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(a ) 520 580 490 550
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Figure 7.8. (a) Glucose biosensor; (b) spectral properties of FITC and Rh. [Reprinted, with permission, from D. Meadows and J. S. Schultz, Talanta 35 (No. 2), 1988, 145 150. FiberOptic Biosensors Based on Fluorescence Energy Transfer . Copyright # 1988 Pergamon Journals Ltd.]
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The emission intensity of FITC is monitored at 520 nm as the analytical signal. When FITC-labeled dextran is bound to Rh ConA, the rhodamine label quenches the 520-nm emission, so that the intensity measured is at a minimum. In the presence of the analyte (glucose), which diffuses across the dialysis ber at the tip of the optical ber, FITC dextran is displaced from Rh ConA by glucose, and emission intensity at 520-nm increases. This sensor allows glucose quantitation at concentrations up to $ 5 mM, and has a relatively slow response due to equilibration of the macromolecular reactions. 7.3.6. Piezoelectric Sensor for Nucleic Acid Detection10 This device employs single-stranded poly(adenylic acid) [poly(A)] as the chemical recognition agent. This species selectively recognizes its complementary polymer, poly(U), through hybridization to form a double-stranded nucleic acid. The poly(A) is immobilized onto the activated surface of a quartz piezoelectric crystal, which is a mass-sensitive transducer. Electric dipoles are generated in anisotropic materials (such as quartz crystals) subjected to mechanical stress, and these materials will
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then undergo dimensional changes, or oscillations, in the presence of an electric eld. The frequency of oscillation is of the order of megahertz (MHz, 106 Hz). This resonant frequency decreases if mass is added to the surface of the crystal; in fact, it has been shown that the resonance frequency is inversely proportional to the surface mass. These transducers are sensitive to humidity, so that measurements must be made under dry or reproducibly humid conditions; in situ
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Figure 7.9. Change in resonant oscillation frequency of a piezoelectric transducer (a) with, and (b) without poly(A) immobilized to the surface. Step 1 is surface modi cation with copolymer, Step 2 is poly(A) immobilization, and Step 3 is hybridization to target poly(U).10 [Reprinted, with permission, from N. C. Fawcett, J. A. Evans, L.-C. Chien, and N. Flowers, Anal. Lett. 21, 1988, 1099 1114. Nucleic Acid Hybridization Detected by Piezoelectric Resonance . Copyright # 1988 by Marcel Dekker, Inc.]
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measurements in solution are not possible because of viscous damping of the oscillations. The nucleic acid sensor was prepared by rst derivatizing the quartz surface with a 3:1 styrene acrylic acid copolymer. Poly(A) was then covalently immobilized onto pendant carboxylic acid groups by amide bond formation with the amino groups on the adenine base. Hybridization occurred during incubation with poly(U). Following each of the three steps, the sensor was rinsed and dried and the resonant frequency of oscillation was measured. Prior to any treatment, the quartz crystals exhibited a resonant frequency of 9 MHz. Because each step in the surface treatment involved the addition of mass to the crystal surface, a frequency decrease was expected after each step. Figure 7.9 shows the actual frequency changes measured for a sensor prepared as described above, as well as for a sensor prepared with no poly(A) included in the second step (i.e., a control sensor). While this sensor employed only a model nucleic acid probe (poly(A)), it demonstrates the principle of detection of the complementary sequence, the analyte poly(U). Figure 7.9(a) shows that a frequency decrease of 600 Hz was observed following the hybridization step, Step 3, and that this decrease was not observed with a control sensor that did not possess surface-bound poly(A). The sensor exhibits a relatively small frequency change that is superimposed on a large initial resonance frequency (9 MHz), and so S/N is low; in addition, dif culties associated with ex situ measurements at constant humidity preclude the use of this device for practical DNA or RNA detection. 7.3.7. Enzyme Thermistors11 A variety of enzyme-based biosensors have been tested using thermistors as the means of signal transduction. Thermistors are similar to electrical resistors, but possess highly temperature dependent resistance values. Since many enzymatic
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