Microchimica Acta

, Volume 169, Issue 3, pp 201–220

Flow based immuno/bioassay and trends in micro-immuno/biosensors


    • Department of Chemistry and Center for Innovation in Chemistry, Faculty of ScienceChiang Mai University
  • Kate Grudpan
    • Department of Chemistry and Center for Innovation in Chemistry, Faculty of ScienceChiang Mai University
Review Article

DOI: 10.1007/s00604-010-0333-1

Cite this article as:
Hartwell, S.K. & Grudpan, K. Microchim Acta (2010) 169: 201. doi:10.1007/s00604-010-0333-1


The term immuno/bioassay refers to analytical techniques that utilize the specific molecular recognition between antibodies and antigens or between biomolecules and specific receptors. Features such as high selectivity and low sample consumption make them very useful for analysis of samples with complex matrices. However, immuno/bioassays involve time-consuming (multi-step) operations which usually consist of steps of multiple incubation and washing. These are tedious and may result in large errors. Automatted immuno/bioassay systems can ease and shorten these processes and thus are highly beneficial. The hyphenation of flow-based techniques (i.e. flow injection related techniques and micro-fluidic systems) with immuno/bioassay protocols paves a new way for performing such assays. Compared to conventional micro-plate formats, flow-based immuno/bioassays can reduce the time needed for analysis, the volumes of samples and reagents consumed, and the need for trained personnel. In order to transform immuno/bioassays from conventional to flow-based formats, the solid surfaces used for the immobilization step has to be changed in order to meet the specific requirements of flow systems. To further develop the on-site analytical systems in micro-fluidic platforms, improvements in detection methods are necessary for high-sensitivity and rapid measurement. This review overviews the advantages and disadvantages of flow-based immuno/bioassay formats, the various types of solid surfaces for immobilization, and the methods of detection. Trends to improve sensitivity, speed and robustness are emphasized.


Flow injectionImmunoassayBioassaySensors

Introduction and practical integration of flow based techniques to immunoassay

Immunoassay is known as a highly specific analytical technique. The basic principle of the technique involves the molecular recognition between an antibody and a specific antigen, similar to the immune system, and this makes it naturally very useful for clinical analysis. The capability of modern laboratories in raising antibodies for various types of antigen including enantiomers [1], compounds containing metal ions [2], or other haptens, has extended its application to many areas of studies such as food technology [35], agricultural products [6, 7], and environmental studies [8, 9]. The typical immunoassay process, especially heterogeneous or label immunoassay, involves many steps. These normally include incubation (among antibody, antigen and relevant reagents) followed by washing/separation to remove excess reagents. Precise timing and precise volume or amount of reagents used for each assay replication are critical for good precision and accuracy. Therefore, it requires a certain level of training to operate these tedious steps. Attempts in automating the immunoassay process have resulted in the development of commercial immunoassay machinery based on robotics or mechanical movable parts that can introduce and remove reagents into or from the standard micro-well plate [10, 11]. These machines are useful for massive sample assays. However, they are at high price and do not give any benefit in shortening up the analysis time or simplifying the operational steps. Most laboratories own only a plate reader for the automatic spectrometric detection step rather than the automatic immunoassay machinery. In many situations, analysis involves low to medium numbers of samples of which results are preferred within a short time to better serve the clients and analysis purposes. Simplifying and shortening of immunoassay procedures would become important.

Strip tests such as those for pregnancy and glucose testing have gained popularity for home healthcare. Its rapidity and self testing serves the privacy needed by clients. These strips are based on immunoassay with pre-immobilization of antibody and relevant reagents onto the specially designed membrane. Results can be visualized with the easy to understand format as one/two lines or positive/negative signs. Similarly, immunoassay in the form of a disposable dipstick for environmental and food testing has also been introduced. They are very convenient, easy to use, rapid and reasonably priced. However, most test strips normally give just a yes/no answer or semi-quantification which is only adequate for the detection of substances that have significant effect or information when they are either present in high amount or absent [1214]. This is not suitable for the analysis of substances where detailed quantities are necessary. Examples are biomarkers of many diseases. These biomarkers are present in the body fluids but the levels are normally different in healthy people as compared to patients with diseases. Therefore, it is important to know how much they are present [1517].

Various flow based techniques (i.e. flow injection (FI), sequential injection (SI) and micro-fluidic systems) can be incorporated with immunoassay to automate the process [18, 19]. Since the year 2000, miniaturized bio-analysis systems such as micro-total analysis system (μ-TAS) [20], and micro-fluidic sensors have emerged as new state of the art developments in chemical/biochemical assays [21, 22]. Numerous developments in micro-fluidic immunoassay chip including unique formats such as credit card, digital style flow lateral unit and compact disc have been reported [2325]. However, the meso- and micro- systems such as FI and SI are still used routinely in many laboratories while the cutting edge technologies of micro-fluidics systems are on-going. Therefore, this review on flow based immunoassay will include flow injection and sequential injection based immunoassays as well as micro-fluidics immunoassays, but with different categorization and emphasis as compared to the previously published reviews [18, 19, 23, 24].

Flow injection technique, where reagents are mixed in small tubings while traveling into the detector, has brought about a new way of performing rapid chemical analysis. Numerous FI applications have been presented through a large number of research articles and commercially available instrumentations that have been accepted in many modern laboratories. Later generations of flow injection technique including stopped-flow injection [26], bead injection [27], sequential injection (SI) with lab-on-valve (LOV) [28, 29] and lab-at-valve (LAV) [30] have expanded the testing possibilities with many unique analysis systems. Most of these systems share a common feature of detection of products or signal changes at non-equilibrium state which is different from the way in which batch-wise process is conducted. Even though at non-equilibrium state, where the degree of reaction is not yet maximized, it is possible to detect the changes using commonly available detectors which now are capable of adequate sensitivity. Without the waiting period for equilibrium to occur, analysis time can be dramatically shortened. SI offers better repeatability with a computer program for system control [1]. Automatic operation of the pump and valve at precise volume and time eliminates the imprecision that may occur when detection is done at non-equilibrium state. When coupling flow based techniques with immunoassay, more detailed quantification of substance can be performed more rapidly with better precision. Automation also helps to minimize the need of having well trained personnel to operate the immunoassay test. FI/SI based immunoassay is therefore a very useful integration.

