本研究成功建構一套結合微流體晶片與重組酶聚合酶擴增（Recombinase Polymerase Amplification, RPA）及側向流動免疫層析（Lateral Flow Dipstick, LFD）之核酸檢測平台，針對泌尿道感染常見UPEC中高度相關之毒力基因 papGII，實現快速、高靈敏度與高特異性的定性與半定量核酸檢測。檢測系統經由引子濃度、反應溫度、時間及鎂離子濃度等參數優化，提升擴增效率與顯色穩定性；同時建立以 RGB色彩空間中 G+B 值總和作為顏色強度指標之影像分析方法，成功建立 105-108 CFU/mL 範圍內呈色強度與菌株濃度間的標準曲線，相關係數高達 0.988，證明系統具備良好半定量分析能力。
此外，本研究所提出之系統檢測極限達104 CFU/mL，對 105 CFU/mL以上樣本可穩定產生明顯顯色訊號;特異性實驗顯示僅papGII基因陽性菌株產生顯色反應，陰性菌株無非特異性訊號。為驗證本研究所提出之檢測系統可行性，在盲測20例未知菌株樣本中，檢測結果為9例真陽性、1例偽陽性、10例真陰性，整體準確率達95%，無偽陰性，且半定量回收率平均為91.15%，其中85%樣本（17例）回收率介於80%至120%間，顯示良好的定量穩定性。人工尿液樣本測試亦展現優異表現，7 例真陽性及3例真陰性均準確判讀，充分展現系統於臨床樣本中之穩定性及準確性。
綜上所述，本研究所開發之微流體核酸檢測平台結合RPA-LFD技術，具備快速、靈敏、特異與操作簡便之優點，適用於泌尿道感染病原菌papGII 基因之現場快速篩檢，未來有潛力擴展至臨床尿液篩檢及其他病原基因快速檢測領域，為提升感染性疾病之早期診斷與即時治療提供有效工具。

This study successfully developed a nucleic acid detection platform combining microfluidic chips with recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) for rapid, highly sensitive, and specific qualitative and semi-quantitative detection of the papGII virulence gene in uropathogenic Escherichia coli (UPEC), a common urinary tract infection pathogen. The system was optimized by adjusting primer concentration, reaction temperature, time, and magnesium ion concentration to enhance amplification efficiency and color stability. An image analysis method using the sum of green and blue (G+B) values in the RGB color space was established, generating a standard curve with a correlation coefficient of 0.988 over the range of 105 to 108 CFU/mL. The detection limit reached 104 CFU/mL, producing clear color signals for samples above 105 CFU/mL. Specificity tests showed color reactions only in papGII-positive strains, with no nonspecific signals in negatives. Blind testing of 20 unknown samples achieved 95% accuracy with no false negatives, and an average semi-quantitative recovery rate of 91.15%, demonstrating good quantitative stability. Testing with artificial urine samples also showed high accuracy and stability. This platform offers a fast, sensitive, specific, and user-friendly tool for onsite rapid screening of papGII in urinary tract infections, with potential for clinical urine screening and broader pathogen gene detection applications.

[1] T. Weichhart, M. Haidinger, W. Hörl, and M. Säemann, "Current concepts of molecular defence mechanisms operative during urinary tract infection," European Journal of Clinical Investigation, vol. 38, pp. 29-38, 2008.
[2] B. Foxman, "Epidemiology of urinary tract infections: incidence, morbidity, and economic costs," The American Journal of Medicine, vol. 113, no. 1, pp. 5-13, 2002.
[3] H. Rubi, G. Mudey, and R. Kunjalwar, "Catheter-associated urinary tract infection (CAUTI)," Cureus, vol. 14, no. 10, 2022.
[4] T. Luu and F. S. Albarillo, "Asymptomatic bacteriuria: prevalence, diagnosis, management, and current antimicrobial stewardship implementations," The American Journal of Medicine, vol. 135, no. 8, pp. e236-e244, 2022.
[5] J. Le, G. G. Briggs, A. McKeown, and G. Bustillo, "Urinary tract infections during pregnancy," Annals of Pharmacotherapy, vol. 38, no. 10, pp. 1692-1701, 2004.
[6] R. Kaur and R. Kaur, "Symptoms, risk factors, diagnosis and treatment of urinary tract infections," Postgraduate Medical Journal, vol. 97, no. 1154, pp. 803-812, 2021.
[7] M. E. Terlizzi, G. Gribaudo, and M. E. Maffei, "UroPathogenic Escherichia coli (UPEC) infections: virulence factors, bladder responses, antibiotic, and non-antibiotic antimicrobial strategies," Frontiers in Microbiology, vol. 8, p. 1566, 2017.
[8] M. Santos, M. Mariz, I. Tiago, J. Martins, S. Alarico, and P. Ferreira, "A review on urinary tract infections diagnostic methods: Laboratory-based and point-of-care approaches," Journal of Pharmaceutical and Biomedical Analysis, vol. 219, p. 114889, 2022.
[9] C.-C. Tseng, J.-J. Wu, H.-L. Liu, J.-M. Sung, and J.-J. Huang, "Roles of host and bacterial virulence factors in the development of upper urinary tract infection caused by Escherichia coli," American Journal of Kidney Diseases, vol. 39, no. 4, pp. 744-752, 2002.
[10] A. L. Flores-Mireles, J. N. Walker, M. Caparon, and S. J. Hultgren, "Urinary tract infections: epidemiology, mechanisms of infection and treatment options," Nature Reviews Microbiology, vol. 13, no. 5, pp. 269-284, 2015.
[11] L. Mody and M. Juthani-Mehta, "Urinary tract infections in older women: a clinical review," Jama, vol. 311, no. 8, pp. 844-854, 2014.
[12] S. Whelan, B. Lucey, and K. Finn, "Uropathogenic Escherichia coli (UPEC)-associated urinary tract infections: the molecular basis for challenges to effective treatment," Microorganisms, vol. 11, no. 9, p. 2169, 2023.
