G ENERAL D ESCRIPTIONS Biosensors can provide rapid and continuous in situ, measurements for on-site or

4.3.1 G ENERAL D ESCRIPTIONS Biosensors can provide rapid and continuous in situ, measurements for on-site or

remote monitoring. Several different transducer types such as optical, electrochem- ical, piezoelectric, and thermometric can be employed. Immunosensors contain specific antibodies for biological recognition and a transducer that converts the binding event of antibody to antigen to a physical signal.

Antibodies may be immobilized on membranes, magnetic beads, optical fibers; or embedded in polymers, or placed on metallic surfaces. In some types of sensors, such as those employing surface plasmon resonance (SPR), evanescent waves, or piezoelectric crystals, the binding of antigen and antibody can be detected directly. With other transducers, an indicator molecule (either a labeled antigen or labeled secondary antibody) is required. An indicator may be fluorescent or it may be an enzyme that alters a colorimetric or fluorescent signal or produces a change in pH affecting the electrochemical parameters.

Optical biosensors may measure fluorescence, fluorescence transfer, fluores- cence lifetime, time-resolved fluorescence, color (either by absorbance or reflect- ance), evanescent waves, or an SPR response. Optical immunosensors are very rapid as they detect the antigen=antibody binding directly without requiring labeled reagents. Data in real time can be generated with devices applied to continuous

Immunoassays

TABLE 4.3 Examples of Biosensors for Determining Pesticides and Metabolites in Biological and Environmental Samples

and Analyte

Sensor Type

Matrix

Range or LOD References Biosensors

Atrazine

[17] Atrazine

Electrochemical immunosensor

Orange juice

0.03 nmol=L

[69] Carbaryl, paraoxon

Electrochemical magnetoimmunosensor

Orange juice

0.027 nmol=L

Disposable screen-printed thick-film electrode

Milk

20 mg=L (carbaryl)

1 mg=L (paraoxon)

Carbofuran

Flow-injection electrochemical biosensor

Fruits, vegetables,

1 –100 nmol

dairy products

Dichlorvos

[72] Dichlorvos

Flow-injection calorimetric biosensor

Water

1 mg=L

[73] Fenthion

Electrochemical biosensor

Wheat

0.02 mg=g

[74] Malathion, dimethoate

Dipstick electrochemical immunosensor

Water

0.01 –1000 mg=L

Malathion: 0.01 –0.59 mM [14] Dimethoate: 8.6 –520 mM OP pesticides

Amperometric biosensor

Vegetables

Fluorescence-based fiber-optic sensor

Buffer

1 –800 mM (paraoxon) [75]

2 –400 mM (DFP a )

OP pesticides and nerve agents

Electrochemical sensor using

Water

1 –3 ng=mL

nanoparticles (ZrO 2 ) as selective sorbents

OP pesticides and nerve agents

Flow-injection amperometric biosensor using

carbon nanotube-modified glassy carbon electrode

Thiabendazole

[16] a Diisopropyl phosphorofluoridate (a nerve agent).

Fluorescence-based optical sensor

Citrus fruits

0.09 mg=kg

110 Analysis of Pesticides in Food and Environmental Samples monitoring situations such as effluent or runoff measurements from hazardous or

agricultural waste streams. Optical immunosensors based on SPR employ immobil- ized specific antibody on a metal layer. When antigen binds, there is a minute change in the refractive index that is measured as a shift in the angle of total absorption of light incident on the metal layer. This technique was used to develop an SPR sensor to detect atrazine at 0.05 ppb in drinking water [78].

Fiber optic biosensors are based on the transmission of light along silica glass or plastic fibers. The advantages of fiber optic sensors are numerous: they are not subjected to electrical interference; a reference electrode is not needed; immobilized reagent does not have to be in contact with the optical fiber; they can be miniaturized; and they are highly stable. A major advantage of these sensors is that they can respond simultaneously to more than one analyte and are useful for remotely monitoring hazardous environments or municipal water supplies.

Electrochemical biosensors offer the advantages of being effective with colored or opaque matrices and do not contain light-sensitive components. In an immuno- sensor format, the binding of antigen to antibody is visualized as an electrical signal. The response may be coupled to signal amplification systems such as an enzyme- conjugated secondary antibody, conferring very low detection limits. Amperometric sensors measure current when an electroactive species is oxidized or reduced at the electrode. Potentiometric sensors detect the change in charge of an antibody when it binds to an antigen. Organophosphorus pesticides may be detected in a number of ways including potentiometric or amperometric methods. In both of these cases, enzymes such as organophosphorus hydrolase or urease may be employed. Depen- dent on the structure of the analyte, the release of hydrogen ions can either be measured via a pH change or a p-nitrophenol (PNP) group may be produced to give a redox compound for an electron shuttle.

Piezoelectric crystals are nonmetallic minerals (usually quartz), which conduct electricity and which develop a surface charge when stretched or compressed along an axis. The crystals vibrate when placed in an alternating electric field. The frequency of the vibration is a function of the mass of the crystal. Antibodies can be immobilized to the surface of piezoelectric crystals and the new vibrational frequency determined as a baseline measurement. The binding of analyte to the immobilized antibody alters the mass and vibrational frequency of the antibody –crystal system. This change in vibration can be measured to determine the amount of analyte detected.

Electroconductive polymer sensors have a specific antibody embedded in a conducting polymer matrix such as polypyrrole. When an analyte binds to the antibody, the ions in the matrix are less free to move, which decreases the ability of the polymer to conduct current. A reagentless electrochemical DNA biosensor has been reported using an Au –Ag nanocomposite material adsorbed to a conducting polymeric polypyrrole [79]. The detection limit was 5.0 3 10 10 M of target oligo-

nucleotides with a response time of 3 s. The integration of nanotechnology and sensor development will provide new analytical platforms and formats. Although new designs may first appear for clinical applications, these advancements will favorably impact the development of sensors for environmental measurements. Table 4.3 summarizes several pesticide biosensors that have been reported for various monitoring situations [14,16,17,69 –77].

Immunoassays and Biosensors 111