Current and emerging commercial optical biosensors

JOURNAL OF MOLECULAR RECOGNITION J. Mol. Recognit. 2001; 14: 261–268 DOI:10.1002/jmr.544 Review Current and emerging commercial optical biosensors Cheryl L. Baird and David G. Myszka* Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132, USA The field of commercial optical biosensors is rapidly evolving, with new systems and detection methods being developed each year. This review outlines the currently available biosensor hardware and highlights unique features of each platform. Affinity-based biosensor technology, with its high sensitivity, wide versatility and high throughput, is playing a significant role in basic research, pharmaceutical development, and the food and environmental sciences. Likewise, the increasing popularity of biosensors is prompting manufacturers to develop new instrumentation for dedicated applications. We provide a preview of some of the emerging commercial systems that are dedicated to drug discovery, proteomics, clinical diagnostics and routine biomolecular interaction analysis. Copyright # 2001 John Wiley & Sons, Ltd. Keywords: SPR; evanescent wave; refractometry; protein array; drug discovery Received 4 July 2001; accepted 26 July 2001 INTRODUCTION Affinity-based biosensors Affinity-based biosensors have sparked a revolution in the science of biomolecular interaction analysis. These instruments employ biological molecules, such as antibodies, receptors, enzymes or nucleic acids, as signal transducers at the interface between solid-state electronics and solutionphase biology. The inherent recognition properties of these biomolecules impart biosensors with high sensitivity and selectivity. As shown in Fig. 1(a), for a typical biosensor assay, the transducer (usually referred to as the ligand, B) is immobilized onto a surface and used to detect a reactant in solution, (referred to as the analyte, A). A detector converts the results of the biological recognition event into an electronic signal, thereby quantifying the amount of analyte/ ligand complex formed at the surface. A key advantage of biosensors is that data may be collected in real time, providing detailed information about a binding reaction. Figure 1(b) depicts the association, equilibrium and dissociation phases recorded for a representative biosensor experiment. Biosensor assays may be designed in a variety of qualitative and quantitative formats, from detecting analyte in crude samples (Link et al., 1999) to obtaining thermodynamic and kinetic rate constants for a molecular interaction (Myszka, 1999a). Recent surveys of the optical biosensor literature demonstrate the wide range of biosensor applications (Myszka, 1999b; Rich and Myszka, 2000a). Biosensor hardware The three main components of a biosensor are the detector, sensor surface and sample delivery system. All of these components vary depending on manufacturer and the intended application. This review focuses on instruments that employ optical detection systems that utilize surface plasmon resonance (SPR) or related spectroscopies (evanescent waveguide or refractometry) to monitor the change in refractive index of the buffer near the sensor surface as complexes form and break down in real time (Squillante, 1998; Huber et al., 1999). These detection methods are label-free, non-invasive, and can measure the amount of complex formed, even in crude preparations. The sensor surface provides the platform on which the ligand is immobilized. Most manufacturers supply instrument-ready surfaces and applicable reagent chemistries for ligand immobilization. Where appropriate, we highlight the availability and utility of these different surfaces. The sample delivery system ensures that the analyte contacts the sensor surface in a rapid and uniform manner. We describe the common sample delivery systems and outline the differences between them (for a detailed review of the two main sample delivery designs consult Ward and Winzor, 2000). *Correspondence to: D. G. Myszka, Center for Biomolecular Interaction Analysis, University of Utah, 50 N. Medical Drive, Room 4A417, Salt Lake City, UT 84132, USA. Email: david.myszka@cores.utah.edu. CURRENT COMMERCIAL SYSTEMS Abbreviations used: LPG, long-period gratings; PWR, plasmon waveguide resonance; SAM, self-assembled mono-layer; SPR, surface plasmon resonance. Six manufacturers currently produce biosensor platforms Copyright # 2001 John Wiley & Sons, Ltd. 262 C. L. BAIRD AND D. G. MYSZKA ranging in price and performance, making it possible for potential users to choose the system most appropriate for a given application. The following section describes each manufacturer’s currently available platform(s). Specifics regarding purchase price and availability can be obtained by contacting the individual manufacturer directly or by visiting their web sites, listed in Table 1. BIACORE1 instruments Figure 1. Biosensor overview. (a) Schematic diagram of a typical optical biosensor assay. The analyte (A) in solution ¯ows over the ligand (B) immobilized on the sensor surface. Formation of AB complex is monitored by a optical detector located on the opposite side of the sensor surface. (b) Characteristic response recorded from a biosensor experiment. An initial baseline signal is recorded as buffer ¯ows over the immobilized ligand (1). During