[13] R. Feynman, "Infinitesimal Machinery. Lecture reprinted in the Journal of Micro electromechanical Systems," ed: Springer publications. Elsevier publications, 1993.
[14] A. Manz, N. Graber, and H. M. Widmer, "Miniaturized total chemical analysis systems: a novel concept for chemical sensing," Sensors and Actuators B: Chemical, vol. 1, no. 1-6, pp. 244-248, 1990.
[15] J. W. Judy, "Microelectromechanical systems (MEMS): fabrication, design and applications," Smart Materials and Structures, vol. 10, no. 6, p. 1115, 2001.
[16] N. Azizipour, R. Avazpour, D. H. Rosenzweig, M. Sawan, and A. Ajji, "Evolution of biochip technology: A review from lab-on-a-chip to organ-on-a-chip," Micromachines, vol. 11, no. 6, p. 599, 2020.
[17] H. Widmer, "Trends in industrial analytical chemistry," TrAC Trends in Analytical Chemistry, vol. 2, no. 1, pp. VIII-X, 1983.
[18] E. Verpoorte and N. F. De Rooij, "Microfluidics meets MEMS," Proceedings of the IEEE, vol. 91, no. 6, pp. 930-953, 2003.
[19] Y. C. Lim, A. Z. Kouzani, and W. Duan, "Lab-on-a-chip: a component view," Microsystem Technologies, vol. 16, pp. 1995-2015, 2010.
[20] B. J. Kirby, Micro-and nanoscale fluid mechanics: transport in microfluidic devices. Cambridge university press, 2010.
[21] Z. Liao, J. Wang, P. Zhang, Y. Zhang, Y. Miao, S. Gao, Y. Deng, and L. Geng, "Recent advances in microfluidic chip integrated electronic biosensors for multiplexed detection," Biosensors and Bioelectronics, vol. 121, pp. 272-280, 2018.
[22] X. Sun, J. J. Wan, and K. Qian, "Designed microdevices for in vitro diagnostics," Small Methods, vol. 1, no. 10, p. 1700196, 2017.
[23] T. M. Hooton, "Uncomplicated urinary tract infection," New England Journal of Medicine, vol. 366, no. 11, pp. 1028-1037, 2012.
[24] S. Salvatore, S. Salvatore, E. Cattoni, G. Siesto, M. Serati, P. Sorice, and M. Torella, "Urinary tract infections in women," European Journal of Obstetrics & Gynecology and Reproductive Biology, vol. 156, no. 2, pp. 131-136, 2011.
[25] E. J. Dielubanza and A. J. Schaeffer, "Urinary tract infections in women," Medical clinics, vol. 95, no. 1, pp. 27-41, 2011.
[26] C. M. Kodner and E. K. T. Gupton, "Recurrent urinary tract infections in women: diagnosis and management," American Family Physician, vol. 82, no. 6, pp. 638-643, 2010.
[27] E. E. Moore, S. E. Hawes, D. Scholes, E. J. Boyko, J. P. Hughes, and S. D. Fihn, "Sexual intercourse and risk of symptomatic urinary tract infection in post-menopausal women," Journal of General Internal Medicine, vol. 23, pp. 595-599, 2008.
[28] J. Rukweza, P. Nziramasanga, M. Gidiri, C. Haruzivishe, and B. Pedersen, "Antibiotic susceptibility of bacterial strains causing asymptomatic bacteriuria in pregnancy: a cross-sectional study in Harare, Zimbabwe," MOJ Immunol, vol. 6, no. 1, p. 00184, 2018.
[29] A. R. Marra, T. Z. S. Camargo, P. Gonçalves, A. M. C. B. Sogayar, D. F. Moura Jr., L. R. Guastelli, C. A. C. Alves Rosa, E. da S. Victor, O. F. P. dos Santos, and M. B. Edmond, "Preventing catheter-associated urinary tract infection in the zero-tolerance era," American Journal of Infection Control, vol. 39, no. 10, pp. 817-822, 2011.
[30] C. Elliott and A. Justiz-Vaillant, "Nosocomial infections: a 360-degree review," International Biological and Biomedical Journal, vol. 4, no. 2, pp. 72-81, 2018.
[31] Z. Zeng, J. Zhan, K. Zhang, H. Chen, and S. Cheng, "Global, regional, and national burden of urinary tract infections from 1990 to 2019: an analysis of the global burden of disease study 2019," World Journal of Urology, vol. 40, no. 3, pp. 755-763, 2022.
[32] W. E. Stamm and S. R. Norrby, "Urinary tract infections: disease panorama and challenges," The Journal of Infectious Diseases, vol. 183, no. Supplement_1, pp. S1-S4, 2001.
[33] S. M. Schappert and E. A. Rechtsteiner, "Ambulatory medical care utilization estimates for 2007," Vital and Health Statistics. Series 13, Data from the National Health Survey, no. 169, pp. 1-38, 2011.
[34] N. D. Stone, M. S. Ashraf, J. Calder, C. J. Crnich, K. Crossley, P. J. Drinka, C. V. Gould, M. Juthani Mehta, E. Lautenbach, M. Loeb, T. MacCannell, P. N. Malani, L. Mody, J. Mylotte, L. E. Nicolle, M. C. Roghmann, S. J. Schweon, A. E. Simor, P. W. Smith, K. B. Stevenson, S. F. Bradley, and the Society for Healthcare Epidemiology Long Term Care Special Interest Group, "Surveillance definitions of infections in long-term care facilities: revisiting the McGeer criteria," Infection Control & Hospital Epidemiology, vol. 33, no. 10, pp. 965-977, 2012.
[35] J. Hrbacek, P. Cermak, and R. Zachoval, "Current antibiotic resistance trends of uropathogens in Central Europe: Survey from a Tertiary hospital urology department 2011–2019," Antibiotics, vol. 9, no. 9, p. 630, 2020.