In this review, various flow based immunoassay formats utilizing different immobilization surfaces and unique ways of sample introduction/detection will be discussed. This will also include bioassays that utilize specific interaction between bio-molecules other than antibody-antigen, but with the same principal operations. Future trends toward miniaturization/micro-fluidics and unique developments including those reported for off-line operation (batchwise, non-flow system), but possible to be adapted for using with on-line (flow based) systems, are emphasized.

Various flow based immuno/bioassay formats

Immunoassays may be categorized in many different ways. Those that are based on the requirement for separation of the free and bound antigen/antibody are classified as either homogeneous or heterogeneous immunoassays. Also, they can be considered according to the order in which the reagents are added. This affects the order of interaction between the reagents and therefore the method can be considered as either a competitive or non-competitive immunoassay. Immunoassays can also be categorized based on the different types of labeling and detection system used. For example, they may be classified as radio isotope immunoassay (RIA), enzyme linked sorbent immunoassay (ELISA), or as chemiluminescence immunoassay. Another way to classify immunoassay techniques is based on the format of the stationary phase, such as micro-plate, bead, membrane and capillary immunoassays. Differences in these solid substrate formats will be discussed and emphasized in this review. It is the transformation of the solid substrate from a conventional micro-plate to various other formats that has made possible automatic flow based immunoassays, while types of labeling and detection unit may remain the same as in the conventional immunoassays. Comparison of various flow based immunoassay formats is summarized in Table 1.
Table 1

Comparison of advantages of various immunoassay formats. The relative amount of advantage of each format per feature is indicated by positive (+) symbol, where relatively more advantageous format is represented by a higher number of (+) symbols. Sensitivity depends mainly on types of label. However, enlargement of surface area and reduction of dilution by performing on-surface detection also affect the amount of detectable product

Advantages/Various immunoassay formats

Adjustable surface area

Ease of replacement

Commercial availability/Ease of modification

Rapidity of process/detection


+ + + + +

+ + + + +

+ + + + +

+ + + + +

By increasing number of beads

By flushing out with flow

Various modified beads commercially available

Detection can be done directly on transparent beads




+ + +

+ + + +

By increasing length or diameter of capillary but may be limited in availability

Need mechanical movement

Various modification methods but not commercially available

Detection can be done directly on transparent capillaries


+ + +


+ + +

+ +

By choosing higher porosity membrane

Need mechanical movement

Several types commercially available

Requires extensive washing




+ +

+ + + + +

By increasing size or surface area

Need mechanical movement

Restriction is on the need of facilitating electron transfer

Detection is done directly on surface

Conventional micro-plate



+ + + + +

+ + +

By selecting bigger size well


Widely available

Excellent for parallel massive sampling but highly time consuming per sample

Bead based immuno/bioassay

Micro-particles are probably the most popular solid surface alternative to the micro-well plate for immobilization of antibody, antigen or relevant reagents. One of the benefits is the high surface area per volume of the micro-particles and the possibility to increase the immobilized surface area as compared to the restricted area of the fixed size micro-well. The ability to accommodate higher numbers of immobilized molecules helps to improve sensitivity and detection limit of the assay. A common way of using micro-particles is in the form of packed column as in chromatography [3136]. This format is also known as immunoaffinity extraction, used mostly to extract the analyte from impurities by immuno-capturing which does not involve as many operational steps as in a normal immunoassay process. A more advanced system was reported using a multi-immunoaffinity column array [37], in which each column was packed with different specific antibodies. With a single flow of sample solution through this array of columns, detection of multi-analytes in the sample can be done simultaneously.

Other formats of bead-based immunoassay take advantages of short diffusion distance between molecules in bulk solution (i.e. antigen) and the immobilized ones on the bead surface (i.e. antibody) suspended in the solution, see Fig. 1a and b for comparison with the conventional micro-well plate. At the early stage of development, the bead-based immunoassay format was operated off-line (non-flow based system). Magnetic beads were used to facilitate the separation step by accumulating the beads at one spot with a magnet while the liquid phase was removed for detection [3841]. The flow injection system could be employed to automate bead separation and improve precision [42].
Fig. 1

Comparison of diffusion distances (d1, d2 and d3) between antigen (◇) in solution and the immobilized antibody (Y) on a the wall of the conventional micro-well plate, b the bead suspended in solution, and c the wall of the narrow capillary

The need for rapid analysis has led to a replaceable bead-based immunoassay with on-line operation. This can be done successfully with bead-injection technique. It is useful for cases where regeneration of bead surfaces is impossible or difficult to be performed on-line. The first generation flow injection system can be coupled with bead injection technique to carry out bioassay with simple operational steps [16]. However, sequential injection is more suitable for immunoassay where many reagents and operational steps are required. Reaction cells, where beads are trapped while solution flows in and out during incubation and washing steps, can be designed in many different ways.

