臺灣博碩士論文加值系統

在本篇研究中，我們開發了利用白金奈米粒子催化生成金奈米粒子偵測凝血藥物與因子的方法，首先，我們利用白金奈米粒子偵測魚精蛋白與肝素，當白金奈米粒子表面吸附了魚精蛋白後會降低還原四氯金酸成金奈米的催化進而使金奈米的粒徑增加，而當溶液中含有魚精蛋白和肝素時，帶正電的魚精蛋白會與帶負電的肝素產生鍵結，將魚精蛋白帶離肝素表面，使其抑制催化效果回復。以此我們便可藉由生成金奈米的吸收波峰的位移作為定量的依據，在魚精蛋白部分，我們設計的檢測系統之線性區間為8 nM 到 80 nM；在肝素部分，我們設計的檢測系統之線性區間為1 nM 到 10 nM。
接著，我們利用白金奈米粒子偵測凝血酶，當白金奈米粒子表面吸附了纖維蛋白原與凝血酶反映後所生成的纖維蛋白後，會使白金奈米粒子產生聚集的現象，因催化表面積的降低，造成催化能力的下降，使得催化出來的金奈米粒子粒徑較大，藉由上述的現象便可透過比色法以及吸收波長位移來進行凝血酶的檢測與定量。凝血酶的檢測方法的線性範圍為0.01 nM~10 nM，偵測極限為0.0003 nM。

In this study, we developed a new method for rapid detection of anticoagulant drugs and clotting factor through the mediated-platinum nanoparticles (PtNPs) as the catalyst to formation the AuNPs in the presence the H2O2.
The catalytic mechanism is the protamine could adsorb on the surface of PtNPs to inhibit the catalyst ability that reducing HAuCl4 to Au0 and further enlarging to AuNPs. In contrast, when the solution containing the protamine was incubated the heparin, negative charge macromolecules, the positive charge of protamine would bind the heparin to inhibit the protamine to adsorb on the surface of PtNPs.Overall, the concept of detection is to use the constant concentration of protamine mixed with heparin; excessive protamine would adsorb on the PtNPs to decrease the catalyst properties of PtNPs to generate the versatile size of AuNPs. By the way, the formation of different size of AuNPs with different SPR that was depended on the degree of inhabitation of PtNPs. We use the abovementioned phenomenon to develop the sensor through recording the wavelength position of AuNPs to monitor the concentration of protamine and heparin. The detection method for protamine and heparin showed the linear range from 8 nM to 80 nM and 1 nM to 10 nM, the detection limit was 2.67 nM and 0.3 nM, respectively.
The mechanism of thrombin detection is to use the combination between fibrinogen and thrombin to coated on platinum nanoparticles and produce aggregation phenomenon. When we add thrombin, it will make the combination of thrombin ,fibrinogen and platinum nanoparticles to reduce the peroxidation catalyst properties of platinum nanoparticles .gold nanoparticles tend to form a larger size, and change the absorption of the wavelength. We use the above phenomenon to develop a colorimetric and absorption of the wavelength to thrombin detection and quantification of the sensor. The detection method for thrombin showed linear range from 0.01 nM to10 nM with detection limit of 0.003 nM.

目錄 I
圖目錄 III
表目錄 VI
1.前言 1
1-1奈米粒子簡介 1
1-2成核理論 2
1-3局部表面電漿共振與文獻探討 6
1-4魚精蛋白與肝素簡介 12
1-5凝血酶簡介 15
1-6紙感測器 18
1-7實驗動機 19
2實驗部分 20
2-1實驗藥品 20
2-2蛋白質分析物 22
2-3實驗儀器 24
2-3藥品配置及合成 26
2-4實驗過程 27
3實驗結果及討論 30
3-1以白金奈米粒子催化生成金奈米粒子偵測魚精蛋白與肝素 30
3-1-1鑑定白金奈米粒子與魚精蛋白與肝素之間的關係 30
3-1-2以白金奈米粒子催化四氯金酸生成金奈米粒子 34
3-1-3PVP-PtNPs、魚精蛋白與肝素實驗條件最佳化 38
3-1-4以白金奈米粒子催化還原四氯金酸定量魚精蛋白 42
3-1-5以白金奈米粒子催化還原四氯金酸定量肝素 45
3-1-6利用白金奈米粒子催化魚精蛋白與肝素之選擇性探討 48
3-1-7真實樣品 50
3-1-8紙感測器定量 51
3-2以白金奈米粒子催化生成金奈米粒子偵測凝血酶 52
3-2-1鑑定白金奈米粒子與血纖維蛋白酶和凝血酶之間的關係 52
3-2-2以白金奈米粒子催化四氯金酸生成金奈米粒子 54
3-2-3白金奈米粒子、血纖維蛋白酶與凝血酶實驗條件最佳化 57
3-2-4以白金奈米粒子催化生成金奈米粒子定量凝血酶 62
3-2-5以白金奈米粒子催化生成金奈米粒子定量凝血酶選擇性探討 64
4結論 65
5參考文獻 66

1. Saunders, A. E.; Sigman, M. B.; Korgel, B. A., Growth Kinetics and Metastability of Monodisperse Tetraoctylammonium Bromide Capped Gold Nanocrystals. The Journal of Physical Chemistry B 2004, 108, 193-199.