[36] C. M. Shih, C. L. Chang, M. Y. Hsu, J. Y. Lin, C. M. Kuan, H. K. Wang, C. T. Huang, M. C. Chung, K. C. Huang, C. E. Hsu, C. Y. Wang, Y. C. Shen, and C. M. Cheng, "based ELISA to rapidly detect Escherichia coli," Talanta, vol. 145, pp. 2-5, 2015.
[37] G. V. Sanchez, R. N. Master, J. A. Karlowsky, and J. M. Bordon, "In vitro antimicrobial resistance of urinary Escherichia coli isolates among US outpatients from 2000 to 2010," Antimicrobial Agents and Chemotherapy, vol. 56, no. 4, pp. 2181-2183, 2012.
[38] G. Kaufman, "Kaufman,.(2011). Antibiotics: mode of action and mechanisms of resistance," Nursing Standard, vol. 25, pp. 42-49.
[39] A. Al-Badr and G. Al-Shaikh, "Recurrent urinary tract infections management in women: a review," Sultan Qaboos University Medical Journal, vol. 13, no. 3, p. 359, 2013.
[40] S. Pulipati, P. S. Babu, M. L. Narasu, and N. Anusha, "An overview on urinary tract infections and effective natural remedies," Journal of Medicinal Plants Studies, vol. 5, pp. 50-56, 2017.
[41] M. Caretto, A. Giannini, E. Russo, and T. Simoncini, "Preventing urinary tract infections after menopause without antibiotics," Maturitas, vol. 99, pp. 43-46, 2017.
[42] C.-N. Shin, "The effects of cranberries on preventing urinary tract infections," Clinical Nursing research, vol. 23, no. 1, pp. 54-79, 2014.
[43] D. K. Govindarajan, N. Viswalingam, Y. Meganathan, and K. Kandaswamy, "Adherence patterns of Escherichia coli in the intestine and its role in pathogenesis," Medicine in Microecology, vol. 5, p. 100025, 2020.
[44] D. K. Govindarajan and K. Kandaswamy, "Virulence factors of uropathogens and their role in host pathogen interactions," The Cell Surface, vol. 8, p. 100075, 2022.
[45] D. K. Govindarajan, B. M. Eskeziyaw, K. Kandaswamy, and D. Y. Mengistu, "Diagnosis of extraintestinal pathogenic Escherichia coli pathogenesis in urinary tract infection," Current Research in Microbial Sciences, vol. 7, p. 100296, 2024.
[46] K. W. Dodson, J. S. Pinkner, T. Rose, G. Magnusson, S. J. Hultgren, and G. Waksman, "Structural basis of the interaction of the pyelonephritic E. coli adhesin to its human kidney receptor," Cell, vol. 105, no. 6, pp. 733-743, 2001.
[47] M. Sung, K. Fleming, H. A. Chen, and S. Matthews, "The solution structure of PapGII from uropathogenic Escherichia coli and its recognition of glycolipid receptors," EMBO reports, 2001.
[48] I. Ambite, D. S. C. Butler, C. Stork, J. Grönberg Hernández, B. Köves, J. Zdziarski, J. Pinkner, S. J. Hultgren, U. Dobrindt, B. Wullt, and C. Svanborg, "Fimbriae reprogram host gene expression–Divergent effects of P and type 1 fimbriae," PLoS pathogens, vol. 15, no. 6, p. e1007671, 2019.
[49] R. A. Welch, V. Burland, G. Plunkett, III, P. Redford, P. Roesch, D. Rasko, E. L. Buckles, S.-R. Liou, A. Boutin, J. Hackett, D. Stroud, G. F. Mayhew, D. J. Rose, S. Zhou, D. C. Schwartz, N. T. Perna, H. L. T. Mobley, M. S. Donnenberg, and F. R. Blattner, "Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli," Proceedings of the National Academy of Sciences, vol. 99, no. 26, pp. 17020-17024, 2002.
[50] M. Biggel, B. B. Xavier, J. R. Johnson, K. L. Nielsen, N. Frimodt Møller, V. Matheeussen, H. Goossens, P. Moons, and S. Van Puyvelde, "Horizontally acquired papGII-containing pathogenicity islands underlie the emergence of invasive uropathogenic Escherichia coli lineages," Nature Communications, vol. 11, no. 1, p. 5968, 2020.
[51] J. A. Snyder, B. J. Haugen, C. V. Lockatell, N. Maroncle, E. C. Hagan, D. E. Johnson, R. A. Welch, and H. L. T. Mobley, "Coordinate expression of fimbriae in uropathogenic Escherichia coli," Infection and immunity, vol. 73, no. 11, pp. 7588-7596, 2005.
[52] C.-C. Tseng, W.-H. Lin, A.-B. Wu, M.-C. Wang, C.-H. Teng, and J.-J. Wu, "Escherichia coli FimH adhesins act synergistically with PapGII adhesins for enhancing establishment and maintenance of kidney infection," Journal of Microbiology, Immunology and Infection, vol. 55, no. 1, pp. 44-50, 2022.
[53] N. Abubakar, M. Aliyu, M. Jibril, and Y. Mohammed, "Polymerase chain reaction detection of haemolysin D gene (hlyD) in uropathogenic Escherichia coli as a novel diagnostic test for urinary tract infection," African Journal of Clinical and Experimental Microbiology, vol. 24, no. 1, pp. 45-50, 2023.
[54] F. Edition, "Guidelines for drinking-water quality," WHO chronicle, vol. 38, no. 4, pp. 104-8, 2011.
[55] K. J. Wojno, D. Baunoch, N. Luke, M. Opel, H. Korman, C. Kelly, S. M. A. Jafri, P. Keating, D. Hazelton, S. Hindu, B. Makhloouf, D. Wenzler, M. Sabry, F. Burks, M. Penaranda, D. E. Smith, A. Korman, and L. Sirls, "Multiplex PCR based urinary tract infection (UTI) analysis compared to traditional urine culture in identifying significant pathogens in symptomatic patients," Urology, vol. 136, pp. 119-126, 2020.
