The Development of Sol-Gel-Derived Array Biosensors

• Main Authors: Hsiao-chung Tsai, 蔡曉忠
• Other Authors: Ruey-an Doong
• Format: Others
• Language: en_US
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/43700185585291982755

博士 === 國立清華大學 === 原子科學系 === 92 === Biosensor is one of the fastest growing analytical techniques for the determination of various targets in a wide variety of fields. Although it is originally designed for the single analyte detection, more and more efforts have been made to develop an array biosensor since the simultaneous determination of multi-analytes is highly demanded. Sol-gel process has been proven to be a remarkable technique for enzyme immobilization and biosensor fabrication. However, the quick gelation problem, detachment of sol-gel from the solid surface, and cracking phenomenon occurred by shrinkage events are major challenges in the development of a sol-gel-derived biosensor array. In this study, a novel sol-gel process suitable for screen-printing technique to fabricate a sol-gel-derived array biosensor containing different enzymes for the simultaneous determination of various analyes is developed. Automated pin-printing and fluorescence detection systems by LabVIEW software are also constructed for the fabrication and detection of the sol-gel-derived array biosensor. To evaluate the applicability and versatility of the developed system, analytical performances in terms of dynamic range, limits of detection (LODs), reproducibility, and standard addition of real samples for the determination of pH, urea, acetylcholine, creatinine, glucose, uric acid, and enzyme inhibitors (heavy metals) were studied. Moreover, a mathematical model for the simulation of buffer capacity was developed to predict the changes in pH and the fluorescence intensity emitted from the hydrolase-based biosensors. The optimization results showed that a 20 % sol solution could minimize the leaching rate of the encapsulated biomolecules. Addition of 0.5 % polyvinylalcohol and 10 % glycerol prevented the cracking events and prolonged the gelation time, respectively. After encapsulating macromolecules, the surface morphology of the sol-gel altered obviously and the surface mean roughness of sol-gel increased from 0.207 to 2.636 nm. High specific surface area was also observed in non-doped sol-gel (515.3 m2/g), depicting the large capacity of sol-gel structures for enzyme loading. The optimized sol-gel process was used to fabricate 1-D and 2-D array biosensor successfully. The 1-D system encapsulated with urease has high sensitivity to Cu(II) and Cd(II) and an analytical range of 10 — 230 uM with a detection limit of 10 uM was achieved. The 2-D array biosensor also showed good analytical performances, which the LODs and dynamic ranges for urea and acetylcholine (ACh) were down to sub-mM level and 2 to 3 orders of magnitude, respectively. The capability of this array biosensor for simultaneous determination of multi-analytes was also demonstrated. Based on the developed array biosensor, two applications including the screening of enzyme inhibitors (heavy metals) and the detection of enzyme substrates were examined. In the determination of Cd(II), Cu(II), and Hg(II), an analytical range between 10 nM to 100 mM was achieved. The standard addition experiment also elucidated the possibility of the array biosensor for real sample analyses. For the substrates detection, the LODs was in the were in the range of 2.5 — 80 uM, while the dynamic ranges were between 2 — 3 orders of magnitude for urea, creatinine, glucose and uric acid, which are satisfactory for the clinical diagnosis. Moreover, a serum sample spiked with various concentrations of substrates could be also determined using the array biosensor and the results obtained were consistent with spectrometric methods, revealing the applicability of the developed array biosensor for the real sample determination. The simulated results are in good agreement with the experimental data of pH and urea, depicting the validation of the proposed algorithm in describing the behavior of pH-based biosensors. Moreover, the developed model also demonstrated the excellent capability in predicting the actual behavior of the reaction in the biosensor system. The non-equilibrium of the CD-based biosensor due to an insufficient reaction time (10 min) and the underestimate of ACh concentration in consequence of its hygroscopic property could be successfully tracked by this model. In summary, the array biosensor exhibits advantages of low cost, easy preparation, usage of trace amount enzyme, and fulfills the requirement of high reproducibility (RSD < 10 %, n = 45), rapid (50 measurements within 15 — 35 min), and trace amount (15 uL) analyses. It not only gives applicability of simultaneous multi-analyte determinations but also provides possibility of real sample analyses in a highly parallel way. Also, the constructed model enables to predict sensor responses and fits well with the experimental results, reflecting the success of this model in predicting the behavior of pH-based biosensors.