer cells from a flowing suspension. The device consists of a square capillary with an inner diameter of roughly five cell diameters, connected via Teflon tubing to a syringe. Aptamers are immobilized on the inner surface of the capillary through biotin-avidin chemistry, the extent of which can be controlled by adjusting the aptamer concentration. Introduction of different cell types into separate devices, as well as mixtures of target and non-target cells, demonstrated that aptamer-target cells can be captured in significantly higher concentrations compared to non-target cells. Once optimized, 91.1 ± 3.5% capture efficiency of target leukemia cells was reported, as well as 97.2 ± 2.8% and 83.6 ± 5.8% for two different colon cancer cell lines. In addition, cells captured in the device were imaged, and the square capillary exhibited better optical properties than standard cylindrical capillaries, leading to the detection of leukemia cells in blood samples. Compared to current microfluidic cell affinity devices, this capture device requires no complicated design or fabrication steps. By providing a simple means of detecting and imaging cancer cells in the blood, this work has potential to directly assist clinicians in determining disease prognosis and measuring therapeutic response.     

Micro-assembly of functionalized particulate monolayer on C18-derivatized SiO2 surfaces

This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C(18)-coated SiO(2) substrate via specific or non-specific bio-mediated interactions. Biotinylated bovine serum albumin pre-adsorbed onto a C(18) surface was used to mediate the surface assembly of streptavidin-coated microbeads (2.8 microm), while a bare C(18) surface was used to immobilize anti-Listeria antibody-coated microbeads (2.8 microm) through hydrophobic interactions. For a C(18) surface pre-adsorbed with bovine serum albumin, hydrophobic polystyrene microbeads (0.8 microm) and positively charged dimethylamino microbeads (0.8 microm) were allowed to self-assemble onto the surface. A monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads. The adsorption characteristics of Escherichia coli and Listeria monocytogenes on these microbead-based surfaces were studied using fluorescence microscopy. Both streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody-coated microbeads showed specific capture of L. monocytogenes, while polystyrene and dimethylamino microbeads captured both E. coli and L. monocytogenes non-specifically. The preparation of microbead-based surfaces for the construction of microfluidic devices for separation, detection, or analysis of specific biological species is discussed.     

High-performance affinity chromatography with immobilization of protein A and L-histidine on molded monolith

Reactive monoliths of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and L-histidine were immobilized on the monoliths directly or through a spacer arm, respectively. The properties of these two kinds of affinity columns were characterized, and the results showed that the columns with coupling of ligands by a spacer arm have some extent of non-specific adsorption for bovine serum albumin. The affinity column based on the monolithic polymer support provided us with good hydrodynamic characteristic, low flow resistance, and easy preparation. These two affinity columns were used for the purification of immunoglobulin G from human serum. The purity of the purified IgG was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The stability of the protein A affinity column was investigated, and its performance remained invariable after half a year. The effects of the nature and the pH of the buffer system on the adsorption capacity of human IgG on histidyl affinity column were also investigated. The protein A affinity column is favorable for rapid analysis of human IgG samples. In contrast, the advantages of mild elution conditions, high stability, as well as low cost provide the histidyl column further potential possibility for fast removal of IgG from human plasma in clinical applications.     

Quantification of chemical–polymer surface interactions in microfluidic cell culture devices

