大多疾病在潛伏期甚至是前期時病徵並非顯而易見，因此無法被輕易的檢測出來，當病發時候往往會導致治療的效率大幅下降。如糖尿病視網膜病變(Diabetic Retinopathy, DR)是糖尿病患者常見的併發症，其主要是因為視網膜上的血管長期處於高血糖的環境導致血管產生病變，是目前造成國人失明的主要原因。目前臨床上常使用螢光血管照影(Fluorescence Angiography, FA)來檢測該疾病，其僅提供血管結構的資訊，但是觀察血管的結構變化是必須靠醫師的臨床經驗去做判斷，並沒有一個定量的依據。當患者察覺有異狀發生時，大多已經步入後期的增生性糖尿病視網膜病變(Proliferative Diabetic Retinopathy, PDR)，因此必須接受手術的治療才能回復部分視力，倘若能即早發現即早治療，則大部分的患者可以避免手術並且維持良好的視力狀況。因此本研究發展一套低濃度的檢測平臺，期望能夠在早期就能檢測定量到疾病，並落實非侵入式的檢測方式，所採用的非侵入式檢體為淚液，從文獻中可以知道淚脂質運載蛋白(Lipocalin 1, LCN1)在淚液中是糖尿病視網膜病變的指標性蛋白(Biomarker)。利用微珠式三明治免疫分析(Bead-based Sandwiched Immunoassay)使抗體抗原間產生專一性鍵結，加上快速光電動圖紋法(Rapid Electrokinetic Patterning, REP)這項光電動技術，讓帶有指標性蛋白的粒子濃縮聚集(Concentration)以提升螢光訊號。目前正常人的淚液中所含的LCN1的濃度約為3150 μg/mL，而糖尿病視網膜病變的指標性蛋白LCN1濃度會隨著疾病越趨嚴重而濃度越高，而本研究的偵測極限可到達15 pg/mL，對於糖尿病視網膜病變能夠達到早期偵測的目的。為了讓檢測平台對疾病的精準度有所提升，利用技術去分選(Sorting)不同大小的粒子，針對糖尿病視網膜病變淚液中不同的指標性蛋白進行交叉比對的檢測。這個檢測平台也可以應用到不同的疾病及檢體，舉凡唾液或尿液，根據不同疾病在不同樣本中所因應到的指標性蛋白去做免疫分析及訊號放大便可以檢測到該疾病。最後，期望此檢測平台可以輔助現今無法定量的眼科醫療現況，並落實居家照護，達到為糖尿病患者視力把關的目的。

Disease diagnosis in incubation period and early stages faces various challenges. As diseases elicit adverse effects upon diagnosis in late stages, therapeutic efficiency is reduced. For example, diabetic retinopathy (DR) is a common complication and major cause of vision loss among patients with diabetes mellitus. In clinical diagnosis, DR is examined through fluorescence angiography (FA). Although this method provide information on blood vessel conformation, the detection of changes in blood vessel conformation without a quantitative basis is dependent on a physician’s clinical experiences. In most cases, a patient’s condition has progressed to proliferative diabetic retinopathy (PDR) when abnormalities are detected. As a consequence, patients must undergo surgery to recover their eyesight. Therefore, signs and symptoms should be detected and treated in early stages to help prevent vision loss. In this research, a low-concentration non-invasive detection platform was developed to detect and quantify DR biomarkers in early stages. Tears were used as a biological sample because they contain DR biomarkers, such as lipocalin 1 (LCN1). A specific bond between an antigen and an antibody was formed through the bead-based sandwiched immunosensing. Rapid electrokinetic patterning (REP), an optoelectrokinetic technology, was employed to focus on immunosensed particles and enhance the fluorescence signal. Results revealed that the LCN1 concentration in the tears of normal control subjects was 3150 μg/mL. The LCN1 concentration in the tears of patients with DR increased with disease severity. The detection limit was 15 pg/mL, and this limit was sufficient to detect DR in early stages. REP was also applied to sort different particle sizes, cross-match different biomarkers in the tears of patients with DR, and enhance the accuracy of the detection platform. Our results suggested that the proposed detection platform could be used to diagnose different diseases and specimens and to detect DR by using biomarkers present in various specimens, such as saliva and urine, through immunosensing and signal amplification. The detection platform could also be utilized to address the quantification limitations in point-of-care ophthalmologic diagnosis. Further research should be conducted to protect the vision of diabetic mellitus patients.

[1] J. D. Wulfkuhle, L. A. Liotta, and E. F. Petricoin, “Proteomic applications for the early detection of cancer,” Nature Reviews Cancer, vol. 3, no. 4, pp. 267-275, 2003.