the association phase analyte is exposed to the ligand surface. An increase in response is generated as analyte/ligand complexes are formed (2). The response plateaus as dynamic equilibrium between complex formation and dissociation occurs (3). During the dissociation phase the surface is washed with buffer and stability of the ligand±analyte interaction can be determined from the decrease in signal observed as the complexes break down over time (4). Remaining complexes are disrupted by washing with a mild chemical disruptant (traditionally mild acid or base), thereby returning the signal to a baseline response and regenerating the ligand surface for the next binding cycle (5). Biacore AB (Uppsala, Sweden) released the first commercial biosensor for biomolecular interaction analysis in 1990 (Jonsson et al., 1991). Since that time, this company has developed a fleet of SPR-based instruments that are designed for diverse user applications. High-quality instrument platforms and supporting material keeps Biacore AB at the forefront of the commercial biosensor market, as 90% of published optical biosensor work is performed using BIACORE technology (Myszka, 1999b; Rich & Myszka, 2000a). These instruments are used to resolve a wide range of affinity and kinetic rate constants (KD = 1 mM to 10 pM, ka = 103 –107 M 1 s 1, kd = 10 6 –10 1 s 1) (Myszka et al., 1998). Currently available platforms include BIACORE 1000, 2000, 3000, Quant, X and J. BIACORE 1000, 2000 and 3000 [Plate 1(a)] are fully automated SPR instruments that can accept samples directly from two 96-well plates. When docked into one of these instruments, a sensor chip is divided into four independent flow cells that can be addressed individually or in series (flow cell volumes range from 60 to 20 nl, depending on the instrument model). This flow cell configuration allows buffer to pass continuously over the sensor chip surface, thereby alleviating the need for time-consuming washing steps when exchanging analyte solution for buffer. In addition, continuous flow systems ensure that the ligand is exposed to a constant analyte concentration for the duration of the binding measurement. Furthermore, the availability Table 1. Optical biosensor manufacturers, instruments and website information Manufacturer Biacore AB Affinity Sensors IBIS Technologies Nippon Laser Electronics Texas Instruments Analytical m-Systems AVIV Instruments Farfield Sensors Ltd Luna Innovations ThreeFold Sensors Graffinity Leica Prolinx HTS Biosystems Quantech Ltd SRU biosystems a Instrument BIACORE 1000, 2000, 3000, X, J, Quant, 8 channel prototype,a S51a IAsys, IAsys Plus, IAsys Auto‡ IBIS I, IBIS II SPR670, SPR Cellia Spreeta BIO-SUPLAR 2 PWR Model 400a AnaLight Bio250a Fiber optic prototypea Label-free prototypea Plasmon Imagera Prototypea OCTAVEa SPR arraya FasTraQ SPR arraya BINDa Website www.biacore.com www.affinity-sensors.com www.ibis-spr.nl www.nle-lab.co.jp/English/ZO-HOME.htm www.ti.com/sc/docs/products/msp/control/spreeta www.micro-systems.de www.avivinst.com www.farfield-sensors.co.uk www.lunainnovations.com http://ic.net/tfs www.graffinity.com www.leica-ead.com www.prolinxinc.com www.htsbiosystems.com www.quantechltd.com www.srubiosystems.com Emerging platforms. Copyright # 2001 John Wiley & Sons, Ltd. J. Mol. Recognit. 2001; 14: 261–268 COMMERCIAL OPTICAL BIOSENSORS Plate 1. Commercially available optical biosensor systems. For perspective, the instruments are scaled roughly to size. (a) BIACORE 3000 from Biacore AB (BIACORE 1000, 2000 and Quant have similar footprints and pro®les). For reference, the BIACORE 3000 occupies approximately 0.3  0.6  0.8 m (depth  height  width). (b) BIACORE X. (c) BIACORE J. (d) IAsys Manual from Af®nity Sensors (IAsys Auto and IAsys Auto‡ have similar footprints and pro®les). (e) IBIS II from IBIS Technologies (IBIS I has a similar footprint and pro®le). (f) SPR670 from Nippon Lasers Electronics (Cellia has a similar footprint and pro®le). (g) BIO-Suplar II from Analytical m-Systems. (h) Spreeta from Texas Instruments. Plate 2. Emerging biosensor technologies. (a) Optical layout of the Aviv PWR Model 400 biosensor. Output at 543.5 (green) and 632.8 nm (red) from the He/Ne lasers are merged at the dichroic mirror. The polarizer, mounted on a rotation stage, permits the alternating production of planepolarized light parallel and perpendicular to the sensor surface. The beam splitter directs light into the reference detector and to the mirror fastened on a linear slide, which allows for manual centering of the light onto the sample block. The sample block, consisting of the prism, sensor surface and detector, is mounted on a rotary table. Rotating this table permits the user to scan the range of excitation angles. (b) The Far®eld vertical planar waveguide sensor structure. Light passes through a sensing waveguide, which contains the immobilized ligand on its surface, and a buried reference waveguide. The light recombines as it exits the waveguides and produces an interference pattern in the far®eld. Binding of analyte to the ligand modi®es the sensor waveguide's optical properties, thereby altering the passage of light through the sensor waveguide relative to that of the reference waveguide. (c) Schematic diagram of the Leica biosensor. The system is similar to a traditional HPLC. Its modular components typically include a solvent-select valve, pump, injector, and SPR detector. (d) Schematic of Luna's LPG-based optical ®ber biosensor. Molecular interactions with an immobilized ligand on the sensor surface cause a change in the refractive index detected by the LPG optical ®ber and alter the passage of light through it. Copyright # 2001 John Wiley & Sons, Ltd. J. Mol. Recognit. 