Aptamer‐based thrombin assay on microfluidic platform

A facile and sensitive aptamer‐based protocol has been developed for protein assay on microfluidic platform with fluorescence detection using an off‐chip microarray scanner. Aptamer‐functionalized magnetic beads were used to capture thrombin that binds to a second aptamer fluorescently labeled by Cy3. Experimental conditions, such as incubation time and temperature, washing time, interfering proteins, and aptamer, etc., were optimized for the microchip method. This work demonstrated there was a good relationship between fluorescence intensity and thrombin concentration in the range of 65–1000 ng/mL with the RSD less than 8%. Notably, an analysis only needs 1 μL volume of sample injection and this system can capture extremely tiny amount thrombin (0.4 fmol). This method has been successfully applied to assay of thrombin in human serum with the recovery of 79.74–95.94%.     

Automated sample preparation method for suspension arrays using renewable surface separations with multiplexed flow cytometry fluorescence detection

In this paper, we describe a new method of automated sample preparation for multiplexed biological analysis systems that use flow cytometry fluorescence detection. In this approach, color-encoded microspheres derivatized to capture particular biomolecules are temporarily trapped in a renewable surface separation column to enable perfusion with sample and reagents prior to delivery to the detector. This method provides for separation of the biomolecules of interest from other sample matrix components as well as from labeling solutions. After sample preparation, the beads can be released from the renewable surface column and delivered to a flow cytometer for direct on-bead analysis one bead at a time. Using mixtures of color-encoded beads derivatized for various analytes yields suspension arrays for multiplexed analysis. Development of this approach required a new technique for automated capture and release of the color-encoded microspheres within a fluidic system. We developed a method for forming a renewable filter and demonstrate its use for capturing microspheres that are too small to be easily captured in previous flow cells for renewable separation columns. The renewable filter is created by first trapping larger beads in the flow cell, and then smaller beads are captured either within or on top of the bed of larger beads. Both the selective microspheres and filter bed are automatically emplaced and discarded for each sample. A renewable filter created with 19.9 μm beads was used to trap 5.6 μm optically encoded beads with trapping efficiencies of 99%. The larger beads forming the renewable filter did not interfere with the detection of color-encoded 5.6 μm beads by the flow cytometer fluorescence detector. The use of this method was demonstrated with model reactions for a variety of bioanalytical assay types including a one-step capture of a biotinylated label on Lumavidin beads, a two-step sandwich immunoassay, and a one-step DNA binding assay. A preliminary demonstration of multiplexed detection of two analytes using color-encoded beads was also demonstrated. The renewable filter for creating separation columns containing optically encoded beads provides a general platform for coupling renewable surface methods for sample preparation and analyte labeling with flow cytometry detectors for suspension array multiplexed analyses.     

Efficiency of a fixed-bed and a gas-lift three-column reactor for continuous production of ethanol by pectate-and alginate-immobilized Saccharomyces cerevisiae cells

Ethanol-tolerant and thermo-tolerant yeast strain Saccharomyces cerevisiae C11-3 cells immobilized in calcium pectate and calcium alginate gels were used for ethanol fermentation in a three-reactor system with a gradient temperature control. The fermentation process has been tested in a fixed-bed and a gas-lift arrangement. The gas-lift system was more efficient due to a better mass transport between the phases. Abrasion was more evident in calcium alginate particles, while calcium pectate beads were not significantly damaged. Two different concentrations of alginate were tested and calcium pectate gel was demonstrated to be more suitable as an immobilization material in comparison with calcium alginate due to its mechanical resistance and favourable diffusion parameters, providing an ethanol production of more than 7.5 g dm⁻³ h⁻¹ over a period of 630 h.     

Comparison of different technologies for alginate beads production

This paper describes the results of the round robin experiment “Bead production technologies” carried out during the COST 840 action “Bioencapsulation Innovation and Technologies” within the 5th Framework Program of the European Community. In this round robin experiment, calcium alginate hydrogel beads with the diameter of (800 ± 100) μm were produced by the most common bead production technologies using 0.5–4 mass % sodium alginate solutions as starting material. Dynamic viscosity of the alginate solutions ranged from less than 50 mPa s up to more than 10000 mPa s. With the coaxial air-flow and electrostatic enhanced dropping technologies as well as with the JetCutter technology in the soft-landing mode, beads were produced from all alginate solutions, whereas the vibration technology was not capable to process the high-viscosity 3 % and 4 % alginate solutions. Spherical beads were generated by the electrostatic and the JetCutter technologies. Slightly deformed beads were obtained from high-viscosity alginate solutions using the coaxial airflow and from the 0.5 % and 2 % alginate solutions using the vibration technology. The rate of bead production using the JetCutter was about 10 times higher than with the vibration technology and more than 10000 times higher than with the coaxial air-flow and electrostatic technology. In memory of our colleague Stefan Rosinski     