[2] M.-Y. Hsu, C.-Y. Yang, W.-H. Hsu, K.-H. Lin, C.-Y. Wang, Y.-C. Shen, Y.-C. Chen, S.-F. Chau, H.-Y. Tsai, and C.-M. Cheng, “Monitoring the VEGF level in aqueous humor of patients with ophthalmologically relevant diseases via ultrahigh sensitive paper-based ELISA,” Biomaterials, vol. 35, no. 12, pp. 3729-3735, 2014.
[3] D. I. Walsh III, G. J. Sommer, U. Y. Schaff, P. S. Hahn, G. J. Jaffe, and S. K. Murthy, “A centrifugal fluidic immunoassay for ocular diagnostics with an enzymatically hydrolyzed fluorogenic substrate,” Lab on a Chip, vol. 14, no. 15, pp. 2673-2680, 2014.
[4] T. Y. Wong, R. Simó, and P. Mitchell, “Fenofibrate–a potential systemic treatment for diabetic retinopathy?,” American Journal of Ophthalmology, vol. 154, no. 1, pp. 6-12, 2012.
[5] N. Cheung, P. Mitchell, and T. Y. Wong, “Diabetic retinopathy,” Lancet, vol. 376, no. 9735, pp. 124-136, Jul 10, 2010.
[6] D. S. Fong, J. D. Cavallerano, L. Aiello, F. L. Ferris, T. W. Gardner, R. Klein, G. L. King, G. Blankenship, and A. D. Assoc, “Diabetic retinopathy,” Diabetes Care, vol. 26, no. 1, pp. 226-229, Jan, 2003.
[7] D. S. W. Ting, G. C. M. Cheung, and T. Y. Wong, “Diabetic retinopathy: global prevalence, major risk factors, screening practices and public health challenges: a review,” Clinical & Experimental Ophthalmology, 2016.
[8] M. R. K. Mookiah, U. R. Acharya, C. K. Chua, C. M. Lim, E. Ng, and A. Laude, “Computer-aided diagnosis of diabetic retinopathy: A review,” Computers in Biology and Medicine, vol. 43, no. 12, pp. 2136-2155, 2013.
[9] R. J. Antcliff, M. R. Stanford, D. S. Chauhan, E. M. Graham, D. J. Spalton, J. S. Shilling, and J. Marshall, “Comparison between optical coherence tomography and fundus fluorescein angiography for the detection of cystoid macular edema in patients with uveitis,” Ophthalmology, vol. 107, no. 3, pp. 593-599, 2000.
[10] N. Rodríguez-Marco, J. Andonegui-Navarro, E. Compains-Silva, A. Rebollo-Aguayo, D. Aliseda-Pérez-de-Madrid, and M. Aranguren-Laflin, “OCT y fototoxicidad macular,” Archivos de la Sociedad Española de Oftalmología, vol. 83, no. 4, pp. 267-272, 2008.
[11] L. Zhou, and R. W. Beuerman, “Tear analysis in ocular surface diseases,” Progress in Retinal and Eye Research, vol. 31, no. 6, pp. 527-550, Nov, 2012.
[12] F. J. Holly, “Formation and rupture of the tear film,” Experimental Eye Research, vol. 15, no. 5, pp. 515-525, 1973.
[13] N. V. U. Hohenstein-Blaul, S. Funke, and F. H. Grus, “Tears as a source of biomarkers for ocular and systemic diseases,” Experimental Eye Research, vol. 117, pp. 126-137, Dec, 2013.
[14] R. Schnetler, W. Gillan, and G. Koorsen, “Immunological and antimicrobial molecules in human tears: a review and preliminary report,” African Vision and Eye Health, vol. 71, no. 3, pp. 123-132, 2012.
[15] J. Tiffany, "The normal tear film," Surgery for the Dry Eye, pp. 1-20, 2008.
[16] J. L. Prabha, “Tear Secretion-A Short Review,” Journal of Pharmaceutical Sciences and Research, vol. 6, no. 3, pp. 155-157, 2014.
[17] T. Sitaramamma, S. Shivaji, and G. N. Rao, “HPLC analysis of closed, open, and reflex eye tear proteins,” Indian Journal of Ophthalmology, vol. 46, no. 4, pp. 239, 1998.
[18] J. Murube, L. Murube, and A. Murube, “Origin and types of emotional tearing,” European Journal of Ophthalmology, vol. 9, no. 2, pp. 77-84, 1999.
[19] N. M. Farandos, A. K. Yetisen, M. J. Monteiro, C. R. Lowe, and S. H. Yun, “Contact lens sensors in ocular diagnostics,” Advanced Healthcare Materials, vol. 4, no. 6, pp. 792-810, 2015.