The jet ring cell concept [43], also known as “leaky tolerance cell” [44], was introduced to trap rather big size particles in the flow line while solution is passed through the small gap, as shown in Fig. 2a. For small size beads i.e. <10 µm that may escape through the small gap, the additional use of larger sized particles to be mixed with the small working beads to create void volume for trapping of the small beads, as illustrated in Fig. 2b, has been reported [44]. A unique micro-fluidic piece, called Lab-on-valve (LOV) [45], that can be integrated onto a sequential injection (SI) valve to accommodate both chemical reaction and detection process is now commercially available. The LOV can also be adapted to trap beads. Direct on-bead measurement could be done with a fiber optic detection unit. This helps to increase sensitivity because of less dilution effect, as compared to downstream solution detection. However, some limitations of bead based immunoassay, especially in the form of packed column and direct on-bead detection, may be encountered. The main problems of beads are irreproducibility of bead packing and light scattering from beads which interferes with the spectrometric based detection [46]. Bead size and uniformity are important factors for reproducible packing. Beads that are too small may clog the valve while beads that are too large would increase void volume, and reduce uniformity of packing.
Fig. 2

Diagrams showing the concept of a jet ring cell or leaky tolerance cell and b the use of large size beads to help trap small working beads

If the bead size is not suitable for operation in a micro-channel or if the LOV piece is not available, a micro-reservoir can be used. The open end of the reservoir eliminates the back pressure which can easily occur in the flow injection-bead based immunoassay system [47]. The micro-reservoir can be attached directly onto one of the ports of the normal multi-port selection valve. The name Lab-at-Valve (LAV) [30] was later introduced for this kind of direct arrangement as an alternative to a LOV.

Beads made of various materials are commercially available. Magnetic beads have gained much popularity because of their ease in being retained in the flow system during washing and incubation steps as compared to non-magnetic beads [48]. The works on magnetic bead based immunoassays, including the pioneering works of the use of magnetic beads with rotating disk electrode (RDE) [3841, 4952] have been reviewed [53]. The effectiveness of online magnetic trapping systems in some applications of flow injection bead based immunoassay using chemiluminescence and electrochemical detections has been demonstrated [54, 55]. A multi-channel flow injection system combined with magnetic collection enables multiple immunoassays [56]. The combination of electrochemical detection with bead-based immunoassay has been increased. The interdigitated electrode array (comb electrodes) enabled redox cycling of the product from enzyme labeled on magnetic beads that were trapped on the array electrodes by a magnet [57]. Although this latter work was performed batch-wise with a drop of the solution, it appears that this method could also be adapted for use within the flow system.

The main point that should be considered for further improvement of flow bead based immunoassays may be how to keep bead suspension from settling down over time. Homogeneity of the bead suspension affects the precision of the number of bead being introduced into the system for each assay.

Capillary based immuno/bioassay

Another solid substrate format that is very useful for flow based technique is capillary. Antigen or antibody can be immobilized onto the inner wall of the capillary. The use of the antibody pre-coated capillary for capturing analyte, by employing capillary force, prior to redissolving and collecting antibody-antigen complex for detection was reported for clinical application [58]. Flow injection capillary immunoassay was started with off-line incubation of reagents in the capillary reactor and followed by injection of product, collected from the capillary, into the flow system for detection [5962]. Another approach is based on incubation of reagents in a test tube or micro-plate and passing the mixture into the flow system (FI or SI) equipped with the protein A coated capillary. Antibody-antigen complex can bind to Protein A on the capillary surface, while antigen alone cannot. After separation of bound and unbound species, the amount of antigen can be estimated [60]. Connecting capillary to the FI system, as part of the tubing flow line, offers the benefit of eliminating back pressure that may occur with bead-based packed column immunoassay [63]. The fused silica capillary, as that used for gas chromatography, and specially treated glass capillary were reported as suitable substrates for covalent binding with biomolecules through chemical reactions that need heat activation [59, 60, 64]. When dealing with chemical reaction without heating process or when physical adsorption is adequate, a lower cost uniformly sized glass capillary such as that used for hematocrit [65, 66] or a plastic/polymer capillary [67, 68] can be employed. Glass capillary is more popular because on-capillary optical detection and extended application such as waveguide immunosensor [69], can be accomplished more easily at lower cost as compared to the more opaque plastic capillary [64, 70].

As compared to beads, capillary has not been used as much. This is probably because capillary does not provide as high of a surface area. In addition, there is no modified surface capillary commercially available to facilitate the immobilization of biomolecules as there is in bead format. Modification and immobilization of biomolecules on the capillary surface normally involve multi-steps and many reagents [64]. Furthermore, removing/replacing the used capillary is not as easy and automatic as removing beads. However, shorter distance between immobilized molecules on the capillary inner wall and the molecules in solution in the narrow capillary, as shown in Fig. 1c, yields better mass transportation efficiency as compared to that of a conventional micro-plate. Capillary also allows for lower volume consumption of solution (i.e. 20 μL vs 300–400 μL) [65, 66]. Antibody immobilized monolithic capillary has also been demonstrated to improve rapidity and reduce flow resistance [71]. In addition, glass capillary is also much lower cost than beads.

Capillary immuno/bioassay is the most convenient format to be transformed into micro-fluidic format. The electrophoresis technique, normally carried out on capillary, can easily be coupled with immunoassay to perform immuno-electrophoresis [72, 73]. Capillary force itself can be utilized for sample/reagents introduction without other sample introduction unit [74, 75].

The main limitation of flow based-capillary immunoassay probably lies on the inconvenience in changing the capillary. A suitable mechanical approach is still needed. Some unique developments for multi-analytes detection were reported and would help to decrease the frequency of capillary changing. A common approach is to fabricate a micro-fluidic platform with embedded multi-capillaries, each of which accommodates different immobilized antibodies [70, 76]. Specific site multi-antibody immobilization is also possible that enables detection of multi-analytes in one capillary [64, 77]. Other convenient alternative ways for rapid changing of capillaries would be helpful.

Membrane based immuno/bioassay

The use of membrane has become popular in immuno/bioassay applications since it mimics the cell membrane, which offers some advantages over other formats of solid phases. The porous nature of a membrane makes available a surface area greater than that of a nonporous plastic surface. Several manufacturers offer micro-well plates with nitrocellulose bottoms and some have assemblies for clamping membranes into micro-plate [78, 79]. The porous characteristics also enable a membrane to adapt well within flow based techniques as it will not cause high back pressure.