[56] M. Kumar, S. Ghosh, S. Nayak, and A. Das, "Recent advances in biosensor based diagnosis of urinary tract infection," Biosensors and Bioelectronics, vol. 80, pp. 497-510, 2016.
[57] J. W. Bartges, "Diagnosis of urinary tract infections," Veterinary Clinics: Small Animal Practice, vol. 34, no. 4, pp. 923-933, 2004.
[58] F. Hajar, M. Taleb, B. Aoun, and A. Shatila, "Dipstick urine analysis screening among asymptomatic school children," North American Journal of Medical Sciences, vol. 3, no. 4, p. 179, 2011.
[59] G. Schmiemann, E. Kniehl, K. Gebhardt, M. M. Matejczyk, and E. Hummers-Pradier, "The diagnosis of urinary tract infection: a systematic review," Deutsches Ärzteblatt International, vol. 107, no. 21, p. 361, 2010.
[60] F. Caméléna, A. Birgy, Y. Smail, C. Courroux, P. Mariani Kurkdjian, S. Le Hello, S. Bonacorsi, and P. Bidet, "Rapid and simple universal Escherichia coli genotyping method based on multiple-locus variable-number tandem-repeat analysis using single-tube multiplex PCR and standard gel electrophoresis," Applied and Environmental Microbiology, vol. 85, no. 6, pp. e02812-18, 2019.
[61] F. Xia, J. Cheng, M. Jiang, Z. Wang, Z. Wen, M. Wang, J. Ren, and X. Zhuge, "Genomics analysis to identify multiple genetic determinants that drive the global transmission of the pandemic ST95 lineage of extraintestinal pathogenic Escherichia coli (ExPEC)," Pathogens, vol. 11, no. 12, p. 1489, 2022.
[62] J. Li, J. Macdonald, and F. Von Stetten, "A comprehensive summary of a decade development of the recombinase polymerase amplification," Analyst, vol. 144, no. 1, pp. 31-67, 2019.
[63] J. R. Johnson, A. L. Stell, F. Scheutz, T. T. O'Bryan, T. A. Russo, U. B. Carlino, C. Fasching, J. Kavle, L. Van Dijk, and W. Gaastra, "Analysis of the F antigen-specific papA alleles of extraintestinal pathogenic Escherichia coli using a novel multiplex PCR-based assay," Infection and immunity, vol. 68, no. 3, pp. 1587-1599, 2000.
[64] H. C. Kunert Filho, D. Carvalho, T. T. Grassotti, B. D. Soares, J. M. Rossato, A. C. Cunha, K. C. T. Brito, L. S. Cavalli, and B. G. Brito, "Avian pathogenic Escherichia coli-methods for improved diagnosis," World's poultry science journal, vol. 71, no. 2, pp. 249-258, 2015.
[65] P. M. Fratamico, C. DebRoy, and D. S. Needleman, "Emerging approaches for typing, detection, characterization, and traceback of Escherichia coli," Frontiers in Microbiology, vol. 7, p. 2089, 2016.
[66] V. M. Sora, G. Meroni, P. A. Martino, A. Soggiu, L. Bonizzi, and A. Zecconi, "Extraintestinal pathogenic Escherichia coli: virulence factors and antibiotic resistance," Pathogens, vol. 10, no. 11, p. 1355, 2021.
[67] L. M. Willis and C. Whitfield, "Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways," Carbohydrate Research, vol. 378, pp. 35-44, 2013.
[68] J. C. Rice, T. Peng, Y.-f. Kuo, S. Pendyala, L. Simmons, J. Boughton, K. Ishihara, S. Nowicki, and B. J. Nowicki, "Renal allograft injury is associated with urinary tract infection caused by Escherichia coli bearing adherence factors," American Journal of Transplantation, vol. 6, no. 10, pp. 2375-2383, 2006.
[69] R. A. Moxley, "Enterobacteriaceae: Escherichia," Veterinary Microbiology, pp. 56-74, 2022.
[70] L. Durant, A. Metais, C. Soulama-Mouze, J.-M. Genevard, X. Nassif, and S. Escaich, "Identification of candidates for a subunit vaccine against extraintestinal pathogenic Escherichia coli," Infection and Immunity, vol. 75, no. 4, pp. 1916-1925, 2007.
[71] S. F. Berlanda, M. Breitfeld, C. L. Dietsche, and P. S. Dittrich, "Recent advances in microfluidic technology for bioanalysis and diagnostics," Analytical Chemistry, vol. 93, no. 1, pp. 311-331, 2020.
[72] D. Figeys and D. Pinto, "Lab-on-a-chip: a revolution in biological and medical sciences," ed: ACS Publications, 2000.
[73] S. Sachdeva, R. W. Davis, and A. K. Saha, "Microfluidic point-of-care testing: commercial landscape and future directions," Frontiers in Bioengineering and Biotechnology, vol. 8, p. 602659, 2021.
[74] W. Jung, J. Han, J.-W. Choi, and C. H. Ahn, "Point-of-care testing (POCT) diagnostic systems using microfluidic lab-on-a-chip technologies," Microelectronic Engineering, vol. 132, pp. 46-57, 2015.
[75] T. Bhardwaj, L. N. Ramana, and T. K. Sharma, "Current advancements and future road map to develop ASSURED microfluidic biosensors for infectious and non-infectious diseases," Biosensors, vol. 12, no. 5, p. 357, 2022.
[76] K. Ren, J. Zhou, and H. Wu, "Materials for microfluidic chip fabrication," Accounts of Chemical Research, vol. 46, no. 11, pp. 2396-2406, 2013.