[20] O. Schirmer, “Studien zur physiologie und pathologie der tränenabsonderung und tränenabfuhr,” Graefe's Archive for Clinical and Experimental Ophthalmology, vol. 56, no. 2, pp. 197-291, 1903.
[21] A. Posa, L. Bräuer, M. Schicht, F. Garreis, S. Beileke, and F. Paulsen, “Schirmer strip vs. capillary tube method: non-invasive methods of obtaining proteins from tear fluid,” Annals of Anatomy-Anatomischer Anzeiger, vol. 195, no. 2, pp. 137-142, 2013.
[22] K. B. Bjerrum, and J. U. Prause, “Collection and concentration of tear proteins studied by SDS gel electrophoresis Presentation of a new method with special reference to dry eye patients,” Graefe's Archive for Clinical and Experimental Ophthalmology, vol. 232, no. 7, pp. 402-405, 1994.
[23] C. Baleriola-Lucas, M. Fukuda, M. Willcox, D. Sweeney, and B. Holden, “Fibronectin concentration in tears of contact lens wearers,” Experimental Eye Research, vol. 64, no. 1, pp. 37-43, 1997.
[24] T. Sitaramamma, S. Shivaji, and G. N. Rao, “Effect of storage on protein concentration of tear samples,” Current Eye Research, vol. 17, no. 10, pp. 1027-1035, 1998.
[25] K. Strimbu, and J. A. Tavel, “What are biomarkers?,” Current Opinion in HIV and AIDS, vol. 5, no. 6, pp. 463, 2010.
[26] R. Mayeux, “Biomarkers: potential uses and limitations,” NeuroRx, vol. 1, no. 2, pp. 182-188, 2004.
[27] O. K. Gasymov, A. R. Abduragimov, P. Prasher, T. N. Yusifov, and B. J. Glasgow, “Tear lipocalin: Evidence for a scavenging function to remove lipids from the human corneal surface,” Investigative Ophthalmology & Visual Science, vol. 46, no. 10, pp. 3589-3596, Oct, 2005.
[28] R. J. Fullard, and D. M. Kissner, “Purification of the isoforms of tear specific prealbumin,” Current Eye Research, vol. 10, no. 7, pp. 613-628, 1991.
[29] E. Csosz, P. Boross, A. Csutak, A. Berta, F. Toth, S. Poliska, Z. Torok, and J. Tozser, “Quantitative analysis of proteins in the tear fluid of patients with diabetic retinopathy,” Journal of Proteomics, vol. 75, no. 7, pp. 2196-2204, Apr 3, 2012.
[30] I. A. Darwish, “Immunoassay methods and their applications in pharmaceutical analysis: basic methodology and recent advances,” Int J Biomed Sci, vol. 2, no. 3, pp. 217-235, 2006.
[31] D. J. Winzor, and J. De Jersey, “Biospecific interactions: Their quantitative characterization and use for solute purification,” Journal of Chromatography B: Biomedical Sciences and Applications, vol. 492, pp. 377-430, 1989.
[32] N. Fujimoto, “Poly (ethylene glycol)-conjugated Alkylamines as Novel Blocking Reagents for Immunoassays,” Citeseer, 2009.
[33] K.-C. Wang, A. Kumar, S. J. Williams, N. G. Green, K. C. Kim, and H.-S. Chuang, “An optoelectrokinetic technique for programmable particle manipulation and bead-based biosignal enhancement,” Lab on a Chip, vol. 14, no. 20, pp. 3958-3967, 2014.
[34] T. Heyduk, “Measuring protein conformational changes by FRET/LRET,” Current Opinion in Biotechnology, vol. 13, no. 4, pp. 292-296, 2002.
[35] R. M. Clegg, “Fluorescence resonance energy transfer,” Current Opinion in Biotechnology, vol. 6, no. 1, pp. 103-110, 1995.
[36] S. A. Hussain, “An introduction to fluorescence resonance energy transfer (FRET),” Syed Arshad Hussain, 2009.
[37] H.-S. Chuang, H.-Y. Ku, F.-T. Li, A. Kumar, J.-C. Wang, and K.-C. Wang, "Optoelectrokinetic Manipulation for Cell Analysis," Essentials of Single-Cell Analysis, pp. 159-194: Springer, 2016.
[38] R. Pethig, and G. H. Markx, “Applications of dielectrophoresis in biotechnology,” Trends in Biotechnology, vol. 15, no. 10, pp. 426-432, 1997.
[39] R. Krishnan, and M. J. Heller, “An AC electrokinetic method for enhanced detection of DNA nanoparticles,” Journal of Biophotonics, vol. 2, no. 4, pp. 253-261, 2009.