Various types of membrane are available. Most unmodified membranes are hydrophobic. Choices should be selected based on suitability with solvent and flow rate required. Nylon and polysulfone work well with aqueous based biological samples while polyvinylidene fluoride (PVDF) is resistant to most solvents [78]. Other membrane materials such as lipid membrane [80] and membranes produced from easy to find raw materials such as shrimp, crab shells (i.e. chitosan) [81] and egg shell [82] are interesting low cost products. Nano-structured membranes have been reported that help to lower detection limit. Some nano-fibrous polymer membranes such as PVDF and PDMS with dendrimer PMMA could be prepared using a low cost electrospinning technique [83, 84]. When nanomaterials such as gold are incorporated onto the nano-pored membrane, antibody could be easily immobilized through disulfide bond [85]. Development of membranes that are stable and more tolerant to the environmental and operational conditions, e.g. air and shear flow, aiming to extend the shelf life and ease the storage, is also an interesting research trend [86]. Silicon membrane with arrays of micropores is one example recently reported [87].

Utilization of membrane in flow injection immunoassay is possible in different ways. Membrane can be used to support immobilization of antibody or bio-receptor to retain analyte of interest, a method known as immuno-filtration [88]. Immobilization can be done in small spots to create a microarray for multiplex assays [89]. Membrane may be placed on the electrode and the change in electrical signal due to antibody-antigen interaction can be measured directly at the electrode surface [90, 91]. Membrane can also be used as part of an extraction unit, similar to liquid–liquid extraction technique. The technique is also known as immuno-supported liquid membrane extraction (ISLM) [92, 93]. The principle is as shown in Fig. 3. Analyte (antigen) in the flowing aqueous stream (donor) is extracted into an organic phase immobilized on the membrane by adjusting of pH. Then, owing to concentration gradient, the analyte is further extracted into another aqueous phase on the other side of the membrane (acceptor) containing a specific bio-ligand such as antibody. The antibody-antigen complex formed will be trapped and accumulated in the acceptor due to its low solubility in organic phase. In a flow injection system, an acceptor can also be conveniently used as an injection loop. The simultaneous enrichment and clean up of analyte from this pre-concentration/extraction process helps to improve sensitivity of the analysis as compared to the FIA system alone.
Fig. 3

Diagram showing extraction of antigen (Ag) from donor to membrane and to acceptor containing antibody (Ab)

Unique applications would depend on the selection of suitable membrane properties. A successful work on the immunoassay of protein isoforms, using a membrane with ion exchange and affinity that is specific for a certain isoform, was reported as membrane assisted isoform immunoassay [94]. Searching for other new membrane with specific properties is still an interesting trend of research.

The porous nature of the membrane offers continuous flow which enables continuous extraction. However, this characteristic may also cause some drawbacks. Large surface area has a higher possibility of nonspecific binding toward proteins. Therefore, when using a membrane in immunoassays, extensive washing steps and blocking agents are essential. The research on minimizing these drawbacks would be very useful.

Label free immuno/bioassay

Label free immuno/bioassay is useful in shortening analysis time by eliminating some incubation steps such as incubations for binding of secondary antibody, label, and substrate. Some problems on alteration of affinity of the biomolecules by the label, may also be reduced. When labels are not used, quantitative analysis must be done through the changes that relate to physical properties such as mass, refractive index, electrical parameters, etc. using various methods of detection (plasmonic, photonic, electronic and mechanic) [9597]. Some new developments with surface plasmon resonance (SPR) technique, the optical detection method commonly used in label free immunoassay, are still on going to improve its optical sensitivity [98], reduce interference signals [99], and to make it suitable for multiplex immunoassay [100, 101]. Other novel developments in optical detections [97, 102] have been reported but not as many as in electrochemical detection. Here, the authors would like to emphasize the aspects of materials and solid surfaces used for immobilization that enhance the performance of the label free immunoassays.

Quantitative binding of analyte onto piezoelectric surface can be measured based on the degree of the resonant frequency shifting due to mass changing. Selectivity of the quantitative analysis is possible by immobilization of specific molecules such as antibody on the surface of the piezoelectric material while sensitivity depends upon viscosity of the solution medium and the immersion angle in the solution [103, 104]. When incorporating immunoassay with the FI technique, a stronger link between the receptor molecules (e.g. antibody) and any solid surface is preferred to prevent the loss of the receptor within the flowing stream of solution. Reported works on piezoelectric detection based immunoassays have placed emphasis on this issue. Various covalent binding procedures such as glass silanization, thiol-gold bonding and bifunctional cross-linking have been reported [105, 106]. However, the processes involve complicated multi-steps and many chemicals. Therefore, trends of research in this area consist of attempts on simplifying the immobilization process while maximizing reusability, by searching for new processes or materials. Examples of reusable piezoelectric immunosensor are amine terminated plasma-polymerized film (PPF) with alginate [107] and conductive polymer entrapment (CPE) [108].

Porous aluminum oxide films can be used as immobilization surface in the interferometric immunosensor [109]. White light reflection from the film gives a spectral pattern that is changed when there are changes due to refractive index or thickness of the film i.e. from binding of the biomolecules on the immobilized receptor on the Al film. It was also found that some aluminium nano-structure when placed close to tryptophan containing proteins can help to enhance fluorescence from proteins without using additional labels [110].

