Peer Reviewed: Can Affinity Sensors Be Used To Detect Food

Can Affinity Sensors Be Used To Detect

C O N TA M I N A N T S ? Immunoanalytical techniques could provide faster and easier analyses of food products.

Ursula Bilitewski Gesellschaft für Biotechnologische Forschung mbH (Germany)

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A N A LY T I C A L C H E M I S T R Y / N O V E M B E R 1 , 2 0 0 0

ood can be contaminated by numerous compounds of different origins. Pesticide residues are found in fruits, vegetables, and grains and their products (1). Eggs, meat, milk, and fish products can contain pesticide residues and fungal mycotoxins that originate from animal feed. Legal or illegal drugs, such as antibiotics, hormones, or other growth promoting agents, have been found in animal products. And occasionally, pathogenic microorganisms or their toxins are found in food of vegetable or animal origin. Many of the current analytical methods for analyzing food require long sample-preparation and analysis times, as well as complex instrumentation. This has led to a call for alternative faster and easier procedures. Immunoanalytical methods are attractive alternatives because antibodies can be developed not only for recognizing proteins, but also for surface antigens of microorganisms and low-molecular-weight compounds. The specificity and high affinity of the antibody–analyte interaction significantly simplifies sample pretreatment. Moreover, because immunoanalysis can be performed with simple optical tests—even in dipstick formats—or by using “one-way optical sensor chips” (2– 4), instrument costs are reduced. In addition, immunoanalysis can be incorporated into sensor systems that can be automated for on-line, high-speed analysis.

N O V E M B E R 1 , 2 0 0 0 / A N A LY T I C A L C H E M I S T R Y

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(a)

Antibodies immobilized on sensor surface Sample and labelled antibodies Sample and labelled analyte–analogue

(c)

Sample

(b)

FIGURE 1.Immunosensor principles based on immobilized antibodies. (a) Direct sensing of the analyte binding reaction; (b) competitive assay formats based on analyte tracers, such as enzymes, fluorophores, or even radioisotopes; (c) sandwich assay formats based on a second labeled antibody.

This article looks at the current state of affinity sensor systems, illustrating their known limitations and potential benefits for food analysis. Recently, the fundamentals of immunoanalysis; the strengths and limitations of the immunoanalytical approach, particularly for pesticide analysis; and the methods for detecting microorganisms have been reviewed (5–7). Thus, this article only briefly describes the fundamentals of important transducer principles. Examples of immunosensor food analysis applications are also presented. However, because food analysis deals with heterogeneous samples, with respect to composition and texture, there have only been a few reports of applying affinity sensor devices to food samples. Thus, the examples are supplemented by relevant environmental (water analysis) and medical (serum or urine analysis) examples. These related examples show that it is not the analytical techniques that need to be developed, but rather suitable food treatment procedures that deliver the analyte in a liquid suitable for the sensor systems.

Some background Most analytes relevant to the food industry can be divided among three categories on the basis of their routine method of analysis. The first includes small chemical compounds, such as pesticides, antibiotics, hormones, and mycotoxins, which are usually determined by multiresidue methods, such as HPLC, GC, and now CE. The second group includes proteins, such as bacterial toxins, antigens, and antibodies, for which enzyme-linked immunosorbent assays (ELISAs) performed in microtiter plates are the established analytical methods. Lastly, relevant microorganisms are usually determined by microbiological methods. Immunosensor systems can handle all three categories of analytes and offer some unique advantages. The most important

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is the possibility of a high degree of automation, which reduces the need for manual operations and allows on-line measurements during procedures such as milking (8) or high-throughput applications. Immunosensors can even analyze turbid samples, because detectors other than photometers can be used. However, immunosensors do not fit the typical definition of sensors. On the one hand, sensors should operate reversibly and in real time. On the other hand, the antigen–antibody reaction, which is the basis of immunosensors, is typically irreversible and usually requires the addition of labeled compounds or tracers, which only allow the indirect determination of the concentration of antigen–antibody complexes. Consequently, immunoanalytical procedures, including immunosensor systems, are based on multistep assays that are performed either manually or automatically and deliver a signal some time after the introduction of the analyte. Basic formats are illustrated in Figures 1 and 2. Only analytical systems, in which the antigen–antibody binding reaction is monitored in real time and one reaction partner is immobilized on the transducer surface, require only the addition of the sample and the regeneration of the sensor surface (Figure 1a). However, any device using an electrode or an optical fiber as the detector is often called a sensor system or sensor, despite the complexity of the analytical procedure and the overall system. It should be pointed out that for the detection of whole cells, there is now an alternative to the immunoanalytical approach. Characteristic DNA sequences can be amplified by a polymerase chain reaction (PCR) followed by analysis using gel electrophoresis, dot blot hybridization of labeled specific probes, or sensor devices (9–11).