[77] S. C. Terry, J. H. Jerman, and J. B. Angell, "A gas chromatographic air analyzer fabricated on a silicon wafer," IEEE Transactions on Electron Devices, vol. 26, no. 12, pp. 1880-1886, 1979.
[78] P. N. Nge, C. I. Rogers, and A. T. Woolley, "Advances in microfluidic materials, functions, integration, and applications," Chemical Reviews, vol. 113, no. 4, pp. 2550-2583, 2013.
[79] J. B. Nielsen, R. L. Hanson, H. M. Almughamsi, C. Pang, T. R. Fish, and A. T. Woolley, "Microfluidics: innovations in materials and their fabrication and functionalization," Analytical Chemistry, vol. 92, no. 1, pp. 150-168, 2019.
[80] A.-G. Niculescu, C. Chircov, A. C. Bîrcă, and A. M. Grumezescu, "Fabrication and applications of microfluidic devices: A review," International Journal of Molecular Sciences, vol. 22, no. 4, p. 2011, 2021.
[81] G. M. Whitesides, "The origins and the future of microfluidics," Nature, vol. 442, no. 7101, pp. 368-373, 2006.
[82] S. Yang and K. Jiang, "Elastomer application in microsystem and microfluidics," Advanced Elastomers-Technology, Properties and Applications, 2012.
[83] J. C. McDonald and G. M. Whitesides, "Poly (dimethylsiloxane) as a material for fabricating microfluidic devices," Accounts of chemical research, vol. 35, no. 7, pp. 491-499, 2002.
[84] I. Miranda, A. Souza, P. Sousa, J. Ribeiro, E. M. S. Castanheira, R. Lima, and G. Minas, "Properties and applications of PDMS for biomedical engineering: A review," Journal of Functional Biomaterials, vol. 13, no. 1, p. 2, 2021.
[85] M. J. Owen and P. J. Smith, "Plasma treatment of polydimethylsiloxane," Journal of Adhesion Science and Technology, vol. 8, no. 10, pp. 1063-1075, 1994.
[86] J. C. McDonald, D. C. Duffy, J. R. Anderson, D. T. Chiu, H. Wu, O. J. A. Schueller, and G. M. Whitesides, "Fabrication of microfluidic systems in poly (dimethylsiloxane)," Electrophoresis: An International Journal, vol. 21, no. 1, pp. 27-40, 2000.
[87] E. Roy, J.-C. Galas, and T. Veres, "Thermoplastic elastomers for microfluidics: Towards a high-throughput fabrication method of multilayered microfluidic devices," Lab on a Chip, vol. 11, no. 18, pp. 3193-3196, 2011.
[88] E. Berthier, E. W. Young, and D. Beebe, "Engineers are from PDMS-land, Biologists are from Polystyrenia," Lab on a Chip, vol. 12, no. 7, pp. 1224-1237, 2012.
[89] M. W. Toepke and D. J. Beebe, "PDMS absorption of small molecules and consequences in microfluidic applications," Lab on a Chip, vol. 6, no. 12, pp. 1484-1486, 2006.
[90] K. Raj M and S. Chakraborty, "PDMS microfluidics: A mini review," Journal of Applied Polymer Science, vol. 137, no. 27, p. 48958, 2020.
[91] A. Bîrcă, O. Gherasim, V. Grumezescu, and A. M. Grumezescu, "Introduction in thermoplastic and thermosetting polymers," in Materials for Biomedical Engineering: Elsevier, 2019, pp. 1-28.
[92] R. Martinez-Duarte and M. Madou, "SU-8 photolithography and its impact on microfluidics," Microfluidics and Nanofluidics Handbook, no. 2006, pp. 231-268, 2011.
[93] H. Sato, H. Matsumura, S. Keino, and S. Shoji, "An all SU-8 microfluidic chip with built-in 3D fine microstructures," Journal of Micromechanics and Microengineering, vol. 16, no. 11, p. 2318, 2006.
[94] S. Metz, R. Holzer, and P. Renaud, "Polyimide-based microfluidic devices," Lab on a Chip, vol. 1, no. 1, pp. 29-34, 2001.
[95] A. Shakeri, N. A. Jarad, S. Khan, and T. F. Didar, "Bio-functionalization of microfluidic platforms made of thermoplastic materials: A review," Analytica chimica acta, vol. 1209, p. 339283, 2022.
[96] H. Becker and C. Gärtner, "Polymer microfabrication technologies for microfluidic systems," Analytical and Bioanalytical Chemistry, vol. 390, no. 1, pp. 89-111, 2008.
[97] C.-W. Tsao and D. L. DeVoe, "Bonding of thermoplastic polymer microfluidics," Microfluidics and nanofluidics, vol. 6, no. 1, pp. 1-16, 2009.
[98] T.-F. Hong, W.-J. Ju, M.-C. Wu, C.-H. Tai, C.-H. Tsai, and L.-M. Fu, "Rapid prototyping of PMMA microfluidic chips utilizing a CO2 laser," Microfluidics and Nanofluidics, vol. 9, no. 6, pp. 1125-1133, 2010.
[99] R. Pelton, "Bioactive paper provides a low-cost platform for diagnostics," TrAC Trends in Analytical Chemistry, vol. 28, no. 8, pp. 925-942, 2009.
[100] X. Li, D. R. Ballerini, and W. Shen, "A perspective on paper-based microfluidics: Current status and future trends," Biomicrofluidics, vol. 6, no. 1, 2012.
[101] A. W. Martinez, S. T. Phillips, M. J. Butte, and G. M. Whitesides, "Patterned paper as a platform for inexpensive, low‐volume, portable bioassays," Angewandte Chemie, vol. 119, no. 8, pp. 1340-1342, 2007.
[102] A. W. Martinez, S. T. Phillips, E. Carrilho, S. W. Thomas III, H. Sindi, and G. M. Whitesides, "Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time, off-site diagnosis," Analytical Chemistry, vol. 80, no. 10, pp. 3699-3707, 2008.
