- 學位論文
創新皮膚疾病早期快速診斷工具－表皮微流體檢測系統之研發
Epifluidics-Based Rapid Diagnostic System for Early Detection of Dermatologic Diseases
若您是本文的作者，可授權文章由華藝線上圖書館中協助推廣。
摘要
經皮傳輸系統能直接透過皮膚表面達到非侵入性給藥或檢體採集的目的，使病患不必承受挨針之苦。表皮微流體系統主要是利用微流體技術從皮膚採集並分析檢體，是近年快速發展的研究領域，目前研究主要應用在採集汗液或細胞間液中的小分子物質，來推斷其在體內循環的濃度，但這樣的設計對疾病診斷來說，皮內檢體的代表性及檢驗的敏感度仍是一大挑戰。然而，對皮膚疾病而言，能幫助診斷的疾病標誌經常存在於皮疹中，也因此皮膚切片是目前診斷皮膚病的主要方法，而非血液檢查。但皮膚切片手術相當具有侵入性，需要專門的設備和醫師處理，不僅耗時，更會在病人皮膚上留下永久性疤痕。使用表皮微流體技術幫助皮膚疾病的診斷是本論文想提出的核心概念，特別是針對皮膚水泡病，於此類疾病能幫助診斷疾病的疾病標誌經常存在於水泡液中，但因為目前缺乏能搭配且可近性高的檢驗工具，所以水泡液檢查的重要性在臨床上經常被低估。隨著精準醫療的快速發展，癌症腫瘤學提出了「液態切片」的概念，而對於皮膚水泡疾病，本論文在此提出「水泡液態切片」的創想，透過非侵入性的水泡液採檢配合新研發的即時檢測工具，期待提高水泡液診斷的臨床應用和價值。 為了實現此目標，本研究使用水痘及帶狀皰疹為作為水泡疾病代表，使用表皮微流體技術研發出全新的快速診斷工具。水痘帶狀皰疹病毒的傳染性和盛行率都相當高，初次感染時會產生水痘，而後病毒潛伏於體內神經節，帶狀皰疹即是由體內的病毒再活化所引起，其特點是沿著皮節出現群聚性水泡和神經痛的症狀，經常使病人痛苦不堪。早期診斷、早期治療對於水痘和帶狀皰疹來說相當重要，由於水泡液具有傳染性、皮疹進展快速，早期給藥可以減少疾病和疼痛的嚴重程度和持續時間，縮減將病毒傳染給別人的機會，更能降低帶狀皰疹後神經痛、病毒擴散至其他器官或產生嚴重併發症的風險。 雖然運用聚合酶連鎖反應來檢測病毒是診斷帶狀皰疹的黃金標準，但由於聚合酶連鎖反應需要專門的實驗室設備、人力且相對耗時，在此檢驗方式可近性不高的狀況下，目前帶狀皰疹大部分都只靠臨床表現來診斷。本研究開發出一種新型水痘帶狀皰疹病毒的快速篩檢試片，運用水痘或帶狀皰疹的水泡液當檢體，可以在十五分鐘內提供具有相當敏感性和特異性的檢驗結果，並且能直接用肉眼作判讀，無需特殊設備即能做疾病診斷。此試片提供了一種簡單方便、非侵入性、無需實驗室設備就能快速檢測水痘或帶狀皰疹的方法。此外，運用反射光譜分析儀或智慧型手機，能更進一步針對試片作病毒濃度的量化分析，以簡易的方式幫助疾病的傳染性評估或追蹤病毒量變化。 另一方面，本研究針對極早期的帶狀皰疹，以微針技術為基礎研發出表皮微流體診斷系統。在帶狀皰疹的初期階段，皮膚表現可能只有一個或數個丘疹或肉眼不易見的微小水泡，因為與許多皮膚疾病的表徵相似，臨床上很可能造成誤判而導致錯誤治療，此階段因為沒有明顯水泡，在診斷不明確的狀況下，多數只能靠皮膚切片來用病理化驗確診。本研究運用特殊設計且生物相容性高的微針來採取皮疹中的微水泡液或細胞間液，並將其結合於聚合酶連鎖反應、病毒快篩試片或螢光免疫分析，此系統成功地在帶狀皰疹的極早期階段檢驗出病毒並診斷疾病。這種安全無痛、操作簡易又可攜帶的全新診斷方法提供了自我居家檢測的願景，這對於傳染力強、盛行率高的水痘及帶狀皰疹來說尤其重要，也為皮膚水泡病的診斷開啟了新的里程碑。總結來說，本研究透過微針與表皮微流體檢測系統之創新研發，提供皮膚疾病一個更早期、快速、準確的定點照護檢驗方式，使疾病能得到更即時有效的治療，可近性高的設計提供了個人居家篩檢的願景，讓偏遠或醫療資源缺乏地區也能擁有精準診斷、精準治療。
並列摘要
The transdermal delivery system, which encompasses both transdermal drug delivery and transdermal sampling, possesses many advantages compared to traditional needle-based procedurals. Epifluidics, also referred to as epidermal microfluidic technology, is a rapidly advancing field that utilizes microfluidic systems to analyze fluids obtained through the skin surface. Traditional epifluidics-based diagnostic methods have primarily been employed to differentiate the dynamics of sweat or interstitial fluid through extraction, capture, and analysis of small molecules to infer their concentration in the systemic circulation. Yet, the representativeness and sensitivity present a significant challenge in their application. In many dermatological diseases, disease markers are mainly present in the skin, making skin biopsy the primary diagnostic method in dermatology, rather than blood tests. However, a skin biopsy is invasive, time-consuming, requires specialized personnel and equipment, and leaves a permanent scar. Our study wanted to apply the technology of epifluidics to dermatologic disease diagnosis, especially for bullous dermatoses, which manifest as blisters on the skin, and the disease markers may be primarily present in the blister fluid. Due to the lack of an accessible coupling diagnostic tool for blister fluid, the importance of blister fluid examination is underestimated. Therefore, we propose the new concept of "blister fluid biopsy," similar to the idea of liquid biopsy in oncology, defined as a non-invasive alternative method for sampling and analyzing blister fluid in bullous dermatoses. To achieve this