Encapsulation of living cells with polymeric systems

Microencapsulation of cells producing recombinant proteins or hormones leads to immunoprotection and immobilization in culture or in vivo. We are investigating three different strategies for the production of calcium cross-linked alginate beads of a small size with immobilized and immunoprotected mammalian cells: a) the AirJet technology (coaxial gas flow extrusion), b) the vibrating nozzle technology, and c) the JetCutter technology. A alginate/poly-L-lysine/alginate complexation was used as the polymeric system. All three methods may be used for production of homogeneous beads with a diameter of approximately 350 μm. While the vibrating nozzle technique was limited to an alginate viscosity of 0.2 Pa·s or less, the AirJet and JetCutter technology were less sensitive to higher viscosities. High frequency Scanning Acoustic Microscopy is used for mechanical characterization of the microspheres as well as for investigation of surface properties.     

Suspended microchannel resonators with piezoresistive sensors

Precision frequency detection has enabled the suspended microchannel resonator (SMR) to weigh single living cells, single nanoparticles, and adsorbed protein layers in fluid. To date, the SMR resonance frequency has been determined optically, which requires the use of an external laser and photodiode and cannot be easily arrayed for multiplexed measurements. Here we demonstrate the first electronic detection of SMR resonance frequency by fabricating piezoresistive sensors using ion implantation into single crystal silicon resonators. To validate the piezoresistive SMR, buoyant mass histograms of budding yeast cells and a mixture of 1.6, 2.0, 2.5, and 3.0 μm diameter polystyrene beads are measured. For SMRs designed to weigh micron-sized particles and cells, the mass resolution achieved with piezoresistive detection (～3.4 fg in a 1 kHz bandwidth) is comparable to what can be achieved by the conventional optical-lever detector. Eliminating the need for expensive and delicate optical components will enable new uses for the SMR in both multiplexed and field deployable applications.     

A chip-based miniaturized format for protein-expression profiling: the exploitation of comprehensively produced antibodies

Numerous antibodies have been developed and validated in recent years, and show promise for use in novel functional protein assays. Such assays would be an alternative to pre-existing comprehensive assays, such as DNA microarrays. Antibody microarrays are thought to represent those functional protein assays. While a variety of attempts have been made to apply DNA microarray technology to antibody microarrays, a fully optimized protocol has not been established. We have been conducting a project to comprehensively produce antibodies against mouse KIAA ("KI" stands for "Kazusa DNA Research Institute" and "AA" are reference characters) proteins. Using our library of antibodies, we established a novel antibody microarray format that utilizes surface plasmon resonance (SPR) technology. A label-free real-time measurement of protein expression in crude cell lysates was achieved by direct readout of the bindings using SPR. Further refinement of the antibody microarray format enabled us to detect a smaller quantity of target proteins in the lysate without the bulk effect. In this review, we first summarize available antibody array formats and then describe the above-mentioned format utilizing updated SPR technology.     

Protein microarrays and multiplexed sandwich immunoassays: what beats the beads?

Protein microarray technology allows the simultaneous determination of a large variety of parameters from a minute amount of sample within a single experiment. Assay systems based on this technology are currently applied for the identification, quantitation and functional analysis of proteins. Protein microarray technology is of major interest for proteomic research in basic and applied biology as well as for diagnostic applications. Miniaturized and parallelized assay systems have reached adequate sensitivity and hence have the potential to replace singleplex analysis systems. However, robustness and automation needs to be demonstrated before this technology will finally prove suitable for high-throughput applications. Miniaturized and parallelized sandwich immunoassays are the most advanced assays formats among the different protein microarray applications. Multiplexed sandwich immunoassays can be used for the identification of biomarkers and the validation of potential target molecules. In this review an overview will be given on the current stage of protein microarray technology with a special focus on miniaturized multiplexed sandwich immunoassays.     

A Magnetically Driven Tandem Chip Enables Rapid Isolation and Multiplexed Profiling of Extracellular Vesicles

The protein phenotypes of extracellular vesicles (EVs) have emerged as promising biomarkers for cancer diagnosis and treatment monitoring. However, the technical challenges in rapid isolation and multiplexed molecular detection of EVs have limited their clinical practice. Herein, we developed a magnetically driven tandem chip to achieve streamlined rapid isolation and multiplexed profiling of surface protein biomarkers of EVs. Driven by magnetic force, the magnetic nanomixers not only act as tiny stir bars to promote mass transfer and enhance reaction efficiency of EVs, but also transport on communicating vessels of the tandem chip continuously and expedite the assay workflow. We designed cyclic surface enhancement of Raman scattering (SERS) tags to bind with target EVs and then release them by exonuclease I, eliminating steric hindrance and amplifying the SERS signal of multiple protein biomarkers on EVs. Due to the excellent assay performance, six breast cancer biomarkers were detected simultaneously on EVs using only 10 μL plasma within 1.5 h. The unweighted SUM signature offers great accuracy in discriminating breast cancer patients from healthy donors. Overall, the dynamic magnetic driving tandem chip offers a new avenue to advance the clinical application of EV-based liquid biopsy.     