2. Pepperberg, I. M., Grey parrot (Psittacus erithacus) numerical abilities: Addition and further experiments on a zero-like concept. J Comp Psychol 2006, 120, 1-11.
3. Aikens, C. M., Electronic Structure of Ligand-Passivated Gold and Silver Nanoclusters. The Journal of Physical Chemistry Letters 2011, 2, 99-104.
4. Hu, Y.; Cheng, H.; Zhao, X.; Wu, J.; Muhammad, F.; Lin, S.; He, J.; Zhou, L.; Zhang, C.; Deng, Y.; Wang, P.; Zhou, Z.; Nie, S.; Wei, H., Surface-Enhanced Raman Scattering Active Gold Nanoparticles with Enzyme-Mimicking Activities for Measuring Glucose and Lactate in Living Tissues. ACS Nano 2017, 11, 5558-5566.
5. Gao, Z.; Xu, M.; Hou, L.; Chen, G.; Tang, D., Irregular-shaped platinum nanoparticles as peroxidase mimics for highly efficient colorimetric immunoassay. Analytica Chimica Acta 2013, 776, 79-86.
6. Sharma, P.; Brown, S.; Walter, G.; Santra, S.; Moudgil, B., Nanoparticles for bioimaging. Advances in Colloid and Interface Science 2006, 123-126, 471-485.
7. Cheng, N.; Tian, J.; Liu, Q.; Ge, C.; Qusti, A. H.; Asiri, A. M.; Al-Youbi, A. O.; Sun, X., Au-Nanoparticle-Loaded Graphitic Carbon Nitride Nanosheets: Green Photocatalytic Synthesis and Application toward the Degradation of Organic Pollutants. ACS Applied Materials & Interfaces 2013, 5, 6815-6819.
8. Xu, X.; Wang, J.; Jiao, K.; Yang, X., Colorimetric detection of mercury ion (Hg2+) based on DNA oligonucleotides and unmodified gold nanoparticles sensing system with a tunable detection range. Biosensors and Bioelectronics 2009, 24, 3153-3158.
9. Martinson, C. A.; Reddy, K. J., Adsorption of arsenic(III) and arsenic(V) by cupric oxide nanoparticles. Journal of Colloid and Interface Science 2009, 336, 406-411.
10. Chen, W.; Fang, X.; Li, H.; Cao, H.; Kong, J., A Simple Paper-Based Colorimetric Device for Rapid Mercury(II) Assay. Scientific Reports 2016, 6, 31948.
11. Wang, Y.; He, J.; Liu, C.; Chong, W. H.; Chen, H., Thermodynamics versus Kinetics in Nanosynthesis. Angew Chem Int Ed 2015, 54, 2022-2051.
12. Thanh, N. T. K.; Maclean, N.; Mahiddine, S., Mechanisms of Nucleation and Growth of Nanoparticles in Solution. Chemical Reviews 2014, 114, 7610-7630.
13. Polte, J.; Ahner, T. T.; Delissen, F.; Sokolov, S.; Emmerling, F.; Thünemann, A. F.; Kraehnert, R., Mechanism of Gold Nanoparticle Formation in the Classical Citrate Synthesis Method Derived from Coupled In Situ XANES and SAXS Evaluation. Journal of the American Chemical Society 2010, 132, 1296-1301.