Electrodes may be modified in various ways to have specific binding with the analyte of interest. Extended applications on study of interaction between protein and small molecules [111] and identification of specific protein in a so called “biobarcode” technique [112] were reported with DNA modified electrode. Direct measurements of the change in conductivity, capacity, and impedance upon binding have been reported successfully in both FI and micro-fluidics formats [113118]. Although the background signal from ions in real sample matrices can present major interferences for the conductivity measurement, this problem could be solved by designing the system in such a way so that the difference between signal from the sample and from the background matrices can be compared in one run [113]. The capability of the newly introduced immuno-transister to detect low molecular weight antigen has been demonstrated [119]. The new label-free immunoassay systems that involve simple preparation are still most wanted.

Electrochemical based immuno/bioassay

Generally, this type of immunoassay refers to the assay that can be detected using electrochemical techniques. An overview on electrochemical immunoassay was presented in 2000. [120] Electrochemical detection can be applied to any of the formats of immunoassay previously mentioned. However, here we will concentrate on the unique modified electrode and the immunoassay where the electrode itself is a support material for immobilization of reagents as part of an immuno-reactor. The label free aspect of the electrochemical based immunoassay was already mentioned in the previous section and will be excluded from this section.

Miniaturization or down scaling of the system is the trend in developing an analysis system. Electrochemical techniques play an important role since the parts needed for the system, such as the conductor, electrode and wire, can all be made very small and the techniques are conveniently adaptable for multiplex immunoassay [121124]. By applying an electrical field during the incubation process, the immuno-binding could also be done in a shorter time [125].

Various types of electrodes have been proposed that can be employed directly as a support element for bioassay. The most popular type is screen printed electrode due to simplicity of preparation, low cost and suitability for mass production [126]. Most works have suggested disposable sensors [123, 127129] while some preffered the renewal of the surface for reusable purposes [130]. The latter comes with the drawback of having a long renewal time which adds to the overall analysis time. If materials used are at low cost, disposable immunosensors such as those made of chitosan membrane [131] can be developed and used without the need of regeneration/reuse which would indirectly help to cut down analysis time.

Nano-materials (such as nano-gold [132, 133] and hydroxyapatite nanopowder [134]), polymer film [135], and membrane with ion-exchange sites [136] can be used to modify the electrode and make it ready for reagentless immobilization. The combination of nano-metal particles such as nano-gold with mediated charge transport reagent helps to catalyze the electrochemical process [137]. The unique 3D nanogold hollow microspheres on chitosan membrane glassy carbon electrode helped to increase immobilization surface area to improve sensitivity [138]. There has been an increased interest of nanotechnology to produce nano-sized electrodes in the forms such as nano-wire [139], nano-tube [140, 141], and nano-pipette [142]. This enables multiplex immunoassay in a reduced size device. Immobilization of bio-molecules onto magnetic material prior to attaching them onto the electrode surface via an external magnet [143] eases the electrode regeneration process, which can be accomplished just by removing the magnet. Overview on the use of metal and semiconductor nanoparticles in electrochemical sensors can be found in the recent review [144].

Chemiluminescence is a detection technique that is very popular for immunoassay because it offers high sensitivity. Its combination with the electrochemical technique to yield electrochemiluminescence was reported to extend its applications. For example, an uncommon enzyme label, i.e. glucose oxidase with low turnover number as compared to peroxidase and alkaline phosphatase, could be utilized as an enzyme label in immunoassay when being used with electrochemiluminescence [145]. Addition of long chain molecules such as dextran [146, 147] or nano-particle such as gold [148] between the electrode surface and the molecules to be immobilized further enhances the performance of immuno/bioassay by increasing flexibility and reducing sterric hindrance in the later steps of immunoassay binding.

Elimination of some steps would help to simplify and shorten analysis time. Examples are the attempts to modify electrode surfaces with materials that undergo redox-cycling i.e. electroactive polymer [127] or substances that act as electron transfer mediator [149] to eliminate addition/incubation of substrate or other electron transfer mediators in the detection process. The ease of the process in which biomolecules are immobilized onto the electrode surface is also in demand. A one-step cross linking reaction was reported for the immobilization of antibody onto electrodes at room temperature, using the mixture of antibody with BSA and glutaraldehyde [126, 138].

Research trends in this area deal with investigations into the seemingly endless alternative possibilitites for immobilization of biomolecules onto the electrode surface with the aim of offering robustness, reusability, and a fast response. The monolayer immobilization is highly desired [150] for improving analysis time and efficiency of analyte transportation to accommodate micro-/nano- technologies. If these electrode modification/immobilization process were used in conjunction with automatic operation of flow based technique, the whole immunoassay process could be much more rapid.

Trends in micro-immuno/biosensors

Similar to chemical analysis, immunoassay is developing toward 1) miniaturization for portable and personal/on-site analysis, 2) multi-analytes detection capability for better information, 3) rapid analysis, 4) regeneratable/reusable device, or 5) disposable device. However, in immunoassay as compared to normal chemical analysis, there are some additional points to be considered due to non-specific binding commonly found with protein based analysis. In many cases, the blocking reagents may not be sufficient due to the pronounced effect of nonspecific binding in a micro-system and the non-permanent nature of a blocking reagent which may be washed off with the flow of reagents. To overcome the nonspecific binding, surface modification and improved detection sensitivity are very important. Therefore, new trends of immunoassay involve not only development of small hardware, but also new chemicals, materials, and techniques that will enhance sensitivity of detection.

Novel sample introduction and flow control systems

A simple flow manipulation system with controllable flow rate but without external power supply is of high demand for use with miniaturized chemical/biochemical analysis systems. Since it is important that sample introduction and flow control systems can be integrated into the same unit with the reaction chamber/reactor and detection unit, having small sized parts is therefore beneficial [151]. Various types of micro-pump and valves for flow injection analysis in micro-fluidic flow injection system, including gravity driven, acoustic wave, ultrasonic and electroactivated pumps, have been reported [152156]. To construct an analysis system that can be used as a stand alone device, pumps that do not require external power supply are of particular interest.

