Capillary-based immunoassays, immunosensors and DNA sensors – steps towards integration and multi-analysis
Introduction
Capillaries have found widespread applications in analytical techniques, including capillary electrophoresis and capillary gas chromatography as columns, flow-injection (FI) analysis as tubing, and biomedical analysis as sampling devices. During the past 10 years, the applications of capillary tubes have been increased in immunoassays (IAs) immunosensors (ISs) and DNA sensors. In binding assays, the capillary serves as the solid support for immobilizing bio-reagents. However, the capillary can also act as a waveguide to enable methods of optical interrogation [1], [2] that permit the development of integrated sensors.
According to IUPAC, a biosensor is defined as a self-contained integrated analytical device, which is capable of providing quantitative or semi-quantitative analytical information using a biological recognition element that is retained in direct spatial contact with a physicochemical transducer [3], [4]. Although this definition underlines the need for intimate contact of the immuno-reactive surface with the physicochemical transducer, some authors include flow-through-based capillary IAs in ISs, highlighting the continuously operating nature of these devices. In this case, though there is no direct spatial contact of the immuno-reactive surface with the physicochemical transducer, contact between immunoreaction and detection is achieved through the flow of immunoreagents. In the context of this review, we report on flow-through-based capillary IAs as assays or as sensors, depending on how they have been reported in the literature.
The extensive use of capillaries in the assays and sensors is due to their advantages over other “traditional” configurations (e.g., microwells and RIA tubes) used in IAs or planar surfaces chosen for the fabrication of sensors, and DNA and protein arrays. The small capillary dimensions permit low bio-reagent consumption and increased surface-to-volume ratio, which are probably the most important advantages [5]. Increased surface-to-volume ratio especially minimizes the role of diffusion, which is the limiting factor for reaction rates, so providing shorter reaction times and greater sensitivity [6]. The combination of short assay time and low bio-reagent consumption is expected to considerably decrease the cost of analysis. In addition, assay automation can be more easily realized, since the cylindrical geometry of the capillary facilitates the incorporation of flow systems.
In this review, we report on methods developed for activation of the inner surface of the capillary as the first step in developing capillary-based binding assays and sensors. We describe flow-through capillary IAs and ISs, capillary IAs based on measuring signal directly on the solid support, and capillary-waveguide ISs and DNA sensors. We also review detection strategies employed for the development of multi-analyte capillary IAs and ISs and DNA-hybridization assays. Finally, we discuss future trends in this field.
Section snippets
Activating capillary inner surfaces and immobilizing probes
The primary function of the capillary tube in immunochemical and DNA-hybridization assays is to serve as solid support for immobilizing bio-probes. Two types of materials have been employed for fabricating capillary tubes (Table 1) {i.e. fused silica or common glass and polymeric materials (e.g., polystyrene [7], poly-(methylpentene) [8], [9], polypropylene [10] and PVC [11], [12])}. Although there are lots of reports on glass capillaries employed in IAs, ISs and DNA biosensors and arrays,
Detecting labels directly on the solid support
This group of capillary IAs includes competitive and non-competitive formats, using enzyme or fluorescent labels. The bound fluorescent label, in the case of fluorescence IAs, or the reaction product between the bound-enzyme label and a suitable chromogenic, luminogenic or fluorogenic substrate, in the case of enzyme IAs, is retained inside the capillary reactor and quantified using an appropriately placed detector.
The first capillary enzyme IA based on this detection principle can be traced
Detecting labels out of the capillary tube
In this group of capillary IAs and ISs, the label or the reaction product between the immobilized enzyme and the substrate is transferred from the capillary to an external detector, in most cases using an appropriate flow system (flow-through assays). The first capillary IA following this detection principle relied on a radioactive label that was displaced after prolonged incubation with an unlabeled analyte, collected and quantified using a scintillation counter [11]. Table 2 shows the
ISs and DNA biosensors based on capillaries acting as waveguides
The IAs and ISs described above employed capillary tubes as solid supports for immobilization of immunoreagents and, in several cases, as flow-through cells. However, the real power of capillaries remained unexploited, since the capability of capillaries to act as waveguides that enable the development of several methods of optical interrogation was not previously utilized [1], [2]. A multifunctional capillary device serving simultaneously as immunoreaction vessel and signal transducer has
Multi-analyte capillary IAs, ISs and DNA-hybridization assays
A long-standing goal in the field of immunoanalytical techniques is development of multiplexed assays where two or more analytes can be determined in the same run so as to decrease considerably the time and the cost of analysis [54].
The current trend in multi-analyte assays is based on spatial resolution of recognition molecules. According to this strategy, different immuno-reactive probes were immobilized on different parts of the same substrate creating an array, thus permitting use of a
Conclusions and future trends
In the past 10 years, the role of the capillary tube in IAs, ISs and DNA-hybridization assays has evolved from being a simple reaction container and solid support for probe immobilization to being a component of an integrated sensor, serving simultaneously as fluidic device, a solid support for spatially-resolved probes and a waveguide of excitation or emission light. Despite these technical advances, a capillary sensor has not yet become commercially available, since improvements in detection
References (71)
Trends Anal. Chem.
(1996)- et al.
Biosens. Bioelectron.
(2001) - et al.
Anal. Chim. Acta
(1994) - et al.
Biosens. Bioelectron.
(1998) - et al.
Biosens. Bioelectron.
(2002) - et al.
Biosens. Bioelectron.
(2002) - et al.
Sens. Actuators, B
(1996) - et al.
Biosens. Bioelectron.
(1999) - et al.
Anal. Biochem.
(2000) - et al.
Anal. Biochem.
(2004)