14. Ma, Z.; Sui, S.-F., Naked-Eye Sensitive Detection of Immunoglubulin G by Enlargement of Au Nanoparticles In Vitro. Angew Chem Int Ed 2002, 41, 2176-2179.
15. Willets, K. A.; Van Duyne, R. P., Localized Surface Plasmon Resonance Spectroscopy and Sensing. Annual Review of Physical Chemistry 2007, 58, 267-297.
16. Link, S.; El-Sayed, M. A., Size and Temperature Dependence of the Plasmon Absorption of Colloidal Gold Nanoparticles. The Journal of Physical Chemistry B 1999, 103, 4212-4217.
17. Woźniak, A.; Malankowska, A.; Nowaczyk, G.; Grześkowiak, B. F.; Tuśnio, K.; Słomski, R.; Zaleska-Medynska, A.; Jurga, S., Size and shape-dependent cytotoxicity profile of gold nanoparticles for biomedical applications. J Mater Sci Mater Med 2017, 28, 92.
18. Sönnichsen, C.; Reinhard, B. M.; Liphardt, J.; Alivisatos, A. P., A molecular ruler based on plasmon coupling of single gold and silver nanoparticles. Nat Biotechnol 2005, 23, 741-745.
19. Li, L.; Li, B.; Cheng, D.; Mao, L., Visual detection of melamine in raw milk using gold nanoparticles as colorimetric probe. Food Chem 2010, 122, 895-900.
20. Chen, Z.; Tan, L.; Hu, L.; Zhang, Y.; Wang, S.; Lv, F., Real Colorimetric Thrombin Aptasensor by Masking Surfaces of Catalytically Active Gold Nanoparticles. ACS Applied Materials & Interfaces 2016, 8, 102-108.
21. Gadzekpo, V. P. Y.; Bühlmann, P.; Xiao, K. P.; Aoki, H.; Umezawa, Y., Development of an ion-channel sensor for heparin detection. Anal Chim Acta 2000, 411, 163-173.
22. Volpi, N.; Maccari, F.; Suwan, J.; Linhardt, R. J., Electrophoresis for the analysis of heparin purity and quality. Electrophoresis 2012, 33, 1531-1537.
23. Yan, H.; Wang, H.-F., Turn-on Room Temperature Phosphorescence Assay of Heparin with Tunable Sensitivity and Detection Window Based on Target-Induced Self-Assembly of Polyethyleneimine Capped Mn-Doped ZnS Quantum Dots. Anal Chem 2011, 83, 8589-8595.
24. Saad, O. M.; Leary, J. A., Heparin Sequencing Using Enzymatic Digestion and ESI-MSn with HOST:  A Heparin/HS Oligosaccharide Sequencing Tool. Anal Chem 2005, 77, 5902-5911.
25. Thirupathi, P.; Park, J.-Y.; Neupane, L. N.; Kishore, M. Y. L. N.; Lee, K.-H., Pyrene Excimer-Based Peptidyl Chemosensors for the Sensitive Detection of Low Levels of Heparin in 100% Aqueous Solutions and Serum Samples. ACS Applied Materials & Interfaces 2015, 7, 14243-14253.
26. Kim, D.-H.; Park, Y. J.; Jung, K. H.; Lee, K.-H., Ratiometric Detection of Nanomolar Concentrations of Heparin in Serum and Plasma Samples Using a Fluorescent Chemosensor Based on Peptides. Anal Chem 2014, 86, 6580-6586.
27. Zhong, Z.; Anslyn, E. V., A Colorimetric Sensing Ensemble for Heparin. J Am Chem Soc 2002, 124, 9014-9015.
28. Wang, S.; Chang, Y.-T., Combinatorial Synthesis of Benzimidazolium Dyes and Its Diversity Directed Application toward GTP-Selective Fluorescent Chemosensors. J Am Chem Soc 2006, 128, 10380-10381.
29. Qi, H.; Zhang, L.; Yang, L.; Yu, P.; Mao, L., Anion-Exchange-Based Amperometric Assay for Heparin Using Polyimidazolium as Synthetic Receptor. Anal Chem 2013, 85, 3439-3445.
30. Shvarev, A.; Bakker, E., Response Characteristics of a Reversible Electrochemical Sensor for the Polyion Protamine. Anal Chem 2005, 77, 5221-5228.