Utilization of the natural vibration of piezoelectric material is an example way to create a miniaturized peristaltic/diaphragm pump [157160]. In the micro-channel, the unique laminar flow characteristics of the liquid and the capillary force enable the transportation of reagents without an external pump or injection system [161, 162]. In the early development of a micro-fluidic system for chemical analysis, H and I shaped systems, example shown in Fig. 4, were introduced for chemical extraction of biomolecules from one liquid stream into another liquid stream that was flowing into the same micro-channel, based on different diffusion rates of small and large sized molecules [163]. Later developments, involving the unique characteristics of fluids interfaces and a modified micro-channel solid surface with immobilized ligands, enabled additional steps such as capturing, washing and detecting in heterogeneous immunoassay [164].
Fig. 4

Example of an H shaped microfluidic system for extraction of small molecules from the mixture of large and small molecules based on the different diffusion rates

High solubility and rapid diffusion of air in polydimethylsiloxane (PDMS), normally considered as a drawback in preparation of PDMS film, was reported to be useful for pumping of solution in a PDMS-glass based micro-channel [165, 166] when moving it in and out of a vacuum desicator. The related work also demonstrated that the diffusion at the boundary of the two streams of laminar flow is sufficient to provide the mixing and capturing process involved in immunoassay [167].

One limitation of pumping systems which are based solely on laminar flow and capillary action is their inability to change flow rate due to the fixed size of the channels. Electro-osmotic flow is a more well known approach for reagent introduction with variable flow rate within the same size channel and this may be a required feature in some cases [168]. However, the limitation of the electro-osmotic flow system lies in the requirement of having an electrical power source and that the liquids being transported as well as the surface of the channel must be charged to some extent to allow the attraction with the oppositely charged electrode to pull the solution in the desired direction. Recently, a time controllable flow in a microchannel using an electrowetting technique was introduced [169]. By embedding working and reference electrodes at 2 sites across the micro-channel and enlarging the glass channel at the specific site designated as a valve, the liquid reagents can travel forward with capillary action and can be stopped at the electrode because of the wider dimension of the channel and hydrophobic nature of the electrode. When the suitable potential was applied to the electrodes, making them to be more hydrophilic, liquid continued to flow past over the electrode surface, and after that the capillary force was resumed, leading the solution to flow into the reactor. As an alternative to electrical power pumps, the magnet can activate the suspension plug of ferromagnetic particles in the channel to create the ferrofluid pump [170].

The search for micro-pumps without the need of electrical supply that offers pulse free, controllable flow rate and flow time with no restriction of types of solution is still on-going.

Rapid/specific immunoassay with immobilization/conjugation through flexible long chain and highly branched molecules

The addition of a chemical chain to extend the immobilized antigen or antibody from the solid surface has increasingly been applied. Extension of the antibody from the surface is thought to provide more freedom of movement and less chance of losing its activity. It is expected that the long flexible structure (spacer arm) can extend the antigen or antibody out into the solution, decreasing the diffusion time for the component in the solution to reach and be captured by the immobilized ones. Another main function of the linker is to orient the antigen or antibody and maximize the accessibility of the binding sites or epitopes. For example, if immobilization of antibody was done through the fragment crystallizable (Fc) region, then the antibody should be oriented in a way that allows binding sites to be available for the next binding steps, as compared to random immobilization which may orient the antibody in such a way that the binding sites cannot be accessed.

There are many linkers, especially with biotin conjugate, commercially available that can be used as spacer arms to link molecules of different functional groups [171]. Other linkers reported for their use in the immobilization step are dextran [146, 147] and polyethylene glycol (PEG) [60, 70, 71]. It has also been reported that PEG added in the label conjugation step, between the thiol-specific quantum dot and the secondary antibody, can also help to increase sensitivity and specificity [172, 173].

Dendrimer or dendron, a highly branched synthetic macromolecule as shown in Fig. 5a, is another interesting tool that can be used to create a high capacity immobilization surface [174, 175] or to accommodate high capacity labels [176, 177]. In combination with some conductive materials such as nano-gold, a dendrimer electrode can be constructed [90, 178]. Dendrimer is different from polymers because it does not have molar mass distribution owing to its symmetrical structure and monodispersion characteristics. Dendrimer is synthesized to have tree-like branches. Each branching point is called a generation (G). More numbers of branches are created when more generations are added. The ability of dendrimer to bind to high numbers of ligand or antibody was reported to be useful as an effective blocking reagent [179]. This should also be useful in increasing sensitivity and rapidity of the assay. The important property of the dendrimer depends on the functional groups at the end of the branches which can be engineered as required. This should make it possible to couple the dendrimer with various ligands or antibodies to enhance immunoassay performance. However, reports on immunoassay using the dendrimer molecule in this way are still rare. Even though the actual dendrimer molecule is not used, its branched structure is usually mimicked to improve sensitivity of the assay as will be described next.
Fig. 5

a A dendrimer. G0–G4 are the generations of branching points from low to high generations b formation of dendrite structure using multi-valence molecules

Highly sensitive immunoassay and possibility of multi-analytes analysis with unique detection systems

Building dendrite structure to accommodate a higher number of labels

Another approach for better detection performance is signal amplification using modified assay procedure. Dendrite structure, not a dendrimer molecule, is used for amplification of signal by accommodating a higher number of labeled molecules. By employing two multi-valent molecules or particles, antibody labeled with dendrite structure could be constructed on the bound antibody-antigen complex. The difference between the dendritic structure Fig. 5b and a dendrimer molecule Fig. 5a is compared. Examples of multi-valent combinations are streptavidin conjugated fluorescence label with biotinylated anti-streptavidin [167], biotinylated liposome with avidin [180], DNA or antibody-modified gold particles [181, 182], and biotinylated protein with streptavidin [183, 184]. The limitation of additional tedious multi-incubation steps during dendrite structure formation has been addressed. Utilization of the unique laminar flow characteristic of liquids in a micro-channel to reduce incubation steps during dendrite construction has been demonstrated [167] by using two continuous laminar streams to mix the reagents and allow the bonding to take place while flowing.