Immunosensor systems requiring no labels The binding reaction between an antibody and the corresponding antigen can be followed in real time if the physical characteristics that change due to the formation of the immunocomplex are determined. The most important of these properties is the increase in mass loading on a transducer surface, which contains an immobilized reaction partner. This increase can be either directly monitored by a microbalance using acoustic sensors or indirectly by using an optical transducer to measure changes in the refractive index of waveguide systems. Both approaches are briefly described in the following. Acoustic sensors. Microbalances that determine an increase in mass due to formation of an immunocomplex are electroacoustic devices. These instruments use piezoelectric materials for the acoustic transduction, coupling mechanical deformation to electric potentials and vice versa. Different types of acoustic waves can be generated, which are detected by either bulk acoustic wave (BAW) sensors or surface acoustic wave (SAW) sensors (12, 13). Although SAW devices are intrinsically more sensitive than BAW devices, the latter are usually used in food analyses, primarily because the transducers are readily available and there are well-established measurement principles. BAW sensors contain quartz wafers in the form of 10- to 16mm disks, squares, or rectangles, which are ~0.15-mm thick

and sandwiched between two gold electrodes (14). These crystals can be used as chemical sensors by covering the electrode surfaces with a selective coating that binds analytes. In immunosensor systems, this coating is a layer of immobilized antibodies (12, 14). Binding of the antigen is detected by a decrease in the resonant frequency. Initially, these sensors were used only for gas-phase measurements; but now, flow-through cells have been developed that put one side of the crystal in contact with the flowing solution (15, 16). This allows realtime monitoring of the affinity reaction (12). Optical immunosensors. Optical immunosensors are based on optical waveguide structures, which are used as substrates for biospecific layers, such as immobilized antibodies. A simple waveguide structure is shown in Figure 3. Although no traveling wave exists in the y-direction in medium 2, a wave travels along the interface, and the electric field component of its electromagnetic wave decays in the y-direction. This decaying component is the evanescent field phenomenon (17). For visible light, the evanescent field “penetrates” 50–500 nm into the y-range. Thus, binding anything along the surface changes the waveguide configuration, particularly, the refractive index profile, and by this, the propagation velocity of the traveling wave. This can be measured, for example, through changes in the coupling conditions (changes of the angle of the light beam leading to the excitation of guided light modes). One increasingly well-known and similar phenomenon is the excitation of surface plasmons in metal layers by laser light. The angle of the light beam leading to surface plasmon resonance (SPR) is the recorded measurement signal. Alternatively, an interferometer can be used for determining the phase differences between two light beams (Mach-Zehnder interferometer). In this approach, a waveguide is divided into two arms, one of which interacts with the sample. The other is covered with a protecting layer. The two light patterns recombine after a certain distance, which leads to a stationary interference pattern. The light intensity at one position in the pattern depends on the phase difference between the light beams, which is the result of changes in the effective refractive index at the surface of the “sample” waveguide. The sensitivity can be improved by increasing the waveguide’s interaction length with the sample—that is, the distance between the splitting and recombination of the laser light (18, 19). All of these approaches measure the binding of molecules to the waveguide surface by changes in refractive index. Thus, the lowest detection limit for a compound depends on the sensitivity of the device to monitor small changes in refractive index and on the compound’s molecular weight. Changes in refractive index are directly proportional to the mass loading due to the binding of the analyte to the waveguide surface (18, 20). Up until now, a procedure as simple as that shown in Figure 1a was only applicable to the detection of low levels of high-

molecular-weight analytes. However, protein binding to the sensor surface can be monitored in real time. This allows, for example, the antibody immobilization to be followed during the immunosensor production, which provides quality control of the preparation. Moreover, kinetic data (e.g., association/dissociation rate constants) and steady-state data suitable for quantitative analysis can be obtained.