31. Huo, H. Y.; Luo, H. Q.; Li, N. B., Electrochemical sensor for heparin based on a poly(thionine) modified glassy carbon electrode. Microchimica Acta 2009, 167, 195.
32. Li, L.; Liang, Y.; Liu, Y., Designing of molecularly imprinted polymer-based potentiometric sensor for the determination of heparin. Anal Biochem 2013, 434, 242-246.
33. Chen, Y.; Liang, R. N.; Qin, W., Potentiometric sensor for sensitive and selective detection of heparin. Chin Chem Lett 2012, 23, 233-236.
34. Rengaraj, A.; Haldorai, Y.; Hwang, S. K.; Lee, E.; Oh, M.-H.; Jeon, T.-J.; Han, Y.-K.; Huh, Y. S., A protamine-conjugated gold decorated graphene oxide composite as an electrochemical platform for heparin detection. Bioelectrochemistry 2019, 128, 211-217.
35. Li, S.; Gao, M.; Wang, S.; Hu, R.; Zhao, Z.; Qin, A.; Tang, B. Z., Light up detection of heparin based on aggregation-induced emission and synergistic counter ion displacement. Chem Commun 2017, 53, 4795-4798.
36. Wang, M.; Zhang, D.; Zhang, G.; Zhu, D., The convenient fluorescence turn-on detection of heparin with a silole derivative featuring an ammonium group. Chem Commun 2008, 4469-4471.
37. Pu, K.-Y.; Liu, B., A Multicolor Cationic Conjugated Polymer for Naked-Eye Detection and Quantification of Heparin. Macromolecules 2008, 41, 6636-6640.
38. Cheng, T.-T.; Yao, J.-L.; Gao, X.; Sun, W.; Shi, S.; Yao, T.-M., A new fluorescence “switch on” assay for heparin detection by using a functional ruthenium polypyridyl complex. Analyst 2013, 138, 3483-3489.
39. Dai, Q.; Liu, W.; Zhuang, X.; Wu, J.; Zhang, H.; Wang, P., Ratiometric Fluorescence Sensor Based on a Pyrene Derivative and Quantification Detection of Heparin in Aqueous Solution and Serum. Anal Chem 2011, 83, 6559-6564.
40. Sun, W.; Bandmann, H.; Schrader, T., A Fluorescent Polymeric Heparin Sensor. Chemistry – A European Journal 2007, 13, 7701-7707.
41. Hu, L.; Liao, H.; Feng, L.; Wang, M.; Fu, W., Accelerating the Peroxidase-Like Activity of Gold Nanoclusters at Neutral pH for Colorimetric Detection of Heparin and Heparinase Activity. Anal Chem 2018, 90, 6247-6252.
42. Ma, X.; Kou, X.; Xu, Y.; Yang, D.; Miao, P., Colorimetric sensing strategy for heparin assay based on PDDA-induced aggregation of gold nanoparticles. Nanoscale Advances 2019, 1, 486-489.
43. Murray, D. J.; Brosnahan, W. J.; Pennell, B.; Kapalanski, D.; Weiler, J. M.; Olson, J., Heparin detection by the activated coagulation time: A comparison of the sensitivity of coagulation tests and heparin assays. Journal of Cardiothoracic and Vascular Anesthesia 1997, 11, 24-28.
44. Raymond, P. D.; Ray, M. J.; Callen, S. N.; Marsh, N. A., Heparin monitoring during cardiac surgery. Part 1: validation of whole-blood heparin concentration and activated clotting time. Perfusion 2003, 18, 269-276.
45. You, J.-G.; Liu, Y.-W.; Lu, C.-Y.; Tseng, W.-L.; Yu, C.-J., Colorimetric assay of heparin in plasma based on the inhibition of oxidase-like activity of citrate-capped platinum nanoparticles. Biosensors Bioelectron 2017, 92, 442-448.
46. Lin, K. Y.; Kwong, G. A.; Warren, A. D.; Wood, D. K.; Bhatia, S. N., Nanoparticles That Sense Thrombin Activity As Synthetic Urinary Biomarkers of Thrombosis. ACS Nano 2013, 7, 9001-9009.
47. Shen, G.; Zhang, H.; Yang, C.; Yang, Q.; Tang, Y., Thrombin Ultrasensitive Detection Based on Chiral Supramolecular Assembly Signal-Amplified Strategy Induced by Thrombin-Binding Aptamer. Anal Chem 2017, 89, 548-551.