[40] P. R. Gascoyne, and J. Vykoukal, “Particle separation by dielectrophoresis,” Electrophoresis, vol. 23, no. 13, pp. 1973, 2002.
[41] A. Ashkin, J. Dziedzic, J. Bjorkholm, and S. Chu, “Observation of a single-beam gradient force optical trap for dielectric particles,” Optics Letters, vol. 11, no. 5, pp. 288-290, 1986.
[42] A. Ashkin, “Optical trapping and manipulation of neutral particles using lasers,” Proceedings of the National Academy of Sciences, vol. 94, no. 10, pp. 4853-4860, 1997.
[43] J. K. Valley, A. T. Ohta, H.-Y. Hsu, S. L. Neale, A. Jamshidi, and M. C. Wu, “Optoelectronic tweezers as a tool for parallel single-cell manipulation and stimulation,” IEEE Transactions on Biomedical Circuits and Systems, vol. 3, no. 6, pp. 424, 2009.
[44] J.-S. Kwon, S. P. Ravindranath, A. Kumar, J. Irudayaraj, and S. T. Wereley, “Opto-electrokinetic manipulation for high-performance on-chip bioassays,” Lab on a Chip, vol. 12, no. 23, pp. 4955-4959, 2012.
[45] P. Y. Chiou, Z. Chang, and M. C. Wu, "A novel optoelectronic tweezer using light induced dielectrophoresis.", Optical MEMS, pp. 8-9, 2003.
[46] S. J. Williams, A. Kumar, and S. T. Wereley, “Electrokinetic patterning of colloidal particles with optical landscapes,” Lab on a Chip, vol. 8, no. 11, pp. 1879-1882, 2008.
[47] N. J. Van Haeringen, “The Lacrimal System,” Adult crying: A Biopsychosocial Approach, vol. 3, pp. 19, 2001.
[48] B. Beigi, D. Gupta, Y. H. L. Luo, M. Saldana, I. Georgalas, G. Kalantzis, and N. El‐Hindy, “Punctal function in lacrimal drainage: the ‘pipette sign’and functional ectropion,” Clinical and Experimental Optometry, vol. 98, no. 4, pp. 366-369, 2015.
[49] Breustedt DA, C.L., Skerra A. 2009; Available from: http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=77136.
[50] M. J. Fischer, “Amine coupling through EDC/NHS: a practical approach,” Surface Plasmon Resonance: Methods and Protocols, pp. 55-73, 2010.
[51] Z. Grabarek, and J. Gergely, “Zero-length crosslinking procedure with the use of active esters,” Analytical Biochemistry, vol. 185, no. 1, pp. 131-135, 1990.
[52] J. V. Staros, R. W. Wright, and D. M. Swingle, “Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions,” Analytical Biochemistry, vol. 156, no. 1, pp. 220-222, 1986.
[53] M. Bruchez, M. Moronne, P. Gin, S. Weiss, and A. P. Alivisatos, “Semiconductor nanocrystals as fluorescent biological labels,” Science, vol. 281, no. 5385, pp. 2013-2016, 1998.
[54] W. C. Chan, and S. Nie, “Quantum dot bioconjugates for ultrasensitive nonisotopic detection,” Science, vol. 281, no. 5385, pp. 2016-2018, 1998.
[55] B. Bhushan, Encyclopedia of nanotechnology: Springer The Netherlands, 2012.
[56] J. Tate, and G. Ward, “Interferences in immunoassay,” The Clinical Biochemist Reviews, vol. 25, no. 2, pp. 105, 2004.
[57] T. Lakshmipriya, M. Fujimaki, S. C. Gopinath, K. Awazu, Y. Horiguchi, and Y. Nagasaki, “A high-performance waveguide-mode biosensor for detection of factor IX using PEG-based blocking agents to suppress non-specific binding and improve sensitivity,” Analyst, vol. 138, no. 10, pp. 2863-2870, 2013.
[58] A. Jain, R. Liu, Y. K. Xiang, and T. Ha, “Single-molecule pull-down for studying protein interactions,” Nature protocols, vol. 7, no. 3, pp. 445-452, 2012.
[59] Y. Nagasaki, H. Kobayashi, Y. Katsuyama, T. Jomura, and T. Sakura, “Enhanced immunoresponse of antibody/mixed-PEG co-immobilized surface construction of high-performance immunomagnetic ELISA system,” Journal of Colloid and Interface Science, vol. 309, no. 2, pp. 524-530, 2007.
[60] R. Stuchell, J. Feldman, R. Farris, and I. Mandel, “The effect of collection technique on tear composition,” Investigative Ophthalmology & Visual Science, vol. 25, no. 3, pp. 374-377, 1984.