Immunosensor systems using labels Adding labels such as radioisotopes, fluorophores, and enzymes to immunosensors often leads to an improvement in sensitivity and an achievable lower detection limit. Moreover, specificity may be improved if the signal transduction is based on a specific property of the label. Thus, immunosensor systems with tracers are often considered a better choice, although they require additional reagents, often additional incubation and washing steps, and, if they are fully automated, more complex devices. Important principles of those systems, particularly with enzymes and fluorophores as labels, are considered next. Fluorescence sensor systems. Fluoroimmunoassays have been studied for almost 20 years as alternatives to radioimmunoassays (21). Fluorescence is rapid, sensitive, cheaper, and safer than radioisotopic methods. Moreover, fluorescence is well established for DNA analysis. Fluorescence sensors use optical fibers or integrated optics. Light propagating through an optical fiber or a planar waveguide generates an evanescent field. Not only does the refractive index change due to binding events occurring within this field, but fluorescent dyes are excited with an evanescent field of light of suitable wavelengths (Figure 4). If the optical properties match, emitted fluorescent light is collected by a waveguide. Thus, the use of optical fibers or waveguides as transducers in fluorescence affinity sensors allows a separation of bound and unbound tracers through the use of the evanescent field. Moreover, because labeling of antibodies with fluorescent probes is an established procedure from immunostaining techniques, most of the fluorescent immunosensor systems are based on either an inhibition assay format (Figure 2b) or a sandwich format (Figure 1c), depending on the analyte to be determined (22–24). However, the use of labeled analyte analogs—compounds chemically similar to the analyte that competes for the antibody-binding sites—has also been reported (Figure 1b) (4, 25). Enzymes as labels. Immunosensor systems based on the use of enzymes as labels could follow the same basic principles as enzyme immunoassays (Figures 1b, 1c, and 2b) (5, 6). The sensor systems differ from “conventional” immunoassays in the degree of automation, the detection principle, the solid phase to which the antibody is bound, and the reusability of the biorecognition layer. Horseradish peroxidase (HRP), alkaline phosphatase (AP), and urease have been used as labels with chromogenic, fluoro-


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genic, and electrochemical detection (26). The sensor system protocol is similar to the corresponding microtiter plate assay—antibody immobilization, the addition of sample, the addition of enzyme tracer (both solutions may be premixed and applied simultaneously as in Figures 1b and 1c), the washing of the sensor, the addition and incubation of enzyme substrates, and detection. Automated devices include a final regeneration step, which allows reuse of the biospecific layer by removing the analyte and tracer that are bound to the immobilized antibodies. This can be achieved by adding solutions, such as ethylene glycol, urea, thiocyanate, or buffers of varying pH (8, 27). However, these regeneration protocols usually affect the binding capacity of the immobilized protein (8, 27–29), leading to a limited reusability of the system. To obtain a constant binding capacity in the system, antibodies are bound to proteins, such as protein A or protein G, allowing the removal by the regeneration protocol and a fresh loading before each assay (27–29). Automation is achieved with flow systems. Systems have been described that often are suitable for direct coupling to a sample line for on-line monitoring of a process (8). The most important element in these automated systems is the affinity reactor, which contains the immobilized antibodies. All solutions are pumped through or incubated in the reactor, where the affinity and enzymatic reactions occur, and the compound to be detected is generated. Various devices can serve as the reactor, including a flow-through column filled with beads; a glass capillary; a silicon-chip reactor; simple, conical propylene cells, in which antibody-containing electrodes are inserted; and microtiter wells with attached optical fibers for monitoring solution optical density or electrodes for determining the amount of electrochemically active products of the enzyme reactions (8, 27–29, 31–33). Even a membrane or the surface of an electrode can serve as affinity reactor (30, 31, 34, 35). Most of the systems described are used for the determination of pesticides in water, but there are also reports on their application to milk (progesterone), poultry (Salmonella typhimurium), and other food analytes (8, 30, 33, 34).

Sensor surface with immobilized antigen Sample and antibodies

(a)

Labelled antibodies and the sample

(b)

FIGURE 2.Immunosensor principles based on immobilized antigens, to which a mixture containing the sample and the antibody is added. (a) Direct sensing of antibody binding; (b) sensing of the antibody binding through the use of a label.