[103] R. H. Tang, M. Y. Xie, M. Li, L. Cao, S. S. Feng, Z. Li, and F. Xu, "Nitrocellulose membrane for paper-based biosensor," Applied Materials Today, vol. 26, p. 101305, 2022.
[104] R. H. Tang, L. N. Liu, S. F. Zhang, X. C. He, X. J. Li, F. Xu, Y. H. Ni, and F. Li, "A review on advances in methods for modification of paper supports for use in point-of-care testing," Microchimica Acta, vol. 186, no. 8, p. 521, 2019.
[105] M. Á. F. de la Ossa, M. López-López, M. Torre, and C. García-Ruiz, "Analytical techniques in the study of highly-nitrated nitrocellulose," TrAC Trends in Analytical Chemistry, vol. 30, no. 11, pp. 1740-1755, 2011.
[106] M. Gao, J. Li, Z. Bao, M. Hu, R. Nian, D. Feng, D. An, X. Li, M. Xian, and H. Zhang, "A natural in situ fabrication method of functional bacterial cellulose using a microorganism," Nature Communications, vol. 10, no. 1, p. 437, 2019.
[107] L. Karstens, N. Y. Siddiqui, T. Zaza, A. Barstad, C. L. Amundsen, and T. A. Sysoeva, "Benchmarking DNA isolation kits used in analyses of the urinary microbiome," Scientific Reports, vol. 11, no. 1, p. 6186, 2021.
[108] L. El Bali, A. Diman, A. Bernard, N. H. Roosens, and S. C. De Keersmaecker, "Comparative study of seven commercial kits for human DNA extraction from urine samples suitable for DNA biomarker-based public health studies," Journal of Biomolecular Techniques: JBT, vol. 25, no. 4, p. 96, 2014.
[109] R. Alaeddini, "Forensic implications of PCR inhibition—a review," Forensic Science International: Genetics, vol. 6, no. 3, pp. 297-305, 2012.
[110] K. A. Stevens and L.-A. Jaykus, "Bacterial separation and concentration from complex sample matrices: a review," Critical Reviews in Microbiology, vol. 30, no. 1, pp. 7-24, 2004.
[111] L. Serioli, T. Z. Laksafoss, J. A. J. Haagensen, C. Sternberg, M. P. Sørensen, S. Molin, K. Zór, and A. Boisen, "Bacterial cell cultures in a lab-on-a-disc: A simple and versatile tool for quantification of antibiotic treatment efficacy," Analytical Chemistry, vol. 92, no. 20, pp. 13871-13879, 2020.
[112] A. Perebikovsky, Y. Liu, A. Hwu, H. Kido, E. Shamloo, D. Song, G. Monti, O. Shoval, D. Gussin, and M. Madou, "Rapid sample preparation for detection of antibiotic resistance on a microfluidic disc platform," Lab on a Chip, vol. 21, no. 3, pp. 534-545, 2021.
[113] M. S. Wiederoder, S. Smith, P. Madzivhandila, D. Mager, K. Moodley, D. L. DeVoe, and K. J. Land, "Novel functionalities of hybrid paper-polymer centrifugal devices for assay performance enhancement," Biomicrofluidics, vol. 11, no. 5, 2017.
[114] Y. Su, "Current state-of-the-art membrane based filtration and separation technologies," in Graphene-based Membranes for Mass Transport Applications, ed. H. Zhu and P. Sun, The Royal Society of Chemistry, 2018, ch. 1, pp. 1-13.
[115] J. J. Ezenarro, J. Mas, X. Muñoz-Berbel, and N. Uria, "Advances in bacterial concentration methods and their integration in portable detection platforms: A review," Analytica Chimica Acta, vol. 1209, p. 339079, 2022.
[116] C.-Y. Wen, Y.-Z. Jiang, X.-Y. Li, M. Tang, L.-L. Wu, J. Hu, D.-W. Pang, and J.-B. Zeng, "Efficient enrichment and analyses of bacteria at ultralow concentration with quick-response magnetic nanospheres," ACS Applied Materials & Interfaces, vol. 9, no. 11, pp. 9416-9425, 2017.
[117] W. Lee, D. Kwon, B. Chung, G. Y. Jung, A. Au, A. Folch, and S. Jeon, "Ultrarapid detection of pathogenic bacteria using a 3D immunomagnetic flow assay," Analytical Chemistry, vol. 86, no. 13, pp. 6683-6688, 2014.
[118] H. O. Yosief, A. A. Weiss, and S. S. Iyer, "Capture of Uropathogenic E. coli by Using Synthetic Glycan Ligands Specific for the Pap‐Pilus," ChemBioChem, vol. 14, no. 2, pp. 251-259, 2013.
[119] J.-C. Liu, P.-J. Tsai, Y. C. Lee, and Y.-C. Chen, "Affinity capture of uropathogenic Escherichia coli using pigeon ovalbumin-bound Fe3O4@ Al2O3 magnetic nanoparticles," Analytical Chemistry, vol. 80, no. 14, pp. 5425-5432, 2008.
[120] Ó. Castillo-Fernandez, N. Uria, F. X. Muñoz, and A. Bratov, "Cell concentration systems for enhanced biosensor sensitivity," Biosensors-Micro and Nanoscale Applications, 2015.
[121] A. Perera, S. Pudasaini, S. S. U. Ahmed, D. T. Phan, Y. Liu, and C. Yang, "Rapid pre‐concentration of Escherichia coli in a microfluidic paper‐based device using ion concentration polarization," Electrophoresis, vol. 41, no. 10-11, pp. 867-874, 2020.
[122] R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim, "Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia," Science, vol. 230, no. 4732, pp. 1350-1354, 1985.
[123] K. B. Mullis, The polymerase chain reaction (no. 5). Springer science & business media, 1994.