Microfluidic cell culture devices have been used for drug development, chemical analysis, and environmental pollutant detection. Because of the decreased fluid volume and increased surface area to volume ratio, interactions between device surfaces and the fluid is a key element that affects the performance and detection accuracy of microfluidic devices, particularly if fluid is recirculated by a peristaltic pump. However, this issue has not been studied in detail in a microfluidic cell culture environment. In this study, chemical loss and contaminant leakage from various polymer surfaces in a microfluidic setup were characterized. The effects of hydrophilic coating with Poly (vinyl alcohol), Pluronic® F‐68, and multi‐layer ionic coating were measured. We observed significant surface adsorption of estradiol, doxorubicin, and verapamil with PharMed® BPT tubing, whereas PTFE/BPT and stainless steel/BPT hybrid tubing caused less chemical loss in proportion to the fraction of BPT tubing in the hybrid system. Contaminants leaching out of the BPT tubing were found to be estrogen receptor agonists as determined by estrogen‐induced green fluorescence expression in an estrogen responsive Ishikawa cell line and also caused interference with an estradiol enzyme‐linked immunosorbent assay (ELISA) assay. Stainless steel/BPT hybrid tubing caused the least interference with ELISA. In summary, polymer surface and chemical interactions inside microfluidic systems should not be neglected and require careful investigations when results from a microfluidic system are compared with results from a macroscale cell culture setup. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Affinity separation of proteins in aqueous three-phase systems

Aqueous polymer three-phase systems composed of dextran-Ficoll-polyethylene glycol-water have been used for affinity partition of proteins. The upper, middle, and lower phases are rich in polyethylene glycol, Ficoll, and dextran, respectively. Affinity partition was performed using the reactive dyes Cibacron Blue F36-A and Remazol Yellow GCL which are known as specific ligands for albumin and prealbumin from human serum. When the ligands were bound alternatively to polyethylene glycol, Ficoll, or dextran the target proteins were directed toward the upper, middle, or lower phase, respectively. In the presence of two ligands immobilized to two different polymers the distribution of two proteins could be steered to different phases at the same time. Serum albumin and prealbumin could be separated by using Cibacron Blue-Ficoll and Remazol Yellow-dextran or Cibacron Blue-polyethylene glycol and Remazol Yellow-dextran as polymer ligands.     

Hexadecanedionic acid-sepharose 4B: A new tool for preparation of albumin-depleted plasma

Serum and plasma are the major sources of human material for clinical molecular diagnostics and drug discovery. However, due to the high abundance of some proteins, of which serum albumin (SA) is most prominent, lower-abundance proteins often remain undetectable in proteomic analysis of these body fluids. We have used hexadecanedionic acid (HDA) immobilized to Sepharose 4B to develop an affinity resin that is effective in the removal of SA from plasma. Two-dimensional gel analysis of the SA-depleted samples shows a significant enhancement of the low-abundance proteins and highly specific capture of serum albumin. The HDA resin shows better performance in terms of specificity than dye-based resins.     

Rare cell chemiluminescence detection based on aptamer-specific capture in microfluidic channels

An aptamer-based "sandwich" approach combined with the chemiluminescence (CL) analysis was developed for the capture and detection of rare cells on a microfluidic chip. Aptamers were immobilized on microfluidic channels to achieve capture and isolation of the specific cells from a cell mixture. The capture efficiency for target cells was more than 70% with the purity greater than 97%, when the content of the target cells was between 0.5% and 10% in the initial cell mixture. Gold nanoparticles (Au NPs) modified with aptamers were then added in to bind on the cells and trigger a CL reaction. A satisfactory linearity of the log/log calibration curve between the CL intensity and the number of target cells was observed with a low detection limit of 30 target cells in a 3 μL cell mixture. Spiked whole blood samples were also used to verify the practicality of the present method. This work demonstrated the potential application of the cheap and rapid CL detection into the early diagnosis of cancers.     

Bead-based microfluidic immunoassays: the next generation

Microfluidic devices possess many advantages like high throughput, short analysis time, small volume and high sensitivity that fulfill all the important criteria of an immunoassay used for clinical diagnoses, environmental analyses and biochemical studies. These devices can be made from a few different materials, with polymers presently emerging as the most popular choice. Other than being optically clear, non-toxic and cheap, polymers can also be easily fabricated with a variety of techniques. In addition, there are many polymer surface modification methods available to improve the efficiency of these devices. Unfortunately, current microfluidic immunoassays have limited multiplexing capability compared to flow cytometric assays. Flow cytometry employ the use of encoded microbeads in contrast with normal or paramagnetic microbeads applied in current microfluidic devices. The encoded microbead is the key in providing multiplexing capability to the assay by allowing multi-analyte analysis. Using several unique sets of code, different analytes can be detected in a single assay by tracing the identity of individual beads. The same principle could be applied to microfluidic immunoassays in order to retain all the advantages of a fluidic device and significantly improve multiplexing capability.     