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Sensor systems for food analysis Low-molecular-weight compounds. The sensitivity of direct reading, label-free sensor systems is insufficient for determining low levels of low-molecular-weight analytes, such as antibiotics, hormones, or pesticides, with the simple assay format as illustrated in Figure 1a. Usually, the analysis is performed using the inhibition format (Figure 2a), in which the analyte or an analyte conjugate is immobilized on the transducer surface. For example, the inhibition format assay has successfully been used to detect triazines in water and sulfamethazin (SMZ) in milk (36). In the latter study, SMZ was covalently immobilized on the surface of a dextran-covered SPR chip. Polyclonal anti-SMZ antibodies were incubated with the milk sample for ~30 min before injection into an SPR system. Defatting the milk samples was necessary before analysis. A detection limit of 400 mV (41). These compounds cause high currents and block the electrode surface. However, these constituents do not interfere with photometric detection. Thus, an immunosensor device with HRP as the enzyme label and photometric (and not electrochemical) detection of HRP activity was designed, resulting in a fast analysis requiring only 8 min (40). This approach offers a strategy for real-time, on-line progesterone monitoring that reduces the time between milking and analysis (8). However, the authors did not suggest their system for routine application because the immunosensor could not be reused very often. Although the antibody-binding sites could be regenerated with thiocyanate, the background signal increased continuously due to residual enzyme on the surface of the reaction wells (8). Nevertheless, in milk samples, when the concentration of progesterone in the physiological range was meas-

y Sample

n2

Waveguide

n1

Substrate

Ey q

Laser

ns

Transmitted light detector

Reflected light detector

FIGURE 3.Measurements based on an optical waveguide structure. The parameters ns, n1, and n2 are the refractive indices of the substrate, waveguide, and solution, respectively, with n1 > n2 and n1 > ns; ␽ is the angle of incidence of the laser beam. A light beam traveling in such a material will be totally reflected at the interface, when its angle of incidence, ␽, is greater than a critical angle. The decay of the electric field as the distance from the waveguide surface, which is known as the evanescent field (Ey), increases, is shown schematically. Depending on the mode of operation, either a detector for the transmitted (e.g., input grating coupler devices) or the reflected (e.g., resonant mirror devices or grating coupler instruments operating in the reflection mode) light is used. The immobilized antibodies only indicate the reaction site of the biochemical recognition.

ured, the sensor showed a linear dependence. Though the 90% prediction interval was wide (0.1–2.5 ng/mL for a 0.5 ng/mL sample), six cows were followed through two reproductive cycles, and three out of four estrus events were detected. An automated affinity sensor system requires complete regeneration and full binding capacity for each new assay. One way to achieve this is with reversible antibody immobilization using protein A or G as an antibody-binding matrix. Protein A or G matrixes were developed for antibody purification in affinity chromatography, and they allow the multiple binding of antibodies even after elution steps. The antibodies can be removed from the system with the enzyme tracer, and fresh antibodies can be bound before each assay. Such systems have been used only with environmental samples and contained flow-through bead-packed columns or capillaries with a surplus of immobilized protein A or G (27–29). However, particle-filled affinity columns are likely to be blocked when systems are applied to food instead of water samples. Thus, open capillaries may be preferred. Moreover, due to the design of the flow systems, sample contact with the electrode could be avoided, thus reducing respective matrix effects (27 ). Determination of proteins. The detection of pathogenic bacteria in food and the medical diagnosis of infected patients is often based on the same principle of analyzing bacterial toxins. For example, the neurotoxin Botulinum toxin (BTX) from Clostridium botulinum, which is a high-molecular-weight (>150 kDa) protein, can be detected using bioassays, immunoassays, or immunosensors (42). Because of the relatively large size of the analyte, label-free sensor principles are applicable, and BTX antibodies were immobilized on the surface of SPR chips. Binding the BTX can be monitored in real time. Using 80 µL of sample and a flow rate of 5 µL/min has led to a reaction time of 16 min and a detection limit of 2.5 µg/mL for BTX. This is 250 times less sensitive than a sandwich immunoassay using the light addressable potentiometric sensor (LAPS)


