Synergies between micropreparative high-performance liquid chromatography and an instrumental optical biosensor

The recent development of an automated surface plasmon resonance technology for the measurement of biomolecular interactions (Pharmacia BIAcore) has provided new opportunities for the detection and analysis of protein-protein interactions. In the BIAcore, detection is based on changes in surface plasmon resonance which are monitored optically. Changes in surface plasmon resonance correspond to changes in surface concentration of macromolecules and can be monitored in real time.

We have found that the detection sensitivity obtainable with this technology (ng/ml concentrations of specific ligands are readily detectable for many applications) is complementary “in a bidirectional manner” to micropreparative HPLC. Thus micropreparative HPLC may be used to purify and characterise reagents for the biosensor, whilst the biosensor may be used to define chromatographic parameters such as elution conditions for affinity chromatography or serve as an affinity detector for fractions obtained during chromatographic purification.

Examples of such applications, including the potential of the biosensor to search for and monitor the purification of unknown ligands for which the target molecule has been identified, are shown. In particular, the use of the biosensor to monitor the purification of soluble epidermal growth factor receptor from A431 cell conditioned media is demonstrated.     

DNA-directed protein immobilization on mixed self-assembled monolayers via a streptavidin bridge

The simultaneous detection of multiple analytes is an important consideration for the advancement of biosensor technology. Currently, few sensor systems possess the capability to accurately and precisely detect multiple antigens. This work presents a simple approach for the functionalization of sensor surfaces suitable for multichannel detection. This approach utilizes self-assembled monolayer (SAM) chemistry to create a nonfouling, functional sensor platform based on biotinylated single-stranded DNA immobilized via a streptavidin bridge to a mixed SAM of biotinylated alkanethiol and oligo(ethylene glycol). Nonspecific binding is minimized with the nonfouling background of the sensor surface. A usable protein chip is generated by applying protein-DNA conjugates which are directed to specific sites on the sensor chip surface by utilizing the specificity of DNA hybridization. The described platform is demonstrated in a custom-built surface plasmon resonance biosensor. The detection capabilities of a sensor using this protein array have been characterized using human chorionic gonadotropin (hCG). The platform shows a higher sensitivity in detection of hCG than that observed using biotinylated antibodies. Results also show excellent specificity in protein immobilization to the proper locations in the array. The vast number of possible DNA sequences combine with the selectivity of base-pairing makes this platform an excellent candidate for a sensor capable of multichannel protein detection.     

Real-time PCR in a plastic chip based on solid state FRET

We show a novel solid state optical detection platform, integrated in a plastic biochip, based on colloidal nanocrystal FRET donors. The approach exploits a "smart" polymeric layer with both optical and biorecognition properties that allows real-time monitoring of biomolecular interactions and quantitative analyses of real-time PCR. The proposed strategy, demonstrated here for DNA detection, may open interesting perspectives for a wide range of applications, such as for proteomic studies.     

Multicolor single-molecule spectroscopy with alternating laser excitation for the investigation of interactions and dynamics

We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. Furthermore, the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously is demonstrated.     

Biofunctionalized gold nanoparticles for SPR-biosensor-based detection of CEA in blood plasma

We report on the use of new biofunctionalized gold nanoparticles (bio-AuNPs) that enable a surface plasmon resonance (SPR) biosensor to detect low levels of carcinoembryonic antigen (CEA) in human blood plasma. Bio-AuNPs consist of gold nanoparticles functionalized both with (1) streptavidin, to provide high affinity for the biotinylated secondary antibody used in the second step of the CEA sandwich assay, and with (2) bovine serum albumin, to minimize the nonspecific interaction of the bio-AuNPs with complex samples (blood plasma). We demonstrate that this approach makes it possible for the SPR biosensor to detect CEA in blood plasma at concentrations as low as 0.1?ng/mL, well below normal physiological levels (approximately nanograms per milliliter). Moreover, the limit of detection achieved using this approach is better by a factor of more than 1,000 than limits of detection reported so far for CEA in blood plasma using SPR biosensors.     

Surface plasmon resonance biosensor for the detection of VEGFR-1—a protein marker of myelodysplastic syndromes

The surface plasmon resonance (SPR) biosensor system with dispersionless microfluidics for the direct and label-free detection of a soluble vascular endothelial growth factor receptor (sVEGFR-1) is described. The detection approach takes advantage of an affinity interaction between sVEGFR-1 and its ligand, vascular endothelial growth factor (VEGF-A), which is covalently immobilized on the surface of the SPR sensor. The ability of the immobilized VEGF-A to specifically bind the sVEGFR-1 receptor is demonstrated in a buffer. The detection of sVEGFR-1 in 2% human blood plasma is carried out by using the sequential injection approach. The detection limit of 25 ng/mL is achieved. In addition, we demonstrate that the functional surface of the sensor can be regenerated for repeated use.     