48. Rezaie, A. R., Tryptophan 60-D in the B-insertion loop of thrombin modulates the thrombin-antithrombin reaction. Biochemistry 1996, 35, 1918-24.
49. Chobanian, H. R.; Pio, B.; Guo, Y.; Shen, H.; Huffman, M. A.; Madeira, M.; Salituro, G.; Terebetski, J. L.; Ormes, J.; Jochnowitz, N.; Hoos, L.; Zhou, Y.; Lewis, D.; Hawes, B.; Mitnaul, L.; O’Neill, K.; Ellsworth, K.; Wang, L.; Biftu, T.; Duffy, J. L., Improved Stability of Proline-Derived Direct Thrombin Inhibitors through Hydroxyl to Heterocycle Replacement. ACS Medicinal Chemistry Letters 2015, 6, 553-557.
50. Deng, Z. J.; Liang, M.; Toth, I.; Monteiro, M. J.; Minchin, R. F., Molecular interaction of poly(acrylic acid) gold nanoparticles with human fibrinogen. ACS Nano 2012, 6, 8962-9.
51. Zauner, G.; Hoffmann, M.; Rapp, E.; Koeleman, C. A.; Dragan, I.; Deelder, A. M.; Wuhrer, M.; Hensbergen, P. J., Glycoproteomic analysis of human fibrinogen reveals novel regions of O-glycosylation. J Proteome Res 2012, 11, 5804-14.
52. Hu, J.; Zheng, P. C.; Jiang, J. H.; Shen, G. L.; Yu, R. Q.; Liu, G. K., Electrostatic interaction based approach to thrombin detection by surface-enhanced Raman spectroscopy. Anal Chem 2009, 81, 87-93.
53. Wermes, C.; Siebke, A.; Wilhelm, C.; Sykora, K.; Glueer, S.; Ganser, A.; Prondzinski, M., Expression of Protease-activated Receptors in Neuroblastoma Cells. 2003; pp 305-311.
54. Zhao, D.; Peng, Y.; Xu, L.; Zhou, W.; Wang, Q.; Guo, L., Liquid-Crystal Biosensor Based on Nickel-Nanosphere-Induced Homeotropic Alignment for the Amplified Detection of Thrombin. ACS Applied Materials & Interfaces 2015, 7, 23418-23422.
55. Palekar, R. U.; Myerson, J. W.; Schlesinger, P. H.; Sadler, J. E.; Pan, H.; Wickline, S. A., Thrombin-Targeted Liposomes Establish a Sustained Localized Anticlotting Barrier against Acute Thrombosis. Mol Pharm 2013, 10, 4168-4175.
56. Fuglestad, B.; Gasper, P. M.; McCammon, J. A.; Markwick, P. R. L.; Komives, E. A., Correlated Motions and Residual Frustration in Thrombin. The Journal of Physical Chemistry B 2013, 117, 12857-12863.
57. Fan, J.; Zhang, Y.; Chang, X.; Zhang, B.; Jiang, D.; Saito, M.; Li, Z., Antithrombotic and fibrinolytic activities of methanolic extract of aged sorghum vinegar. J Agric Food Chem 2009, 57, 8683-7.
58. Hsieh, C.-L.; Peng, C.-H.; Chyau, C.-C.; Lin, Y.-C.; Wang, H.-E.; Peng, R. Y., Low-Density Lipoprotein, Collagen, and Thrombin Models Reveal that Rosemarinus officinalis L. Exhibits Potent Antiglycative Effects. J Agric Food Chem 2007, 55, 2884-2891.
59. Römhildt, L.; Pahlke, C.; Zörgiebel, F.; Braun, H.-G.; Opitz, J.; Baraban, L.; Cuniberti, G., Patterned Biochemical Functionalization Improves Aptamer-Based Detection of Unlabeled Thrombin in a Sandwich Assay. ACS Applied Materials & Interfaces 2013, 5, 12029-12035.
60. Liu, X.; Hua, X.; Fan, Q.; Chao, J.; Su, S.; Huang, Y.-Q.; Wang, L.; Huang, W., Thioflavin T as an Efficient G-Quadruplex Inducer for the Highly Sensitive Detection of Thrombin Using a New Föster Resonance Energy Transfer System. ACS Applied Materials & Interfaces 2015, 7, 16458-16465.