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y

Ey

Waveguide or optical fiber

Filter

Laser

Detector

FIGURE 4.Schematic of a fluorescence immunosensor based on an inhibition-type assay. Excitation of the fluorescence occurs through the evanescent field of the waveguide (or optical fiber) by coupling laser light into the waveguide. Due to the exponential decay of the evanescent field (Ey), only fluorophores bound to the waveguide surface are excited. The emitted fluorescence intensity is recorded with a suitable detector (e.g., photomultiplier tube or charge-coupled detector camera).

principle with urease as the enzyme label. However, the latter requires a 2-h reaction time plus several manual filtration and washing steps and additions of reagents (42). Reduced sensitivity and problems from unspecific binding are common in label-free detection systems. Both problems may be decreased by using tracers, such as fluorescent-labeled or enzyme-labeled antibodies. Because of the size of the analytes, sandwich-type formats (Figure 1c) and competitive formats (Figure 1b) can be used. Fluorescence sensor systems can be rather simple because the fluorophore-labeled secondary antibody is pre-incubated with the sample, and the whole immunocomplex captured by the first antibody is immobilized on the waveguide (Figure 1c). For example, the simultaneous determination of staphylococcal enterotoxin B, MS2 bateriophage, and Bacillus globigii has been accomplished by incubating biotinylated antibodies on a NeutrAvidin-coated waveguide surface using a corresponding patterned flow cell (24). Flow channels were placed perpendicular to the antibody lines so that the sample could react with all the antibodies simultaneously. Assay time for all three analytes was 35 min. This investigation demonstrated that it is possible to simultaneously detect three very different analytes by a rather simple sensor setup. It was applied only to qualitative analysis of spiked serum samples to prove that the sensor works, even for complex mixtures. Moreover, previous investigations by the same group on one of the analytes (SEB) had already shown that the same principle (sandwich immunoassay with fluorescence detection by waveguides) can be applied to food samples, such as spiked ham extract. They found that, whereas the detection limit was almost unchanged compared to

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standard solutions in buffer, recovery rates of the quantitative analysis (which in practice is only of minor importance for the chosen analyte) were only 69% (43). No matrix effects were observed with an enzyme tracer-based system using electrochemical detection to determine African Swine Fever virus in serum from pigs (44). The system is very sensitive and can identify positive samples even after serum dilution. The high sensitivity is because of the particle-filled affinity column. Samples were premixed with biotinylated virus–antigen and HRP-labeled antivirus–antigen antibodies. The largest signals were observed when neither the virus–antigen nor the pig’s own antibodies against the virus–antigen were present. The analysis time of ~15 min was mainly due to the liquid transportation and washing steps. Determination of whole cells (bacteria). Because of the size of whole cells, label-free mass-sensitive detection methods, with specific antibodies immobilized on the transducer surface, should be ideal. However, acoustic sensors are of only limited reusability, and lower detection limits are in the 105- to 107cells/mL range (14). Moreover, immobilized antibodies may have only limited access to cell-surface antigens. Displacement assays for determining pathogens have been reported. In one study, heat-killed Listeria monocytogenes cells were immobilized on piezoelectric crystals (45). Adding antiListeria antibodies caused the formation of an immunocomplex, which was accompanied by a decrease in sensor frequency (Figure 5b). The presence of Listeria cells in the sample caused the displacement of the previously bound antibodies from the sensor surface, which was monitored by an increase of the resonant frequency. Alternatively, SPR has been used to detect bacteria and monitor bacterial interactions to immobilized affinity proteins (46, 47 ). These reports used commercially available SPR devices and standard sensor chips with a carboxymethylated dextran layer on top of the gold layer of the transducer. The dextran layer was activated according to the manufacturer’s protocol, and the binding proteins—antibodies, protein A, protein G , or fibronectin—were covalently bound (46, 47 ). Addition of the bacterial suspension, E. coli 0157:H7 or Staphylococcus aureus, led to a rapid increase in signal, which allowed the evaluation of kinetic data (Figure 1a) (46, 47 ). Although primary signals were not as large as expected from the size of the cells, they could be enhanced by the addition of a secondary antibody (sandwich-type sensor, Figure 1c) (46). This led to a detection limit for E. coli of 5–7 ⫻ 10 7 cfu/mL and a total assay time of ~25 min using 30 µL of the bacterial suspension. The sensor surface was regenerated by a short, 1-min pulse of 6 M guanidine-HCl (pH 1.0), and it could be reused for ~50 measurements. However, because sensitivity was not sufficient, the sensors were not applied to real food samples. Enzyme tracers are often used to im-