Single-frequency impedance analysis of biofunctionalized dendrimer-encapsulated Pt nanoparticles-modified screen-printed electrode for biomolecular detection

We report the fabrication of polyamidoamine (PAMAM) dendrimer with 128 carboxyl group-encapsulated Pt nanoparticle-modified screen-printed carbon electrode, as an impedimetric biosensor, for the quantitative detection of human cardiac biomarker troponin-I (cTnI). PAMAM-Pt was electrochemically deposited over SPCE and its 128 terminal carboxyl groups were used as anchors for the site-specific biomolecular immobilization of protein antibody, anti-cTnI. The biosensor was characterized by contact angle measurements, transmission electron microscopy, UV-visible spectroscopy, and electrochemical techniques. A single-frequency impedance analysis study was utilized for the biomolecular sensing by monitoring the changes in the phase angle obtained at an optimized frequency resulting from antigen-antibody interactions. An optimized frequency of 100 Hz was obtained at which maximum changes in the phase angle were observed after immunoreactions for a given concentration of analyte. A concentration-dependent increase in the phase angle of the biosensor was observed with increasing cTnI concentration in the range of 1 pg mL^?1 to 100 ng mL^?1. Based on the concentration response data, the dissociation constant was found to be 0.51 pM reflecting high affinity of biosensor towards cTnI analyte arising due to high anti-cTnI loading with a better probe orientation on the 3-dimensional PAMAM-Pt structure.     

Biosensor chip mass spectrometry: a chip-based proteomics approach

Rapid advances in genomic sequencing, bioinformatics, and analytical instrumentation have created the field of proteomics, which at present is based largely on two-dimensional electrophoresis (2-DE) separation of complex protein mixtures and identification of individual proteins using mass spectrometry. These analyses provide a wealth of data, which upon further evaluation leads to many questions regarding the structure and function of the proteins. The challenge of answering these questions create a need for high-specificity approaches that may be used in the analysis of biomolecular recognition events and interacting partners, and thereby places great demands on general protein characterization instrumentation and the types of analyses they need to perform. Over the past five years we have been actively involved in interfacing two general, instrumental techniques, surface plasmon resonance-biomolecular interaction analysis (SPR-BIA) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, into a single concerted approach for use in the functional and structural characterization of proteins. Reviewed here is the recent progress made using biomolecular interaction analysis - mass spectrometry (BIA-MS) in the detailed characterization of proteins and protein-protein interactions and the development of biosensor chip mass spectrometry (BCMS) as a new chip-based proteomics approach.     

A 96-well microplate incorporating a replica molded microfluidic network integrated with photonic crystal biosensors for high throughput kinetic biomolecular interaction analysis

A nanoreplica molding process has been used to produce polymer microfluidic channels, with integrated label-free photonic crystal biosensors as the bottom surface of the channels. Multiple flow channels are gathered in parallel so that an imaging detection instrument may simultaneously monitor the binding kinetics of many biomolecular interactions. In this work, the flow channel pattern has been adapted to a 96-well microplate format in which, for each 12-element row of the microplate, a single well serves as a common access port for 11 flow channels that are connected to separate microplate wells. Application of pneumatic pressure or suction to the common well serves to drive forward or backward flow to the channels. The system is demonstrated by measuring the kinetic binding interaction of protein A with IgG molecules of high, medium, and low affinity. The approach offers a means for minimizing the volume of reagent required to functionalize the biosensor surface, while retaining compatibility with the microplate assay fluid-handling methods that are most commonly used in biological research.     

Biosensor Based on Electrocodeposition of Carbon Nanotubes/Polypyrrole/Laccase for Neurotransmitter Detection

A new biosensor has been developed by a simple electrocodeposition of multiwalled carbon nanotubes (MWCNT), polypyrrole (PPy) and laccase (Lac) on the platinum (Pt) electrode surface. The neurotransmitter biosensor was applied to the detection of dopamine in urine samples using differential pulse voltammetry (DPV). The Pt/MWCNT/PPy/Lac biosensor exhibited a detection limit of 0.14 µM, which is an adequate level for monitoring dopamine in urine. Reproducibility and repeatability values of 2.9 % and 1.7 %, respectively, were obtained compared to the conventional procedure. The proposed biosensor was successfully applied in the determination of dopamine in urine, and the obtained results were in full agreement with those from the HPLC procedure.     