61. Liu, X.; Shi, L.; Hua, X.; Huang, Y.; Su, S.; Fan, Q.; Wang, L.; Huang, W., Target-induced conjunction of split aptamer fragments and assembly with a water-soluble conjugated polymer for improved protein detection. ACS Appl Mater Interfaces 2014, 6, 3406-12.
62. Wang, Y.; Liu, B., Conjugated polyelectrolyte-sensitized fluorescent detection of thrombin in blood serum using aptamer-immobilized silica nanoparticles as the platform. Langmuir 2009, 25, 12787-93.
63. Zhang, D.; Zhao, Q.; Zhao, B.; Wang, H., Fluorescence anisotropy reduction of allosteric aptamer for sensitive and specific protein signaling. Anal Chem 2012, 84, 3070-4.
64. Xue, L.; Zhou, X.; Xing, D., Sensitive and homogeneous protein detection based on target-triggered aptamer hairpin switch and nicking enzyme assisted fluorescence signal amplification. Anal Chem 2012, 84, 3507-13.
65. Tennico, Y. H.; Hutanu, D.; Koesdjojo, M. T.; Bartel, C. M.; Remcho, V. T., On-chip aptamer-based sandwich assay for thrombin detection employing magnetic beads and quantum dots. Anal Chem 2010, 82, 5591-7.
66. Wang, Y.; Bao, L.; Liu, Z.; Pang, D. W., Aptamer biosensor based on fluorescence resonance energy transfer from upconverting phosphors to carbon nanoparticles for thrombin detection in human plasma. Anal Chem 2011, 83, 8130-7.
67. Huang, D.; Niu, C.; Li, Z.; Ruan, M.; Wang, X.; Zeng, G., A sensitive strategy for label-free and time-resolved fluorescence assay of thrombin using Tb-complex and unmodified gold nanoparticles. Analyst 2012, 137, 5607-13.
68. Zhang, L.; Lei, J.; Liu, L.; Li, C.; Ju, H., Self-Assembled DNA Hydrogel as Switchable Material for Aptamer-Based Fluorescent Detection of Protein. Anal Chem 2013, 85, 11077-11082.
69. Lin, B.; Sun, Q.; Liu, K.; Lu, D.; Fu, Y.; Xu, Z.; Zhang, W., Label-Free Colorimetric Protein Assay and Logic Gates Design Based on the Self-assembly of Hemin-Graphene Hybrid Nanosheet. Langmuir 2014, 30, 2144-2151.
70. Chang, C.-C.; Chen, C.-P.; Wu, T.-H.; Yang, C.-H.; Lin, C.-W.; Chen, C.-Y. Gold Nanoparticle-Based Colorimetric Strategies for Chemical and Biological Sensing Applications Nanomaterials (Basel, Switzerland) PubMed. 2019,45,1265-1278
71. Cunningham, J. C.; Brenes, N. J.; Crooks, R. M., Paper Electrochemical Device for Detection of DNA and Thrombin by Target-Induced Conformational Switching. Anal Chem 2014, 86, 6166-6170.
72. Lin, J.-H.; Huang, K.-H.; Zhan, S.-W.; Yu, C.-J.; Tseng, W.-L.; Hsieh, M.-M., Inhibition of catalytic activity of fibrinogen-stabilized gold nanoparticles via thrombin-induced inclusion of nanoparticle into fibrin: Application for thrombin sensing with more than 104-fold selectivity. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2019, 210, 59-65.
73. Vidal, E.; Lorenzetti, A. S.; Lista, A. G.; Domini, C. E., Micropaper-based analytical device (μPAD) for the simultaneous determination of nitrite and fluoride using a smartphone. Microchem J 2018, 143, 467-473.
74. Wu, S.; Li, D.; Gao, Z.; Wang, J., Controlled etching of gold nanorods by the Au(III)-CTAB complex, and its application to semi-quantitative visual determination of organophosphorus pesticides. Microchimica Acta 2017, 184, 4383-4391.
75. Gabriel, E. F. M.; Garcia, P. T.; Cardoso, T. M. G.; Lopes, F. M.; Martins, F. T.; Coltro, W. K. T., Highly sensitive colorimetric detection of glucose and uric acid in biological fluids using chitosan-modified paper microfluidic devices. Analyst 2016, 141, 4749-4756.