基于微流控的真菌单细胞捕获和培养

【背景】真菌单细胞培养在研究细胞异质性及细胞生长特性等方面十分重要,因此需要建立简单便捷的方法对真菌单细胞进行培养与观察。【目的】基于微流控建立一种真菌单细胞的捕获及培养方法,同时直观地对单细胞进行定位和实时观察。【方法】利用L-edit设计芯片图案并利用等离子键合的方法制备微流控芯片;通过注射泵将红酵母菌溶液及里氏木霉孢子溶液进样以实现单细胞捕获;采用台盼蓝染色法测定酵母细胞的存活率;利用显微镜对酵母单细胞及木霉孢子的萌发、生长、繁殖过程进行观察。【结果】所制备的芯片形状完好,可实现酵母或孢子的单细胞捕获;酵母的捕获率为25.00%±1.38%;分别于0、2、4、6h对酵母进行观察,可看到酵母出芽过程;培养至48h,芯片上酵母细胞的存活率与游离培养条件下的存活率无显著性差异;分别于0、3、6、9 h对单个孢子进行观察,可以看到孢子萌发以及菌丝生长情况,且直至120h菌丝仍在生长。【结论】设计并制备了一种用于真菌单细胞捕获及定位培养的微流控芯片,这是此种芯片在真菌单细胞培养中的首次应用。细胞可在此微流控芯片上正常生长至少2 d,并可实现5 d及更长时间的培养,此方法可对真菌单细胞进行直观、定位的实时观察,有望用于多种微生物单细胞的生理、遗传性状研究,以及原生质体融合育种研究。     

Impact of adsorbents selection on capture efficiency of cell culture derived human influenza viruses

The study aims on affinity matrix selection for a cell culture derived influenza virus capture step in downstream processing. Euonymus europaeus lectin (EEL) was used as an affinity ligand. Human influenza A/Puerto Rico/8/34 (H1N1) virus produced in MDCK cells was chosen as a model strain. The chromatographic separation characteristics of reinforced cellulose membranes and different matrices such as agarose, cellulose, polymer and glass particles with immobilized EEL have been determined. Results obtained were compared to affinity matrices, which are currently used in large-scale vaccine manufacturing. Mass balances for the viral membrane protein hemagglutinin showed that EEL affinity chromatography results in higher recoveries than conventional processes using Cellufine sulphate and heparinized agarose. The most efficient media, a polymer and a cellulose membrane, have been further characterized by protein and host cell DNA measurements. Separations based on the polymer matrix and the cellulose membrane removed contaminating DNA to 0.2 and 1%, respectively. Total protein contents were decreased to 50 and 31%, respectively. The EEL-membrane showed the highest influenza virus binding capacity. These characteristics demonstrate that EEL affinity chromatography is a promising candidate for capturing influenza viruses from MDCK cell culture broths in addition to currently applied chromatographic media.     

Micrometer-sized supported lipid bilayer arrays for bacterial toxin binding studies through total internal reflection fluorescence microscopy

In this article, we present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy. The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated distearoylphosphatidylcholine:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside G(T1b) or G(M1). The ganglioside-populated SLB arrays were then exposed to either cholera toxin B subunit or tetanus toxin C fragment. Binding was assayed on planar substrates by total internal reflection fluorescence microscopy down to 100 pM concentration for cholera toxin subunit B and 10 nM for tetanus toxin fragment C. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is influenced by the microenvironment of the SLB and the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions.     