2001; 14 C. L. BAIRD AND D. G. MYSZKA Plate 3. Biacore S51 platform from Biacore AB. (a) Biacore S51 instrument (approximate size 60  60  60 cm). (b) Schematic of S51 hydrodynamic addressing the ¯ow cell. By controlling the ¯ow rate from two separate inlets (in 1 and in 2), reagents may be exposed to one, two or all three detection spots within the same ¯ow cell. (c) Binding data collected for the drug melagatran (429.5 Da) injected over two different density surfaces of thrombin. The red line represents the global ®t of the data from both surfaces to a bimolecular interaction model. Data supplied by Johanna Deinum (AstraZeneca). Plate 4. Image of a 315-spot protein array from Biacore AB. In this setup, one analyte solution addresses all the ligands and interactions are simultaneously detected at speci®c ligand spots within a single ¯ow cell. The colored sensorgrams (labeled i±iv) demonstrate the possibility of collecting high-resolution information from the individual spots simultaneously and independently. Copyright # 2001 John Wiley & Sons, Ltd. J. Mol. Recognit. 2001; 14 COMMERCIAL OPTICAL BIOSENSORS of four flow cells permits the user to immobilize three different ligands and maintain a reference surface within the same sensor chip. BIACORE 2000 and 3000 models are capable of monitoring binding interactions within all four flow cells simultaneously. The delivery of samples to each surface in series allows in-line reference subtraction and improves data quality (Myszka, 1999a; Rich and Myszka, 2000b). The detector used in the BIACORE 3000 instrument has a wider dynamic range than that of the 1000 and 2000 instruments. This increased detector range allows for the immobilization of higher densities of ligands and enables binding reactions to be studied in buffers with higher refractive indexes. BIACORE 3000 also provides a microrecovery feature that allows bound material to be eluted off the chip surface in small volumes (3–5 ml), which are convenient for subsequent analysis by mass spectrometry (Nedelkov and Nelson, 2000; Nelson et al., 2000). Additional advances in the BIACORE 3000 auto-sampler reduce sample delivery times. BIACORE 2000 and 3000 are capable of collecting binding data from 4 to 40 °C. The integrated instrument software contains helpful ‘Wizards’ for automating common tasks. In addition, customized scripts can be written to execute more complex experiments. BIACORE Quant is a fully automated biosensor instrument dedicated to the determination of vitamin concentrations in fortified food products. Integrated software automatically calculates results and generates final reports. In addition, a variety of Qflex kits are available from Biacore AB for vitamin quantitation (for details see www.biacore.com/food). BIACORE X [Plate 1(b)] is designed to support basic research in a multi-user laboratory. Samples are loaded into the injection port by manual pipetting, but they are automatically injected into the instrument, providing precise control of the association and dissociation phases. Two flow cells may be addressed independently or in series, permitting in-line referencing. BIACORE X offers a dynamic range similar to that of BIACORE 3000 and manual sample recovery from the sensor surface (Nedelkov and Nelson, 2000; Nelson et al., 2000). BIACORE J [Plate 1(c)] is a manually operated SPR instrument designed to be accessible as a general laboratory tool. Two flow cells allow in-line referencing and buffer is delivered continuously over the surfaces using a patented pulsation-free peristaltic pump. The transition between buffer and sample is slow relative to the injection systems described above. This slow switching limits the ability of the J to resolve very rapid kinetics. However, the sensitivity and reliability of the system makes it capable of determining the amount of active biomolecules in solution, as well as affinity constants (Rich and Myszka, 2001a,b). Biacore AB currently provides 10 different sensor surfaces, some of which are tailored for specific applications (Rich and Myszka, 2000a,b). The most commonly used surface is coated with a carboxymethyl dextran matrix. This matrix provides a convenient handle for applying a variety of surface immobilization chemistries. The matrix also helps maintain the activity of immobilized ligands and reduces non-specific binding to the sensor surface itself. Modified versions of the dextran matrix are also available, including one with a shorter matrix, one with less charge, Copyright # 2001 John Wiley & Sons, Ltd. 263 and a version precoated with either streptavidin for biotin capture or a nickel-chelation ligand for His-tagged protein capture. Biosensor surfaces are also available for immobilizing liposomes as bilayers or hybrid monolayers. Both of these lipophilic surfaces can be used to mimic biological membranes and simplify investigations involving membrane-associated systems (Cooper & Williams, 1999a,b). Flat