[124] K. J. Breslauer, R. Frank, H. Blöcker, and L. A. Marky, "Predicting DNA duplex stability from the base sequence," Proceedings of the National Academy of Sciences, vol. 83, no. 11, pp. 3746-3750, 1986.
[125] W. Rychlik, W. Spencer, and R. Rhoads, "Optimization of the annealing temperature for DNA amplification in vitro," Nucleic acids Research, vol. 18, no. 21, pp. 6409-6412, 1990.
[126] N. Chester and D. R. Marshak, "Dimethyl sulfoxide-mediated primer Tm reduction: a method for analyzing the role of renaturation temperature in the polymerase chain reaction," Analytical Biochemistry, vol. 209, no. 2, pp. 284-290, 1993.
[127] G. Schochetman, C.-Y. Ou, and W. K. Jones, "Polymerase chain reaction," The Journal of infectious diseases, vol. 158, no. 6, pp. 1154-1157, 1988.
[128] J. Ye, G. Coulouris, I. Zaretskaya, I. Cutcutache, S. Rozen, and T. L. Madden, "Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction," BMC Bioinformatics, vol. 13, no. 1, p. 134, 2012.
[129] C. Dieffenbach, T. Lowe, and G. Dveksler, "General concepts for PCR primer design," PCR methods appl, vol. 3, no. 3, pp. S30-S37, 1993.
[130] Z. Yang, J. T. Le, D. Hutter, K. M. Bradley, B. R. Overton, C. McLendon, and S. A. Benner, "Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems," Biology Methods and Protocols, vol. 5, no. 1, p. bpaa004, 2020.
[131] T. C. Lorenz, "Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies," Journal of Visualized Experiments: JoVE, no. 63, p. 3998, 2012.
[132] T. Notomi, H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, and T. Hase, "Loop-mediated isothermal amplification of DNA," Nucleic acids research, vol. 28, no. 12, pp. e63-e63, 2000.
[133] K. Nagamine, T. Hase, and T. Notomi, "Accelerated reaction by loop-mediated isothermal amplification using loop primers," Molecular and Cellular Probes, vol. 16, no. 3, pp. 223-229, 2002.
[134] L. Becherer, N. Borst, M. Bakheit, S. Frischmann, R. Zengerle, and F. von Stetten, "Loop-mediated isothermal amplification (LAMP)–review and classification of methods for sequence-specific detection," Analytical Methods, vol. 12, no. 6, pp. 717-746, 2020.
[135] D.-G. Wang, J. D. Brewster, M. Paul, and P. M. Tomasula, "Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification," Molecules, vol. 20, no. 4, pp. 6048-6059, 2015.
[136] K. G. Ptitsyn, S. A. Khmeleva, L. K. Kurbatov, O. S. Timoshenko, E. V. Suprun, S. P. Radko, and A. V. Lisitsa, "Lamp Primer Designing Software: The Overview," Biomedical Chemistry: Research and Methods, vol. 7, no. 4, pp. e00226-e00226, 2024.
[137] R. Ramezani, Z. K. Parizi, N. Ghorbanmehr, and H. Mirshafiee, "Rapid and simple detection of Escherichia coli by loop-mediated isothermal amplification assay in urine specimens," Avicenna Journal of Medical Biotechnology, vol. 10, no. 4, p. 269, 2018.
[138] N. Saengsawang, T. Ruang-Areerate, P. Kesakomol, T. Thita, M. Mungthin, and W. Dungchai, "Development of a fluorescent distance-based paper device using loop-mediated isothermal amplification to detect Escherichia coli in urine," Analyst, vol. 145, no. 24, pp. 8077-8086, 2020.
[139] O. Piepenburg, C. H. Williams, D. L. Stemple, and N. A. Armes, "DNA detection using recombination proteins," PLoS biology, vol. 4, no. 7, p. e204, 2006.
[140] I. M. Lobato and C. K. O'Sullivan, "Recombinase polymerase amplification: Basics, applications and recent advances," Trac Trends in Analytical Chemistry, vol. 98, pp. 19-35, 2018.
[141] X. Xia, Y. Yu, M. Weidmann, Y. Pan, S. Yan, and Y. Wang, "Rapid detection of shrimp white spot syndrome virus by real time, isothermal recombinase polymerase amplification assay," PLoS One, vol. 9, no. 8, p. e104667, 2014.
[142] M. Tan, C. Liao, L. Liang, X. Yi, Z. Zhou, and G. Wei, "Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications," Frontiers in Cellular and Infection Microbiology, vol. 12, p. 1019071, 2022.
[143] J. Chen, Y. Xu, H. Yan, Y. Zhu, L. Wang, Y. Zhang, Y. Lu, and W. Xing, "Sensitive and rapid detection of pathogenic bacteria from urine samples using multiplex recombinase polymerase amplification," Lab on a Chip, vol. 18, no. 16, pp. 2441-2452, 2018.
[144] B. Raja, H. J. Goux, A. Marapadaga, S. Rajagopalan, K. Kourentzi, and R. C. Willson, "Development of a panel of recombinase polymerase amplification assays for detection of common bacterial urinary tract infection pathogens," Journal of Applied Microbiology, vol. 123, no. 2, pp. 544-555, 2017.
[145] J. R. Brody and S. E. Kern, "History and principles of conductive media for standard DNA electrophoresis," Analytical Biochemistry, vol. 333, no. 1, pp. 1-13, 2004.
[146] N. C. Stellwagen, A. Bossi, C. Gelfi, and P. G. Righetti, "DNA and buffers: are there any noninteracting, neutral pH buffers?," Analytical Biochemistry, vol. 287, no. 1, pp. 167-175, 2000.
[147] R. C. Allen and B. Budowle, Gel Electrophoresis of Proteins and Nucleic Acids: Selected Techniques. Berlin and New York: Walter de Gruyter, 1994, 352 pp., ISBN: 978-3-11-013896-2.