Detection of bisphenol A using a novel surface plasmon resonance biosensor

We present a compact surface plasmon resonance (SPR) biosensor for the detection of bisphenol A (BpA), an endocrine-disrupting chemical. The biosensor is based on an SPR sensor platform (SPRCD) and the binding inhibition detection format. The detection of BpA in PBS and wastewater was performed at concentrations ranging from 0.05 to 1,000 ng/ml. The limit of detection for BpA in PBS and wastewater was estimated to be 0.08 and 0.14 ng/ml, respectively. It was also demonstrated that the biosensor can be regenerated for repeated use. Results achieved with the SPR biosensor are compared with those obtained using ELISA and HPLC methods.     

A novel low-cost and easy to develop functionalization platform. Case study: Aptamer-based detection of thrombin by surface plasmon resonance

A novel low-cost platform to assess biomolecular interactions was investigated using surface plasmon resonance and an aptamer-based assay for thrombin detection. Gold SPR surface functionalized with a carboxylated cross-linked BSA film (cBSA) and commercially available carboxymethylated dextran chip (CM5) were used as immobilization platforms for the thrombin binding aptamer. The high end commercial instrument Biacore 3000 and a custom made FIA set-up involving TI Spreeta sensor (TSPR2K23) were used to assess different concentrations of thrombin within the range 0.1-150 nM both in buffer and in a complex matrix (plasma) using the obtained aptasensors. Based on data derived from both CM5 and cBSA platforms, the cBSA aptasensor exhibited good selectivity, stability and regeneration ability, both in buffer and in complex matrices (plasma), comparable with CM5.     

Facile fabrication of branched-chain carbohydrate chips for studying carbohydrate-protein interactions by QCM biosensor

A novel approach for fabricating branched-chain (BC) carbohydrate chips to study carbohydrate-protein interactions using quantz crystal microbalance (QCM) biosensor was developed. This approach utilizes functional alkynyl-branch molecule modified chip surfaces, which is functionalized with terminal alkynyl group for covalent linking of unprotected azide-carbohydrates via click chemistry.     

Sports drug testing using immuno-MS: clinical study comprising administration of human chorionic gonadotropin to males

The applicability of a mass spectrometry (MS)-based method for determination of various forms of human chorionic gonadotropin (hCG) in doping analysis was demonstrated. A clinical study involving the hCG-containing pharmaceuticals Pregnyl and Ovitrelle was carried out, comprising a single injection of one pharmaceutical per participant to a total of 24 healthy male voluntaries. Hereafter, serum and urine samples were collected over a period of 14 days. The analysis of the samples using immuno-MS demonstrated elimination profiles of intact hCG for both pharmaceuticals, with last day of detection following administration at day 7 in serum, and at day 10 in urine, at limit of detections as defined by the World Anti-Doping Agency. Furthermore, the method allowed detection and differentiation of the various forms of hCG known to be present in serum and urine as a function of metabolism. For both pharmaceuticals, only the intact hCG was detected in serum, whereas in urine the injection of Pregnyl as hCG source (containing urinary hCG, i.e., most hCG variants) was shown to generate a more complex hCG variant pattern compared to Ovitrelle (contains only intact hCG). By detecting hCG using this MS-based approach in doping analysis, strong analytical evidence is provided minimizing the risk of false-positive and false-negative results.     

Development of an electrochemical flow injection immunoassay (FIIA) for the real-time monitoring of biospecific interactions

Not only are sensors a revolution in analysis; they themselves are also experiencing a revolution brought about by parallel developments in sensor fabrication techniques and materials, polymer chemistry, signal processing methodologies, the increased use of biomolecular processes as a means of analyte detection, and the coupling of sensors to other techniques such as flow injection analysis. Many of these developments have been incorporated into the present study, which we are undertaking in the development of our immunosensor technology. The system described here utilises screen-printed electrodes which are low-cost, disposable devices that are simple to fabricate. Incorporated into our sensor is the electroactive polymer, polyaniline, which brings about mediatorless redox coupling between the electrode and biomolecular components attached to the polymer surface. This system also utilises enzyme-labelled antibodies as the biomolecular recognition component for the analysis of the test analyte, biotin. The system has also been integrated into a flow injection system. This has led to the monitoring of real-time antibody-antigen interactions using electrochemical methods and foreshadows the development of single-step immunosensors.     