Trace element uptake in liver cells. 2. Effect of different proteins in the medium on the uptake of copper and zinc by hepatoma cells

Cultured rat hepatoma cells (HTC-cells) were used to study the uptake of copper and zinc from a minimal salt-glucose medium, supplemented with albumin from different species or with ovalbumin. Competitive equilibrium dialysis showed that at low molar ratios of metal/protein (less than 1) the affinity for copper of human and bovine albumin was about equal, but that of dog albumin or ovalbumin was much lower. Only a small difference in affinity for zinc could be detected between human albumin and ovalbumin. Supplementing the medium with the different proteins the rate of copper uptake in the cell at a given molar Cu/protein ratio increased as follows: human albumin congruent to bovine albumin less than dog albumin less than ovalbumin. When the molar Cu/protein ratio was increased, a discontinuity was seen with all three albumin species at a ratio of about 1. In contrast, the zinc uptake mimics that of Cu/ovalbumin, and no discontinuity was observed using different molar Zn/protein ratios. These results indicate that the rate of copper and zinc uptake depends strongly on its affinity for the protein: a low affinity leads to a high uptake. The results suggest further that at physiologic concentrations zinc is taken up by a mechanism different from that for copper.     

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.      

Affinity of human serum albumin for bilirubin varies with albumin concentration and buffer composition: results of a novel ultrafiltration method

Albumin binding is a crucial determinant of bilirubin clearance in health and bilirubin toxicity in certain disease states. However, prior attempts to measure the affinity of albumin for bilirubin have yielded highly variable results, reflecting both differing conditions and the confounding influence of impurities. We therefore have devised a method based on serial ultrafiltration that successively removes impurities in [(14)C]bilirubin until a stable binding affinity is achieved, and then we used it to assess the effect of albumin concentration and buffer composition on binding. The apparent binding affinity of human serum albumin for [(14)C]bilirubin was strongly dependent on assay conditions, falling from (5.09 +/- 0.24) x 10(7) liters/mol at lower albumin concentrations (15 microm) to (0.54 +/- 0.05) x 10(7) liters/mol at higher albumin concentrations (300 microm). To determine whether radioactive impurities were responsible for this change, we estimated impurities in the stock bilirubin using a novel modeling approach and found them to be 0.11-0.13%. Formation of new impurities during the study and their affinity for albumin were also estimated. After correction for impurities, the binding affinity remained heavily dependent on the albumin concentration (range (5.37 +/- 0.26) x 10(7) liters/mol to (0.65 +/- 0.03) x 10(7) liters/mol). Affinities decreased by about half in the presence of chloride (50 mm). Thus, the affinity of human albumin for bilirubin is not constant, but varies with both albumin concentration and buffer composition. Binding may be considerably less avid at physiological albumin concentrations than previously believed.     

A dynamic model for bilirubin binding to human serum albumin

Site-directed mutagenesis of human serum albumin was used to study the role of various amino acid residues in bilirubin binding. A comparison of thermodynamic, proteolytic, and x-ray crystallographic data from previous studies allowed a small number of amino acid residues in subdomain 2A to be selected as targets for substitution. The following recombinant human serum albumin species were synthesized in the yeast species Pichia pastoris: K195M, K199M, F211V, W214L, R218M, R222M, H242V, R257M, and wild type human serum albumin. The affinity of bilirubin was measured by two independent methods and found to be similar for all human serum albumin species. Examination of the absorption and circular dichroism spectra of bilirubin bound to its high affinity site revealed dramatic differences between the conformations of bilirubin bound to the above human serum albumin species. The absorption and circular dichroism spectra of bilirubin bound to the above human serum albumin species in aqueous solutions saturated with chloroform were also examined. The effect of certain amino acid substitutions on the conformation of bound bilirubin was altered by the addition of chloroform. In total, the present study suggests a dynamic, unusually flexible high affinity binding site for bilirubin on human serum albumin.