[148] X. Mao, W. Wang, and T.-E. Du, "Rapid quantitative immunochromatographic strip for multiple proteins test," Sensors and Actuators B: Chemical, vol. 186, pp. 315-320, 2013.
[149] B. B. Dzantiev, N. A. Byzova, A. E. Urusov, and A. V. Zherdev, "Immunochromatographic methods in food analysis," TrAC Trends in Analytical Chemistry, vol. 55, pp. 81-93, 2014.
[150] J. Singh, S. Sharma, and S. Nara, "Evaluation of gold nanoparticle based lateral flow assays for diagnosis of enterobacteriaceae members in food and water," Food Chemistry, vol. 170, pp. 470-483, 2015.
[151] J. Hu, S. Wang, L. Wang, F. Li, B. Pingguan Murphy, T. J. Lu, and F. Xu, "Advances in paper-based point-of-care diagnostics," Biosensors and Bioelectronics, vol. 54, pp. 585-597, 2014.
[152] E. B. Bahadır and M. K. Sezgintürk, "Lateral flow assays: Principles, designs and labels," TrAC Trends in Analytical Chemistry, vol. 82, pp. 286-306, 2016.
[153] J. Budd, B. S. Miller, N. E. Weckman, D. Cherkaoui, D. Huang, A. T. Decruz, N. Fongwen, G.-R. Han, M. Broto, C. S. Estcourt, J. Gibbs, D. Pillay, P. Sonnenberg, R. Meurant, M. R. Thomas, N. Keegan, M. M. Stevens, E. Nastouli, E. J. Topol, A. M. Johnson, M. Shahmanesh, A. Ozcan, J. J. Collins, M. F. Suarez, B. Rodriguez, R. W. Peeling, and R. A. McKendry, "Lateral flow test engineering and lessons learned from COVID-19," Nature Reviews Bioengineering, vol. 1, no. 1, pp. 13-31, 2023.
[154] S. Goudarzi, A. Ahmadi, M. Farhadi, S. K. Kamrava, S. Saghafi, and K. Omidfar, "Development of a new immunochromatographic assay using gold nanoparticles for screening of IgA deficiency," Iranian Journal of Allergy, Asthma and Immunology, pp. 105-112, 2015.
[155] S. Yang, J. Yang, G. Zhang, X. Wang, S. Qiao, D. Zhao, Y. Zhi, X. Li, G. Xing, J. Luo, J. Fan, and D. Bao, "Development of an immunochromatographic strip for the detection of antibodies against foot-and-mouth disease virus serotype O," Journal of Virological Methods, vol. 165, no. 2, pp. 139-144, 2010.
[156] R. Kumar, C. K. Singh, S. Kamle, R. P. Sinha, R. K. Bhatnagar, and D. N. Kachru, "Development of nanocolloidal gold based immunochromatographic assay for rapid detection of transgenic vegetative insecticidal protein in genetically modified crops," Food Chemistry, vol. 122, no. 4, pp. 1298-1303, 2010.
[157] C. Parolo, A. de la Escosura-Muñiz, and A. Merkoçi, "Enhanced lateral flow immunoassay using gold nanoparticles loaded with enzymes," Biosensors and Bioelectronics, vol. 40, no. 1, pp. 412-416, 2013.
[158] C. Fang, Z. Chen, L. Li, and J. Xia, "Barcode lateral flow immunochromatographic strip for prostate acid phosphatase determination," Journal of Pharmaceutical and Biomedical Analysis, vol. 56, no. 5, pp. 1035-1040, 2011.
[159] K. E. McCracken and J.-Y. Yoon, "Recent approaches for optical smartphone sensing in resource-limited settings: a brief review," Analytical Methods, vol. 8, no. 36, pp. 6591-6601, 2016.
[160] G. M. Fernandes, W. R. Silva, D. N. Barreto, R. S. Lamarca, P. C. F. L. Gomes, J. F. S. Petruci, and A. D. Batista, "Novel approaches for colorimetric measurements in analytical chemistry–a review," Analytica Chimica Acta, vol. 1135, pp. 187-203, 2020.
[161] V. Chernov, J. Alander, and V. Bochko, "Integer-based accurate conversion between RGB and HSV color spaces," Computers & Electrical Engineering, vol. 46, pp. 328-337, 2015.
[162] G. Saravanan, G. Yamuna, and S. Nandhini, "Real time implementation of RGB to HSV/HSI/HSL and its reverse color space models," in 2016 International Conference on Communication and Signal Processing (ICCSP), 2016: IEEE, pp. 0462-0466.
[163] C.-H. Ko, C.-C. Tseng, S.-Y. Lu, C.-C. Lee, S. Kim, and L.-M. Fu, "Handheld microfluidic multiple detection device for concurrent blood urea nitrogen and creatinine ratio determination using colorimetric approach," Sensors and Actuators B: Chemical, vol. 422, p. 136585, 2025.
[164] J. R. Johnson and J. J. Brown, "A novel multiply primed polymerase chain reaction assay for identification of variant papG genes encoding the Gal (α1–4) Gal-binding PapG adhesins of Escherichia coli," Journal of Infectious Diseases, vol. 173, no. 4, pp. 920-926, 1996.
[165] G. Bradski and A. Kaehler, Learning OpenCV: Computer Vision with The OpenCV Library. " O'Reilly Media, Inc.", 2008.
[166] A. Cuénod, J. Agnetti, H. M. B. Seth-Smith, T. Roloff, D. Wälchli, D. Shcherbakov, R. Akbergenov, S. Tschudin-Sutter, S. Bassetti, M. Siegemund, C. H. Nickel, J. Moran-Gilad, T. G. Keys, V. Pflüger, N. R. Thomson, and A. Egli, “Bacterial genome-wide association study substantiates papGII of Escherichia coli as a major risk factor for urosepsis,” Genome Medicine, vol. 15, no. 1, p. 89, 2023.