Fluorescence energy transfer-based multiplexed hybridization assay using gold nanoparticles and quantum dot conjugates on photonic crystal beads

A multiplexed assay strategy was developed for the detection of nucleic acid hybridization. It is based on fluorescence resonance energy transfer (FRET) between gold nanoparticles (AuNPs) and multi-sized quantum dots (QDs) deposited on the surface of silica photonic crystal beads (SPCBs). The SPCBs were first coated with a three-layer primer film formed by the alternating adsorption of poly(allylamine hydrochloride) and poly(sodium 4-styrensulfonate). Probe DNA sequences were then covalently attached to the carboxy groups at the surface of the QD-coated SPCBs. On addition of DNA-AuNPs and hybridization, the fluorescence of the donor QDs is quenched because of the close proximity of the AuNPs. However, the addition of target DNA causes a recovery of the fluorescence of the QD-coated SPCBs, thus enabling the quantitative assay of hybridized DNA. Compared to fluorescent dyes acting as acceptors, the use of AuNPs results in much higher quenching efficiency. The multiplexed assay displays a wide linear range, high sensitivity, and very little cross-reactivity. This work, where such SPCBs are used for the first time in a FRET assay, is deemed to present a new and viable approach towards high-throughput multiplexed gene assays.

A multiplexed bead assay for profiling glycosylation patterns on serum protein biomarkers of pancreatic cancer

A multiplexed bead-based immunoassay was developed to simultaneously profile glycosylation patterns of serum proteins to investigate their usefulness as biomarkers for pancreatic cancer. The multiplex assay utilized protein-specific capture antibodies chemically coupled individually to beads labeled with specific amounts of fluorescent dye. Captured proteins were detected based on the extent and specific type of glycosylation as determined by successive binding of fluorescent lectin probes. Advantages to this technique include the fact that antibodies coupled to the beads had minimal nonspecific binding to the lectins ConA/SNA, avoiding the step of chemically blocking the antibody glycans and the bead assays were performed in a 96-well filter plate enabling high-throughput screening applications with improved reproducibility. The assay was tested with ConA and SNA lectins to examine the glycosylation patterns of α-1-β glycoprotein (A1BG) and serum amyloid p (SAP) component for use as potential biomarkers for the detection of pancreatic cancer based on the results from prior biomarker studies. The results showed that the SNA response on the captured A1BG protein could distinguish chronic pancreatitis samples from pancreatic cancer with a p-value of 0.035 and for the SAP protein with SNA, a p-value of 0.026 was found between the signal of normal controls and the pancreatic cancer samples. For the ConA response, a decline in the signal for both proteins in the serum samples was found to distinguish pancreatic cancer from normal controls and renal cell carnoma samples (A1BG, p<0.05; and SAP, p<0.0001).     

Automated analytical microarrays: a critical review

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple analytes in a single experiment. The specific affinity reaction of nucleic acids (hybridization) and antibodies towards antigens is the most common bioanalytical method for generating multiplexed quantitative results. Nucleic acid-based analysis is restricted to the detection of cells and viruses. Antibodies are more universal biomolecular receptors that selectively bind small molecules such as pesticides, small toxins, and pharmaceuticals and to biopolymers (e.g. toxins, allergens) and complex biological structures like bacterial cells and viruses. By producing an appropriate antibody, the corresponding antigenic analyte can be detected on a multiplexed immunoanalytical microarray. Food and water analysis along with clinical diagnostics constitute potential application fields for multiplexed analysis. Diverse fluorescence, chemiluminescence, electrochemical, and label-free microarray readout systems have been developed in the last decade. Some of them are constructed as flow-through microarrays by combination with a fluidic system. Microarrays have the potential to become widely accepted as a system for analytical applications, provided that robust and validated results on fully automated platforms are successfully generated. This review gives an overview of the current research on microarrays with the focus on automated systems and quantitative multiplexed applications. Figure MCR 3: A fully automated chemiluminescence microarray reader for analytical microarrays     

Decolorization of Some Azo Dyes by Immobilized <Emphasis Type="Italic">Geotrichum</Emphasis> sp. Biomass in Fluidized Bed Bioreactor

Geotrichum sp. strain, which is able to decolorize azo dyes enzymatically, was used in this study for decolorization of synthetics solutions contaminated by toxic azo dyes orange G, trypan blue, azorubine, and methyl red. The biomass of Geotrichum sp. was immobilized in calcium alginate and polyacrylamide gels and used for the decolorization of tested azo dyes in fluidized bed bioreactor. The highest specific decolorization rate was obtained when the fungal biomass was entrapped in calcium alginate beads. Immobilized biomass in calcium alginate continuously decolorized azo dyes after eight repeated batch decolorization experiments without significant loss of activity whereas polyacrylamide immobilized biomass retained only 10% of its activity after 4 days of incubation. The effects of some physicochemical parameters such as temperature, pH, and dyes concentration on decolorization performance of isolated fungal strain were also investigated.     