prove sensitivity. In some cases, (a) Sample the procedures resemble conventional ELISAs, in which the test solution is incubated with antibodies in conventional microRemoval of analyte analogues titer plates (e.g., anti-Salmonella [33]). The detection of alkaline phosphatase activity is done elec(b) trochemically and the assay had 4 a detection limit of 10 cfu/mL, Sample both in buffer and in various types of food. This was achieved, however, only when an 18-h preenrichment step was included. Removal of antibodies A detection limit of 119 cfu/test was reported for Salmonella using a commercially available system (34). Spiked chicken carcasses were filtered and cen- FIGURE 5.Displacement assays. trifuged. The residues were incu- (a) Immobilized antibodies preloaded with fluorescent-labeled, analyte analogs, or (b) immobilized with analyte analogs bated with a biotin- and a FITC- preloaded with antibodies. The displacement of labeled analyte analogs by the analyte can be detected either downlabeled antibody pair, leading to stream from the immobilized antibodies (signals increasing with analyte concentration) or through a fluorescence sensor a “sandwich” (Figure 1c); mixed (signals decreasing with analyte concentration). The displacement of antibodies in (b) is usually detected through labelfree detection principles, such as piezoelectric crystals. with streptavidin; and filtered through a biotinylated membrane, which captured the immunocomplex through the strepta- plate, beads, or the surface of an optical fiber or SPR chip (11, vidin bridge. A urease-labeled anti-FITC antibody was bound to 48–50). Fluorophores or digoxigenin are inserted to aid the dethe immunocomplex on the membrane. Detection was made termination of the corresponding products. For example, Listeria was detected after the amplification of a possible via a silicon, chip-based LAPS due to pH changes that characteristic 200-mer fragment of the flaA gene using a biotin- and a fluorescein amadite (FAM)-labeled primer (11). The double-stranded products were separated from the sample via streptavidin-coated beads. The strands were denatured, and the FAM-labeled single strands in the supernatant were removed and incubated with an optical fiber coated with short oligonucleotides, complementary to a sequence from the 200-mer fragment. Hybridization could be occurred when the added urea was hydrolyzed by urease. The monitored in real time. A similar principle is followed in “DNA cell suspension could be used without pre-enrichment; however, exact detection limits were not given. All quantitative data were chips” (51). Fluorescent dyes are incorpogiven with respect to a single test and not with respect to the rated into the PCR products via correoriginal samples. Thus, even sensor systems using sandwich-type spondingly labeled primers or nucleotides, formats with signal amplification through enzyme tracers cannot and the hybridization of these products to immobilized oligonucleotides is detected. improve detection limits significantly over label-free systems. The detection of particular DNA sequences is the current Spatially resolving difmethod for the identification of viruses and microorganisms. ferent immobilized oliEstablished methods of DNA analysis involve the amplification gonucleotides on a glass of target DNA sequences using PCR (8). The analysis of a PCR support allows for the simixture can be simplified by using labeled primers that yield la- multaneous detection of beled PCR products or labeled probes that hybridize to target several DNA sequences. sequences. Suitable labels include biotin, fluorescein, or other The reliability of the fluorophores (e.g., Cy5), and digoxigenin (11, 48, 49). Biotin analysis is enhanced, in is usually integrated to allow the immobilization of the oligonu- particular, for the idencleotide to a streptavidin-coated surface, such as a microtiter tification of certain mi-

Adding labels to immunosensors often leads to improved sensitivity and


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crobial strains. Such devices, however, have been applied mainly to medical diagnoses and pharmaceutical screening programs and not yet to food analysis. However, DNA chips for food analysis are under development, and applications will certainly appear soon. Because binding reactions can be monitored without the need for a label (Figures 1a and 2a), the hybridization of complementary oligonucleotide sequences can be monitored in real time using SPR or grating couplers without requiring a second label (50–53). Recently, PCR products from the verotoxin 2gene of E. coli 0157:H7 were analyzed using an SPR device with a specific oligonucleotide immobilized on the sensor surface (52). The major problem is that it is difficult to generate the single-stranded sequences that hybridize to the immobilized oligonucleotide. This problem was solved using a specific version of asymmetric PCR. With a 50-µL sample and a flow rate of 5 µL/min, as little as 1.5 ⫻ 10–7 M of the PCR product could be detected. A common feature of all these sensor systems is the analysis of PCR products with respect to their concentration. However, quantitative data for the original sample are usually not given due to PCR’s nonlinear behavior. Moreover, in sensor systems, PCR products are not investigated with respect to their correct size or length. As a result, it would not be known that PCR amplified the “wrong” DNA sequence, leading to the wrong product with, typically, a length different than the targeted product. PCR, only together with separation by gel electrophoresis, iden-