Present and future of surface plasmon resonance biosensors

Surface plasmon resonance (SPR) biosensors are optical sensors exploiting special electromagnetic waves—surface plasmon-polaritons—to probe interactions between an analyte in solution and a biomolecular recognition element immobilized on the SPR sensor surface. Major application areas include detection of biological analytes and analysis of biomolecular interactions where SPR biosensors provide benefits of label-free real-time analytical technology. This paper reviews fundamentals of SPR affinity biosensors and discusses recent advances in development and applications of SPR biosensors.     

Electrical biochip technology—a tool for microarrays and continuous monitoring

Based on electrical biochips made in Si-technology cost effective portable devices have been constructed for field applications and point of care diagnosis. These miniaturized amperometric biosensor devices enable the evaluation of biomolecular interactions by measuring the redox recycling of ELISA products, as well as the electrical monitoring of metabolites. The highly sensitive redox recycling is facilitated by interdigitated ultramicroelectrodes of high spatial resolution. The application of these electrical biochips as DNA microarrays for the molecular diagnosis of viral infections demonstrates the measurement procedure. Self-assembling of capture oligonucleotides via thiol-gold coupling has been used to construct the DNA interface on-chip. Another application for this electrical detection principle is continuous measuring with bead-based biosensors. Here, paramagnetic nanoparticles are used as carriers of the bioanalytical interface in ELISA format. A Si-micromachined glucose sensor for continuous monitoring in interstitial fluid ex vivo shows the flexibility of the electrical platform. Here the novel approach is a pore membrane in micrometer-dimensions acting as a diffusion barrier. The electrochemical detection takes place in a cavity containing glucose oxidase and a Pt-electrode surface. The common hydrogen peroxide detection, together with Si technology, enable precise differential measurements using a second cavity.     

Label-free cell-based assays using photonic crystal optical biosensors

Biosensor technologies that have been primarily used in the past for characterizing biomolecular interactions are now being used to develop new approaches for performing cell-based assays. Biosensors monitor cell attachment to a transducer surface, and thus provide information that is fundamentally different from that provided by microscopy, as the sensor is capable of monitoring temporal evolution of integrin-surface interactions that are difficult to measure by other means. Label-free biosensor technologies are especially advantageous for monitoring the behavior of cells because they do not require stains that typically result in cell death, and are not subject to effects such as photobleaching. As a result, cells can be quantitatively monitored in their culture environment over an extended period of time while processes such as proliferation, apoptosis, cytotoxicity, chemotaxis, ion channel activation, and membrane-bound protein activation are modulated by the introduction of a variety of chemical or biological stimuli. This review describes the application of photonic crystal optical biosensor microplates to a variety of cell-based assays. Detection instruments for photonic crystals measure the aggregate behavior of large cell populations, or, using recently developed biosensor imaging detection, independent monitoring of individual cells. These technological developments offer the ability to perform assays with a limited number of available cells for applications such as high throughput screening with primary cells or stem cells.     

Field effect of screened charges: electrical detection of peptides and proteins by a thin-film resistor.

For many biotechnological applications the label-free detection of biomolecular interactions is becoming of outstanding importance. In this Article we report the direct electrical detection of small peptides and proteins by their intrinsic charges using a biofunctionalized thin-film resistor. The label-free selective and quantitative detection of small peptides and proteins is achieved using hydrophobized silicon-on-insulator (SOI) substrates functionalized with lipid membranes that incorporate metal-chelating lipids. The response of the nanometer-thin conducting silicon film to electrolyte screening effects is taken into account to determine quantitatively the charges of peptides. It is even possible to detect peptides with a single charge and to distinguish single charge variations of the analytes even in physiological electrolyte solutions. As the device is based on standard semiconductor technologies, parallelization and miniaturization of the SOI-based biosensor is achievable by standard CMOS technologies and thus a promising basis for high-throughput screening or biotechnological applications.     

Electrochemical Biosensing Platform Using Carbon Nanotube Activated Glassy Carbon Electrode

Carbon nanotube enhanced electrochemically activated glassy carbon electrode (GCE) has been prepared and applied for sensitive electrochemical determination of DNA and DNA bases. The results indicate that the relative activation could efficiently enhance electron transfer at the pretreated GCE so that this carbon nanotube activated glassy carbon electrode could provide relatively low detection limit with good reproducibility for the respective biomolecular determination. Besides, greatly enhanced sensitivity could be obtained for the relevant electrochemical detection of the bio‐recognition process including DNA biosensing by using the carbon nanotube activated GCE. This approach provided a detection limit of 7.5 nM for guanine and 150 ng/mL for acid denatured DNA. These observations suggest that the carbon nanotube activated glassy carbon electrode could be utilized as a very sensitive and stable biosensor for some specific biological process.