Removal of Cobalt Ions from Aqueous Solution by Adsorption onto Cross-Linked Calcium Alginate Beads

The removal of cobalt ions from dilute aqueous solutions using cross linked calcium alginate beads as the adsorbent is reported in this article. The influence of various experimental parameters such as pH, initial metal ion concentration, contact time and solid to liquid ratio were studied. The adsorption data were applied to Langmuir and Freundlich isotherm equations and various static parameters were calculated. It was observed that the uptake of cobalt ions was found to increase with time and that maximum adsorption was obtained within the first 60 minutes of the process. These results indicate that the cross linked calcium alginate beads have potential for removing cobalt ions from industrial waste water.     

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1.     

Discovery of a quorum sensing modulator pharmacophore by 3D small-molecule microarray screening

The screening of large arrays of drug-like small-molecules was traditionally a time consuming and resource intensive task. New methodology developed within our laboratories provides an attractive low cost, 3D microarray-assisted screening platform that could be used to rapidly assay thousands of compounds. As a proof-of-principle the platform was exploited to screen a number of quorum sensing analogs. Quorum sensing is used by bacterium to initiate and spread infection; in this context its modulation may have significant clinical value. 3D microarray slides were probed with fluorescently labeled ligand-binding domains of the LuxR homolog CarR from Erwinia carotovora subsp. carotovora. The 3D microarray platform was used to discover the biologically active chloro-pyridine pharmacophore, which was validated using a fluorometric ligand binding assay and ITC. Analogs containing the chloro-pyridine pharmacophore were found to be potent inhibitors of N-acyl-homoserine-lactone (AHL) mediated quorum sensing phenotypes in Serratia (IC(50) = ～5 μM) and Pseudomonas aeruginosa (IC(50) = 10-20 μM).     

Detection of benzimidazole carbamates and amino metabolites in liver by surface plasmon resonance-biosensor

Two surface plasmon resonance (SPR) biosensor screening assays were developed and validated to detect 11 benzimidazole carbamate (BZT) and four amino-benzimidazole veterinary drug residues in liver tissue. The assays used polyclonal antibodies, raised in sheep, to detect BZTs and amino-benzimidazoles. A modified Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) extraction method was developed to isolate benzimidazole carbamate residues. Liver samples were extracted using an acetonitrile extraction method. BZTs were purified by dispersive solid phase extraction (d-SPE) using C(18) sorbent. Residues of amino-benzimidazoles were effectively cleaned-up using a simple cyclohexane defatting step. The assays were validated in accordance with the performance criteria described in 2002/657/EC. The BZT assay limit of detection was calculated to be 32 μg kg(-1), the detection capability (CCβ) was determined to be 50 μg kg(-1) and the mean recovery of analytes was in the range 77-132%. The amino-benzimidazole assay limit of detection was determined to be 41 μg kg(-1), the CCβ was determined to be 75 μg kg(-1) and analyte recovery was in the range 103-116%. Biosensor assay performance was tested by analysing liver tissue from animals treated with benzimidazole drugs and comparing the results with an ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) confirmatory method. All non-compliant samples were identified using the biosensor assays.     

Optical technologies for the read out and quality control of DNA and protein microarrays

Microarray formats have become an important tool for parallel (or multiplexed) monitoring of biomolecular interactions. Surface-immobilized probes like oligonucleotides, cDNA, proteins, or antibodies can be used for the screening of their complementary targets, covering different applications like gene or protein expression profiling, analysis of point mutations, or immunodiagnostics. Numerous reviews have appeared on this topic in recent years, documenting the intriguing progress of these miniaturized assay formats. Most of them highlight all aspects of microarray preparation, surface chemistry, and patterning, and try to give a systematic survey of the different kinds of applications of this new technique. This review places the emphasis on optical technologies for microarray analysis. As the fluorescent read out of microarrays is dominating the field, this topic will be the focus of the review. Basic principles of labeling and signal amplification techniques will be introduced. Recent developments in total internal reflection fluorescence, resonance energy transfer assays, and time-resolved imaging are addressed, as well as non-fluorescent imaging methods. Finally, some label-free detection modes are discussed, such as surface plasmon microscopy or ellipsometry, since these are particularly interesting for microarray development and quality control purposes.