goal, our study developed an epifluidics-based diagnostic tool for examining blister fluid in bullous dermatoses, using varicella and herpes zoster as model diseases. Varicella and herpes zoster are common human infectious diseases caused by the varicella-zoster virus (VZV). Primary VZV infection results in varicella, and followed by latent infection in ganglionic neurons. Herpes zoster, caused by the endogenous reactivation of VZV, is characterized by grouped vesicles along one or adjacent dermatomes and neuralgia. The diagnosis of varicella and herpes zoster is a pressing issue due to the contagious nature of blister fluids, the rapid progression of the disease, and the clear benefits of early treatment, including pain relief, reduction of rash duration, lowering the risk of post-herpetic neuralgia or severe complications, and prevention of viral transmission. Although polymerase chain reaction (PCR) is the gold standard for diagnosing herpes zoster, it is inaccessible in most settings due to reliance on specialized equipment and its relatively time-consuming nature. In our study, we developed a novel rapid test strip that can detect VZV in blister fluid, providing results with adequate sensitivity and excellent specificity within 15 minutes, even with direct visual interpretation. Importantly, it offers a non-invasive, lab-free method for quick diagnosis of varicella and herpes zoster. Additionally, quantitative evaluations of VZV concentrations can be accomplished using a reflectance spectral analyzer or a smartphone. Currently, the diagnosis of herpes zoster is mainly established through clinical inspection, due to the lack of accessible point-of-care testing. However, clinical diagnosis is insufficient in the early stage, because the cutaneous manifestations may be limited to erythematous papuloplaques with or without microvesicles, which resemble other skin conditions. In this study, we established a new microneedle-based epifluidic diagnostic system. With specifically-designed biocompatible microneedles to extract interstitial or microvesicular fluid in the skin, and coupling it with PCR, rapid test strips, or immunoassay, our system successfully identified VZV during the early stage of herpes zoster. This safe and painless methodology facilitates the vision of at-home, self-performed point-of-care testing in remote and low-resource settings. In conclusion, our study represents an evolution in the early and rapid diagnosis of herpes zoster. These innovative concepts have the potential to be extrapolated to other dermatological diseases, especially bullous dermatosis.
參考文獻
1. Vestita M, Tedeschi P, Bonamonte D. Anatomy and Physiology of the Skin. Textbook of Plastic and Reconstructive Surgery. Springer; 2022. p. 3-13.
2. Menon GK, Cleary GW, Lane ME. The structure and function of the stratum corneum. International journal of pharmaceutics. 2012;435(1):3-9.
3. Prausnitz MR, Langer R. Transdermal drug delivery. Nature biotechnology. 2008;26(11):1261-8.
4. Dharadhar S, Majumdar A, Dhoble S, Patravale V. Microneedles for transdermal drug delivery: a systematic review. Drug development and industrial pharmacy. 2019;45(2):188-201.
5. Lee Y, Hwang K. Skin thickness of Korean adults. Surgical and radiologic anatomy. 2002;24(3):183-9.