Direct reading, label-free sensors are not sensitive enough to measure low levels of antibiotics, tification by blotting, and probe hybridization, is used, for example, in the detection of genetically modified organisms.

Future directions A number of analytical procedures and suitable affinity sensor devices, some of which are even commercially available, have been reported. However, applications to food analysis are rare. A common feature of all affinity-based approaches is the need for known analytes. Only predetermined analytes can be detected by methods that require antibodies and known, relevant DNA sequences. Thus, all discussions of the value of immunoanalytical techniques in residue analysis, with regard to the specificity and cross-reactivity of antibodies, also relate to immunosensors.

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Similar aspects are also discussed in environmental analysis; however, water, in particular drinking water, is much easier to handle than food matrixes. Nevertheless, immunosensor systems show some distinct advantages over conventional immunoassays because they offer reduced analysis times for single samples, which is particularly important for on-site or on-line analysis, and they can be automated, which reduces experimental error and speeds analysis. However, automated devices appear to be rather bulky as long as conventional tubing, pumps, and valves are used. The affinity reactor is usually the smallest part in the setup. Thus, microtechnology could significantly contribute to more user-friendly devices. The transfer of immunoassays onto microchips has already been described for medical applications and may prove valuable for food analysis (54). Microtechnology leads to integrated systems with short flowing lines and thus further accelerates analysis. In addition, it offers the possibility of including capillary electrophoresis—a method that does not require the predefinition of the target analyte. At present, microanalytical systems struggle with “real-world” samples. This leads to additional requirements for sample pretreatment and sample purity. Matrix effects are another issue to consider; they can influence the antibody–antigen interaction or the detection (unspecific binding). Because the antibody–antigen interaction is the basis of all immunoanalytical approaches, it is not possible to give a strategy for those interferences. The easiest approach is a dilution of the sample, which also reduces the concentration of the interferants. This, however, is only possible if the sensor system is sensitive enough to meet the required concentration ranges (e.g., maximum residue levels) after sample dilution. If matrix effects arise from interactions of sample constituents with the transducer surface (e.g., blocking the surface), a treatment such as coverage with a repelling polymer can reduce the problem. Alternatively, the detection strategy can be changed to one that avoids direct contact between the transducer and sample, to a photometric system or a method that uses a label. Unfortunately, most studies are not aimed at finding the best detector for a given problem in the food industry. Moreover, all the steps to improve a system are seldom reported. DNA-based analytical methods, including gene sensors, will probably find the most rapid application. DNA analysis seems to be the only method for detecting genetic modifications and is the most sensitive approach for detecting microorganisms. However, these methods require the extraction of DNA from the sample and, at present, specialized personnel and equipment. Before they can be used routinely, these challenges will need to be addressed. In the short term, microtiter-plate-based tests could provide routine DNA analysis. In the long run, microtechnology might be able to integrate DNA extraction, amplification, and analysis. As this article shows, there are already examples of affinity

sensors that detect contaminants in food, usually with little sample pretreatment, providing faster analysis times and automation. However, there are areas of food chemistry that do not yet benefit from these sensors. For example, the quantitative multiresidue analysis of numerous related compounds requires an antibody for each analyte, and pathogenic microorganisms require time-consuming precultivation steps because immunosensors cannot detect single cells. Thus, the application of affinity sensors to food analysis is not yet fully exploited, and many more collaborations between analytical chemists and food chemists are needed. Ursula Bilitewski heads the Bioanalytical Group within the Division of Bioprocess Engineering at the German Research Center for Biotechnology. Her research interests include bioanalytical tools for describing the state of cells in a fermenter or as a consequence of incubation with bioactive compounds. Address correspondence to Bilitewski at Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124, Braunschweig, Germany ([email protected]).

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