surfaces composed of carboxyl groups or plain gold surfaces are also available. These two surfaces can be modified using a variety of coating methods, such as spincoating with polymeric films from dilute solutions or coating with organic self-assembled mono-layers (SAMs). Alternatively, polymers could be adsorbed to the plain gold surface via various functional groups. IAsys1 instruments Affinity Sensors (Franklin, MA) markets the IAsys line of optical biosensor instruments, which are based on resonant mirror technology. Approximately 10% of the 1998–1999 optical biosensor peer-reviewed publications reported using IAsys instruments (Myszka, 1999b; Rich & Myszka, 2000a). These instruments use evanescent waves to monitor the refractive index of the buffer near the sensor surface (Cush et al., 1993). Current instruments employ a stirred micro-cuvette (50–150 ml) to deliver analyte to the sensor surface. The open cuvette format permits the analysis of particulate suspensions (e.g. membranes, viruses and whole cells) and is designed to minimize potential sample aggregation, surface fouling and blockage that may occur in flow cell-based systems. In addition, it has recently been reported that the IAsys biosensor can be adapted to accommodate a flow cell configuration (Despeyroux et al., 2000). The IAsys Manual System [Plate 1(d)] is an entry-level single-well optical biosensor. The user controls sample delivery times by pipetting and aspirating material into and out of the cuvette. The IAsys Plus is a dual-well cuvette system that allows for in-line referencing to correct for the effects of bulk refractive index changes, non-specific binding, thermal drift and evaporation. The IAsys Plus can be upgraded to an automated system with the Auto‡ Advantage add-on option. The IAsys Auto‡ Advantage System is fully automated and designed for the medium- to high-throughput laboratory. The Auto‡ Advantage integrates the resonant mirror detection system utilized in the IAsys Plus with a fully functioning robotic system, Peltier thermostatted dual sample stage, and robot-accessible buffer and reagent racks. System operation can be manual using Point and Transfer (PAT1) software or automated using Adaptive Sequence Kontrol (ASK1) software. In the ASK software the user programs a variety of analytical steps, thereby increasing instrument throughput and productivity. IAsys cuvettes are available with a choice of derivatized sensor surfaces. Currently available cuvette surfaces include a carboxymethyl dextran matrix (not available in the USA), or planar surfaces containing amino, carboxylate, biotin, or hydrophobic functional groups. J. Mol. Recognit. 2001; 14: 261–268 264 C. L. BAIRD AND D. G. MYSZKA IBIS1 instruments Bio-Suplar II1 The IBIS line of instruments, distributed by IBIS Technologies BV (The Netherlands), is based on a scanning mirror surface plasmon resonance detector. The IBIS I is a singlechannel SPR instrument equipped with an auto-sampler. The IBIS II [Plate 1(e)] is a double-beam SPR instrument that provides fully synchronized, real-time analysis with reference channel subtraction. Both IBIS instruments can be configured as a cuvette- or flow cell-based system. In the cuvette mode, the inlet of the cuvette is connected to the auto-sampler for sample injection and the outlet is connected to the drain pump for sample removal. Sample in the cuvette is stirred using an automatic aspirate/dispense system that is driven by a syringe pump. The volume of the cuvette ranges from 10 to 150 ml, although 50 ml is recommended. This cuvette set-up uses minimal sample volumes and offers flexibility in sample analysis, for measurement times may range from seconds to hours. In the flow cell mode, the sample and/or reference solutions are also delivered through syringe pumps and the flow cell outlets can be configured to recycle the sample or send it to waste. Flow rates can be as slow as 0.2 ml/s. Besides SPR measurements in aqueous solutions (Wink et al., 1999; de Mol et al., 2000), the dynamic range of IBIS instruments can accommodate measurements in some organic solvents. For example, the self-assembly of thiols onto gold substrates from ethanol can be studied in real time. IBIS manufactures a broad selection of interchangeable sensor surfaces (SensorDisks1) for different applications. Sensor Disks are available with a plain gold surface or derivatized with planar functional groups (amine or carboxylic acid). Higher-capacity surfaces consisting of a flexible, water-swelling polymer layer functionalized with carboxylic acid groups are available. IBIS Technologies also offers a spin-coater, which enables a basic IBIS-Gold SensorDisk to be coated with a reproducible, thin layer of user-defined polymer. Analytical m-Systems (Regensburg, Germany) markets the Bio-Suplar II [Plate 1(g)], a computer-controlled SPR biosensor intended for research studies in the fields of surface chemistry and biomolecular interactions. The BioSuplar II is unique in that it uses a prism installed on a swivel carriage to focus light onto the sensor chip surface. Rotation of the carriage can be performed manually or automatically by the computer-controlled rotating mechanism. The device can operate with either a retro-reflector or symmetric transmission prism. Analytical m-Systems offers a range of