Multi-enzymes or substrates

For the assay with enzyme label, multi-enzymes or multi-substrates can be used to amplify the detection signal. These appear to be used in static formats such as in micro-plate immunoassay format offline or in a specially made reactor, however, online use should also be possible. Some unique examples are described next.

The employments of two substrates or enzymes, named as bienzyme-substrate-recycle, were reported [185187]. This helped to increase the detection signal by producing an intermediate product that reacts with the 2nd enzyme and can re-start the production of the final product again and again. The detection could be done when more enzyme-substrate product had accumulated in order to obtain a better sensitivity. By adding enzyme as part of the electrode, a unique way to integrate enzyme amplification with electrochemical detection was achieved [188]. This approach is different from the bienzyme-substrate-recycle in that amplification is due to the direct electron transfer process occurring at the electrode surface with the embedded enzyme rather than by enzyme-substrate-recycling.

Encapsulated labels

Another way to amplify the detection signal is by encapsulating the vast amount of detectable molecules (such as fluorophores) and release them at the detection step. Liposome and Nano-encapsulated organic micro/nanocrystalline particle labels are described here.

Liposome is a highly complex, microscopic lipid sphere with a membrane, composed of phospholipids and a cholesterol bilayer, and core that usually contains aqueous solution. It has been applied as a technology for drug delivery. The use of liposomes in immunoassay was reviewed in 1997 [189]. Since that time, work on liposomal immunoassay has progressed.

Liposome can be formed naturally or artificially. It can bind specifically to antibodies, specific antigen or vitamin by insertion of biomolecules with specific interaction to those targets into the phospholipids bilayer during liposome synthesis. Alternatively, conjugation of liposome label with antibody could also be done via non-covalent binding between streptavidin and biotin. Matrix for detection is encapsulated inside the cavity core. This requires the liposome disruption, normally by adding detergent, in the detection step. In the past, liposome-released peroxidase was reported with potentiometric detection [190]. Currently, liposomes filled with dye or chemiluminescent/bioluminescent solution are mainly used for better sensitivity. Because a liposome vesicle contains a large number of molecules, it can be detected with very high sensitivity as compared to a conventional fluorophor label [191], see Fig. 6. The exceptional performances of liposome immunoassays with some that offer detection limit as low as zeptomole (10−21) level have been demonstrated [192194].
Fig. 6

Comparison of a conventional labeled antibody and b encapsulated labeled antibody that can accommodate a large number of labels. (◊ is antigen, Y is antibody, ✯ is label)

Nano-encapsulated organic microcrystalline particle label is based on the similar idea as the liposome label where a large amount of fluorescent molecules are released from the label in the detection step, except that the materials used for encapsulation are not the same. For example, instead of a phospholipids layer, ultra-thin poly-electrolyte layers such as poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate) are used to trap the fluorescein molecules, using a layer-by-layer technique [195]. These layers can be covalently bound to antibodies that are needed to be labeled. By adding DMSO and NaOH, trapped particles can be released for detection. The attempts to simplify the encapsulating process have been reported using amphiphilic polymers such as distearoylphosphatidyl- ethanolamine modified with amino(polyethylene glycol), and alkylated poly(ethylene imine) to coat the nanocrystalline fluorescence particles [196, 197]. The technique can replace the layer-by-layer technique but with the lower sensitivity due to smaller overall size and therefore, lower numbers of trapped fluorescein molecules. The interaction of the nano-encapsulated nanocrystalline label and the antibodies was also through adsorption which is less specific as compared to the covalent bonding when using the layer-by-layer coating. However, the incorporation of non-covalent yet strong and specific bonding between Avidin/Streptavidin/Neutravidin and biotinylated biomolecules can overcome this problem [198, 199].

One limitation of using a large amount of fluorescence particles or aggregates is known as the self quenching effect. A new class of photoluminescence particulate label called nanocrystalline siole such as hexaphenylsiole has been introduced to be used as an alternative to fluorescein molecules [200]. Siole has an increased luminescence intensity when presented in aggregate form while has no emission when dissolved in organic solvent at room temperature. Therefore, it can be used as nanocrystalline labels without quenching effect. Sensitivity of up to 40–140 folds as compared to the direct FITC-label was reported. DNA prime probe encapsulating liposome has recently been reported with some unique detection methods such as rolling circle amplification (RCA) where repeated sequence of DNA helps to improve sensitivity and “biobarcode” where unique sequences of DNA can be used to label each particular analyte [201]. The research trends in liposome FI-immunoassays still focus on searching for compounds to be entrapped inside the liposome for the best detection power. Enzyme label can be a good candidate due to its amplification power but it is important to stabilize its native structure for the best efficiency. A cross-linked 3D hydrogel network was claimed to be suitable for entrapping enzyme label because it helps to reserve the micro-environment for immobilization of enzyme [202].

Quantum dot labels

Semiconductor nano-particles known as quantum dots (QDs) have gained popularity for sensitive labels. They are composed of atoms from groups II–VI or groups III–V of which CdSe, CdTe and ZnS are commonly used [203, 204]. QDs have unique properties of size dependent fluorescence which means a single excitation wavelength can excite the same type of QD of various sizes and yield different emission wavelengths. QDs have been used as alternatives to organic fluorophores for molecular and cellular labeling in biological imaging, in studying of virus localization in living cells and immunoassay [205]. Strong fluorescent emission enhances sensitivity of the assay while multiple emission wavelengths enable multiplex immunoassay in various types of sample [206212]. QDs are also more tolerant to harsh conditions such as heat [213]. However, the limitation and poor performance of QDs as compared to the organic fluorophores were also reported when using QDs with a normal flow cytometry on an environmental sample. This might be due to interference from other fluorescence species in the matrices, irreversible aggregation and reduction in stability of QDs in aqueous environments [214, 215]. The trends of research in immunoassay with QD labeling include attempts to overcome the difficulties involving the simultaneous detection of fluorescent signals, to improve solubility of QD in aqueous solution by using proper coated (capped) material, and to explore alternative detection enhancement methods.