chemicals for the preparation of chemical and biological sensor surfaces. The self-assembling coatings are based on stabilized long-chain alkanethiols and are strongly adhesive to many metallic surfaces such as gold, silver and platinum. Stable monolayer coatings with carboxyl groups or avidin on the surface are also available. The manufacturer will also immobilize custom-specified molecules such as antibodies, antigens, and oligonucleotides on the sensor surfaces. SPR6701 and Cellia1 instruments Nippon Laser Electronics (Nagoya, Japan) manufactures SPR-based optical biosensors with a variety of available options (Toyama et al., 2000). The series SPR670 [Fig. 2(f)] and Cellia instruments can be configured with dual-channel simultaneous detection of two flow cells or in a cuvette mode. The Cellia line of instruments supports flow cell units with different dimensions for protein and cell binding studies (Senzaki et al., 1999; Nishimura et al., 2000). Models with auto-samplers that accept 96-well plates are available. Both instruments contain a buffer-degassing mechanism and are robust enough to tolerate some organic solvents. Binding reactions can also be monitored across a wide temperature range (4–95 °C). Nippon Laser Electronics distributes plain gold surfaces, which can be modified with diverse chemistries (e.g. carboxyl, thiol, hydrophobic, biotin, nickel chelators or oriented Langmuir-Blodgett films) for specific applications. Stripping the sensor chips with the UV/ozone cleaning system provided by this manufacturer allows for the repeated use of the chips. Copyright # 2001 John Wiley & Sons, Ltd. Spreeta2 Texas Instruments (Dallas, TX) has developed Spreeta, a handheld, battery-operated SPR detector (Woodbury et al., 1998). The detector itself is smaller than a matchbox [Plate 1(h)] and contains a fully integrated light source, sensor surface and detector on a microchip. Currently, the Spreeta sensor surface is only available with a plain gold coating. Spreeta is primarily marketed for applications in diagnostics, manufacturing, and food/beverage quality and safety (Elkind et al., 1999). However, the detector must be configured around a user-defined instrument. APT Instruments (Litchfield, IL; www.aptinstruments.com) and Nomadics (Stillwater, OK; www.nomadics.com) each offer an evaluation module that integrates the Spreeta sensor with a flow cell and computer to produce a low-cost SPR biosensor for laboratory researchers. APT instruments has also integrated the Spreeta sensor into a portable hand-held refractometer, the HR300, which can be used for research and quality control applications. EMERGING BIOSENSOR SYSTEMS At the time of this writing, several instrument manufacturers provided information on systems that were currently under development. In the following section, we highlight key features of these emerging systems. Aviv1 PWR model 400 Aviv Instruments (Lakewood, NJ) is developing a new biosensor based on plasmon waveguide resonance (PWR; Salamon et al., 1997). PWR (also known as coupled plasmon waveguide resonance, CPWR) is similar to SPR in that both rely on the generation of surface plasmons. Unlike SPR, however, PWR employs two He/Ne lasers to probe the sensor surface and a polarizer to produce plane-polarized J. Mol. Recognit. 2001; 14: 261–268 COMMERCIAL OPTICAL BIOSENSORS light-oriented either parallel or perpendicular to the sensor surface [Plate 2(a)]. These features of the Aviv instrument offer unique opportunities to examine biomolecules in isolation or in concert with their binding partner(s). For example, the anisotropic examination of the surface yields information about ligand conformational reorganization upon changes in the environment (e.g. pH, salinity or crowding) and analyte binding. Anisotropic examination of the surface is particularly applicable in the study of planar lipid bilayers. In addition to measuring the thickness and ordering of pure lipid membranes, conformational changes in membrane-associated proteins can be followed with the Aviv technology. For example, PWR was used to demonstrate that agonists and antagonists induce different conformational states in dopioid receptor assembled on the sensor surface (Salamon et al., 2000). This capability could benefit drug discovery efforts centered on seven-transmembrane receptors. 1 Analight 265 Luna1 innovations Luna Analytics Inc. (Blacksburg, VA) is developing a fiber optic biosensor system based on long-period gratings (LPGs). The LPG is a spectral loss element that scatters light out of an optical fiber at a particular wavelength. In the Luna instrument, light scattering is dependent on grating period, fiber refractive index, and the refractive index of the surrounding environment. LPG-based biological/chemical sensors operate with specially designed affinity coatings or swellable polymers that cause selective, quantitative changes in the refractive index detected by the LPG in the presence of target molecules [Plate 2(d)]. Unlike prismbased systems, these optical fiber components do not require critical alignment with the detector and are therefore suitable for robust operations. Also, the fiber cable can be easily multiplexed and automated, making it a promising technology for high-throughput applications. ThreeFold Sensors1 Bio250 Farfield Sensors Ltd (Manchester, UK) has developed a new biosensor to detect molecular interactions, as well as the conformational changes associated with a molecular binding event (Cross et al., 1999). The sensor is based on an interferometer that uses planar waveguides instead of the traditional slits. The sensor consists of an exposed ‘sensing’ waveguide and a buried ‘reference’ waveguide [Plate 2(b)]. The sensing waveguide is engineered so that the optical properties of the surface change upon analyte binding. These changes alter the passage of light through the sensing waveguide relative to the reference waveguide. This difference is recorded as an interference pattern in the farfield by a photodiode array. Because the waveguides are optically coupled, simultaneous examination of multiple wavelengths or polarizations can be performed. Farfield Sensors offers a variety of prepared biosensor chip surfaces that enable detection of molecular interactions occurring at different distances from the sensor surface. A basic glass surface for custom functionalization is also available. Leica1 biosensor Leica Microsystems Inc. (Wetzlar, Germany) is developing an SPR-based affinity biosensor. This instrument is an extension of Leica’s core technology of reflected light critical angle refractometers. At the current stage of development, the instrument is a single-channel system configured like a traditional HPLC with modular components and HPLCcompatible fluidics [Plate 2(c)]. The fluidics system can be custom configured to allow for special applications, such as recirculation of ligand during immobilization or analyte during the binding study. The sensor surface area is relatively large and should allow interfacing with a mass spectrometer. Plain gold sensor slides are currently available and a number of different sensor surfaces based on SAMs are being developed. The manufacturer plans to begin development of multi-channel instruments in the future. Copyright # 2001 John Wiley & Sons, Ltd. ThreeFold Sensors (Ann Arbor, MI), which currently markets a hand-held fiber optic evanescent biosensor for real-time monitoring of interactions between fluorophorelabeled molecules in solution and a binding partner immobilized on a sensor fiber (Smith et al., 1900), is preparing to release a label-free instrument in late 2001. This new instrument utilizes a nitrogen laser to provide evanescent excitation of a fluorophore present on the sensor fiber. Changes in fluorescence are detected when an unlabeled analyte in solution binds to an unlabeled ligand immobilized adjacent to the fluorophore. Biacore1 S51 Biacore AB has developed an entirely new biosensor platform, Biacore S51 [Plate 3(a)]. This system is designed for increased sensitivity, higher throughput, greater automation, and enhanced ease of use over previously released BIACORE instruments. Improved referencing in Biacore S51 is achieved through proprietary hydrodynamic addressing, which allows one reference and two ligand surfaces to be created within a single flow cell [Plate 3(b)]. A Y inlet uses a buffer stream to limit sensor surface coupling chemistry to the two side regions of the flow cell, with the middle of the sensor surface isolated from coupling conditions by the buffer flow rate. Three detection spots are simultaneously addressed in each of two flow cells, thereby eliminating lag time between reference and sample and improving resolution of fast kinetics. Simultaneous sample delivery also ensures constant analyte concentrations across the surface and reduces systematic noise due to temperature and pumping artifacts. The Biacore S51 detection unit has been designed with state-of-the-art optics, light source and detectors. Automated and integrated loading of sensor chips within the optical detection area permits a closed detection environment for improved thermal stability. Higher throughput is made possible by incorporating 384-well microtiter plate compatibility and fast sample loading via a new injection J. Mol. Recognit. 2001; 14: 261–268 266 C. L. BAIRD AND D. G. MYSZKA technique in which the injection needle is fixed directly to the flow cell and the sample racks move to provide for sample addressing. The result is decreased sample carryover, faster sample loading and the potential for future parallel sample processing possibilities. Plate 3(c) shows an example of binding data collected from the S51 for different concentrations of the drug melagatran (429.5 Da) injected over thrombin immobilized at two different densities within the same flow cell. These data demonstrate the potential to monitor the binding kinetics for low-molecular-weight analytes, as well as the ability to collect reliable response data from Biacore S51 below 1 RU in magnitude (1 RU corresponds to 1 pg/mm2 of protein). Biacore S51 has been designed with a focus on drug discovery applications. To increase throughput, additional automation is incorporated in the new platform. Largevolume reagent flasks are used for dedicated washing functions to handle poorly soluble compounds. Continuous buffer degassing improves sensitivity and enables extended sample runs. New instrument and analysis software simplifies instrument operation and speeds up data evaluation. Software is provided for predefined assay formats for screening, lead characterization, and in vitro ADME (absorption, distribution, metabolism and excretion) studies. Because of these novel features, Biacore S51 represents a significant advance in the application of SPR biosensors in the pharmaceutical and biotechnology industries. EMERGING ARRAYS Eight-channel parallel array With the continued success of biosensors has come the demand for instrumentation with higher throughput. One approach is to deliver separate samples in parallel to an array of sensor surfaces. While simple in concept, implementing such a system requires a substantial amount of technological development, which can only be driven by a clear commercial need. Recently, a consortium within the European community established ‘FoodSense’, a project to demonstrate the applicability of optical biosensors for routine monitoring of chemical residues in foods (for detailed information on the project see www.slv.se/foodsense2). The group has tested a prototype parallel-array instrument assembled by Biacore AB. This platform consisted of a compact SPR optical detection unit, a sensor chip containing Eight parallel channels (each with its own injection needle) and an integrated commercial robot for liquid handling and sample preparation. The instrument had an operation throughput of approximately 200 samples per hour. Prototypes of the eight-channel array instrument have been evaluated at various sites under realistic conditions such as a pig abattoir in Northern Ireland and a dairy plant in Germany. A number of prototype kits have also been developed and evaluated, including kits for the detection of veterinary drug residues in pig bile, bovine urine and bovine milk. Applications of the project extend to the analysis of vitamins, pesticides, mycotoxins and pathogens in food products. Validating the utility of biosensors for routine Copyright # 2001 John Wiley & Sons, Ltd. food analysis along with establishing the applications and protocols will help to drive the development of these higher throughput parallel array systems. Spreeta 2k1 Prolinx1 Inc. (Bothell, Washington), in collaboration with Texas Instruments Corporation (TI), is developing “OCTAVE” an optical biosensor incorporating Prolinx’s proprietary surface chemistry technology, Versalinx, with a new version of the TI Spreeta Liquid Sensor, Spreeta 2k. The new sensor is 4.5 times smaller than the current Spreeta system and contains on-board memory for storage of identification, calibration values and use history. The system will exploit an agitated well design that is robot friendly. Parallel analyses on eight sensors will be possible with the system, enabling a user to simultaneously acquire the data needed for a kinetic analysis under identical conditions of agitation, temperature and timing. The miniaturized sensor is small enough to array sensor elements on the 9 mm centers corresponding to a standard 96-well plate. EMERGING TWO-DIMENSIONAL ARRAYS Now that the complete sequences of several genomes are known there is a need to understand the functions of the encoded proteins. Protein profiling and interaction analysis will require large-scale experimentation necessitating very high-throughput, yet sensitive, methods of detection. To address this need, biosensor detectors and two-dimensional array surfaces are being developed that can support the analysis of hundreds to thousands of proteins at one time. BIACORE 2D SPR array Scientists at Biacore AB have recently demonstrated the possibility of immobilizing ligands at multiple spots within a single flow cell. Plate 4 illustrates an example of a prototypical flow cell containing 315 individual spots of immobilized protein. SPR detectors can be aligned to collect high-resolution binding information from each spot simultaneously and independently. Biacore AB is currently collaborating with Millennium Pharmaceuticals (Cambridge, MA) to develop the reagents and hardware required to support these SPR arrays in proteomics applications focused on drug discovery. Graffinity1 SPR microarray Graffinity (Heidelberg, Germany) is developing the Plasmon Imager1 for parallel, label-free binding detection. In contrast to most other SPR biosensors, the Plasmon Imager employs a wavelength-dependent measurement of the SPR effect to detect binding of biomolecules to their respective ligands. Inside the instrument a two-dimensional sensor J. Mol. Recognit. 2001; 14: 261–268 COMMERCIAL OPTICAL BIOSENSORS array is imaged onto a spatially resolving detector. The wavelength of the light illuminating the sensor array is scanned and the resonance spectrum of each sensor field on the array is recorded. A shift in the resonance spectrum is observed at the position of those sensor fields that contain a ligand that binds the analyte in solution. The magnitude of the wavelength shift reflects the binding affinities of the immobilized ligands. Sensor chip surfaces for this instrument are designed to be inexpensive and disposable. The Plasmon Imager has been used successfully by Graffinity to produce a characteristic binding fingerprint for a protease against a set of tethered organic ligands and a negative control. Parallel measurements of this kind can be done with 1536-9216 compounds on the sensor. A protein can therefore be screened for interactions with a library of immobilized compounds using only a few milliliters of protein solution. The threshold of detection for a given ligand requires a protein surface coverage of about 200 pg/mm2. HTS1 SPR array Quantech (Eagan, Minnesota), which is developing SPRbased biosensors for medical diagnostics, has teamed with PE Biosystems to establish HTS, a company focused on developing label-free optical detection systems for the scientific market. Both Quantech and HTS utilize gratingcoupled SPR (GCSPR) in their biosensors instead of prismbased SPR technology. The GCSPR technology is inexpensive yet sensitive and can be produced in various formats depending on sample requirements. GCSPR utilizes a plastic molded transducer, which is easily reconfigured to integrate additional features such as channels or reaction chambers without additional costs. HTS’s first product line, the FLEX CHIP1 Kinetic Analysis System, will be based upon the company’s proprietary SPR technology. HTS is advancing GCSPR to produce a high-density two-dimensional SPR Array Detector (SPRAD). The SPRAD array format is designed to screen one sample against more than 5000 unique elements on 100 mm square chemistry pixels and has the potential to expand to 90 000 100 mm pixels. Such array formats are inexpensive and easy to manufacture, making the FLEX CHIP highly amenable to high-throughput screening where disposable, single-use surfaces are desired. The first FLEX CHIP product will have a linear array format and is expected to be available for sale to the pharmaceutical and genomics research markets by mid2001. A two-dimensional high-density format FLEX CHIP System will be available by the end of 2001. BIND1 biosensor SRU Biosystems (Woburn, MA) is developing a novel biosensor based on their BIND (Biomolecular INteraction Detector) technology. Similar to the Graffinity and HTS systems, BIND technology makes use of a special-purpose optical grating, a subwavelength structured surface, to Copyright # 2001 John Wiley & Sons, Ltd. 267 create a very sharp optical resonant reflection at a particular wavelength that can be accurately traced as biological material is attached to the surface. This surface can be inexpensively mass-produced and is easily adapted for various assay formats. The readout system consists of a white light lamp that illuminates a small spot of the grating at normal incidence through a fiber optic probe and a spectrometer that collects the reflected light through a second fiber. This detection technique is capable of resolving changes of 1 Å thickness of protein binding (corresponding to a fractional monolayer of a small molecule), and can be performed with the grating surface either immersed in fluid or dry. A single reading can be performed in several milliseconds, making it possible to measure a large number of reactions taking place in parallel on the surface and to monitor reaction kinetics in real time. Currently, a laboratory prototype instrument can scan a 96-well plate in 30 s. Initially, SRU Biosystems is focusing on developing an instrument for high-throughput screening and pharmaceutical research, proteomics and infectious disease diagnosis. However, the technology can also be generally applied to a variety of biochemical assays, including DNA microarrays, protein microarrays, and even as a replacement for the ELISA assay. A commercially available instrument is expected in June 2002. CONCLUSION It is exciting to witness the rapid evolution of affinity-based biosensor technology. In 10 years we have gone from being able to study molecular interactions in real time without labeling, to the verge of having the ability to analyze thousands of interactions at once. However, there will be substantial hurdles to overcome. Unlike nucleic acid arrays, protein arrays cannot be generated by direct synthesis on the chip surface. We will therefore need to produce and purify the ligands that are critical for creating the biological recognition surface we wish to employ. In many instances, we will need to overcome the limitations of the amount of material to be detected. For instance protein expression profiling that relies on biological detection such as antibodies is inherently limited by the affinity of the surface ligand, as well as by the concentration of analyte in solution. Finally, data interpretation will present new challenges. As we have witnessed in the recent past for nucleic acid arrays, processing and interpreting large quantities of data will pose an additional challenge, and, unlike expression profiling, with protein arrays we may be interested in resolving the kinetic properties of each interaction, as well as determining absolute concentrations of reactants. It is interesting to note how the development of commercial biosensors has expanded the science of molecular interaction analysis. The availability of commercial instruments and the accompanying accessories and reagents (e.g. sensor surfaces and coupling chemistries) has brought optical biosensors from the realm of the specialist to the applied world of the general laboratory scientist. J. Mol. Recognit. 2001; 14: 261–268 268 C. L. BAIRD AND D. G. MYSZKA REFERENCES Cooper MA, Williams DH. 1999a. Binding of glycopeptide antibiotics to a model of a vancomycin-resistant bacterium. Chem. Biol. 6: 891±899. Cooper MA, Williams DH. 1999b. 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