Normally, QDs are capped with carboxylic functional groups such as mercapto acetic acid, mercaptosuccinic acid or dihydrolipoic acid to provide negative charges on the QDs surfaces for bio-conjugation with the positive charges of the protein. To make QDs safer to handle and to make them water soluble for easier conjugation with protein, amphiphilic polymers such as poly(maleic anhydride) were used for coating instead [203, 216]. Bio-conjugation, which can be achieved via the carboxylic group of the polymer coated onto QDs and the amino group of protein, is more stable than electrostatic bonding. Apart from polymers, silica has also been used as an interesting capping material to prevent the oxygen accelerated photobleaching process and to prevent the loss of fluorescent properties in common buffer solutions, which are normally encountered with semiconductor based QDs [204, 217]. The transparent nature of SiO2 enhances and stabilizes fluorescence. Other materials such as gold particles [218] and InP/ZnS [219] have also been proposed to be used as QDs as alternatives to potentially toxic semiconductive materials.

The most popular detection method for QDs labeling is fluorescence spectrometry. Improving optical resolution of the fluorescent detection is desired, especially for the research trends in simultaneous detection of multiplexes [220]. Coupling of QDs onto micro-particles such as latex beads, referred to as “nano-on-micro” (NOM) was suggested [221]. This work utilized the improved light scattering intensity of the NOM for additional immuno-reaction detection along with identification of multi-analytes from fluorescent emission signals.

Alternative ways of detection other than fluorescence spectrometry is also of interest. Electrochemical immunoassay such as ones that detected electrical signal changes due to the releasing of Cd from the Cd semiconductor based QDs, [222224], or one with the combined electrochemistry with chemiluminescent spectroscopy (electrochemiluminescent) [225] have been reported. Instead of using them as conjugated labels, QDs can also be used to modify the electrode surface leading to the development of label-free immunoassay for the measurement of the change in signal due to sterric hindrance of binding [226]. Other less common ways of detection for QDs labels such as acoustic optic-tunable filter [215] and atomic force microscopy [227] are also possible.

Robust and longer shelf life solid substrate using molecular imprinting

The objective of molecular imprinting is to create a mold of a specific antigen onto a substrate that will act as an artificial antibody or enzyme. With this manipulation as an alternative way to produce specific binding for an analyte, the ethical and time consuming issues involved in raising antibodies in living animals can be avoided. The longer shelf life, the high tolerance to matrix effects, the easy storage condition and the robustness of the artificial antibodies are also important benefits.

As illustrated in Fig. 7, by adding the molecules of a target analyte (template) into the polymer during its production process and then extracting those molecules out, the polymer that is formed will have cavities to bind with the target molecules through electrostatic attractions and/or complimentary physical shape. Successful imprinting of variously sized molecules has been reported and some are commercially available [228]. Most of the developed molecular imprinting polymers are in the form of microbeads which are used as packing materials in a micro-column for the separation of analyte.
Fig. 7

Diagram showing molecular imprinting production process and its use for binding the target analyte

Now that the development of biosensors are increasingly of interest, the recent trends of research in molecular imprinting have been focused on improving the production of protein imprints, especially in the thin film and nanotube formats that can be easily adapted with the sensor device. This will also enable the imprinted molecules to be used more conveniently with the flow based system. So far molecular imprinting based immunoassays incorporated with the flow based system are still rare. One report [229] indicated the benefits of reducing non-specific binding by utilizing epitope imprinting rather than protein- engaged imprinting. The work was applied with quartz crystal microbalance (QCM) for detection of Dengue virus. Another work [230] reported the polymerization of acrylic based imprinting onto the inner wall of glass capillary. The advantage of reuseability of the capillary was demonstrated. This resulted in a low cost and automatic system with long term stability that is suitable for unattended monitoring [228, 230].

New materials other than polymer that are compatible with the easier imprinting process are also in demand. Sol-gel material has been reported as successfully used to create imprints [231233]. Generally, sol-gel is a wet chemical process used for fabrication of metal oxide materials. Meso-porous sol-gel glass could be used for immobilization of biomolecules [231]. Sol-gel bioglass can be produced in various forms such as monoliths, thin film, powder and fiber. The advantages of using sol-gel are owing to the ability to create the imprint at ambient conditions and in aqueous solution. This greatly helps to eliminate the problems of losing biological activity of the protein being imprinted which results when using high temperature and harsh organic media.


The combination of various flow based systems with immunoassay has led to improved performances as compared to the conventional batch-wise immunoassays. This integration requires major changes on the format of the solid surface for immobilization of bio-reagents. Rapidity and precision are the major benefits gained, making the highly selective immunoassay technique even more superior. However, the ability of the conventional micro-plate immunoassay to perform massive samples in parallel still remains the great benefit of the conventional batch-wise system [234]. The FI/SI based immunoassays are suitable for small to medium number of samples where the results are required in a short time. Research trends in flow based chemical analysis have led to Lab-on-Chip and down scaling micro-fluidics devices, many of which can be applied to perform immuno/bioassays. Attempts in developing small sized liquid introduction, flow control and detection systems as well as high sensitivity detection systems have been continuously progressing to the same goal of making a cost effective and easy to operate single unit immuno/bioassay based micro-total analysis device.


The authors thank the Thailand Research Fund (TRF), the Commission on Higher Education (CHE) and the Center for Innovation in Chemistry (PERCH-CIC) for support.

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© Springer-Verlag 2010