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Hava örnekleme sistemi ile biyoaerosol tespiti için mikroakışkan tabanlı tanı sistemi geliştirilmesi

Development of a microfluidics-based diagnostic system for bioaerosol detection using an air sampling system

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  1. Tez No: 977835
  2. Yazar: HÜLYA ERASLAN GÜLTEKİN
  3. Danışmanlar: DOÇ. DR. ALİ FUAT ERGENÇ
  4. Tez Türü: Doktora
  5. Konular: Biyomühendislik, Biyoteknoloji, Mekatronik Mühendisliği, Bioengineering, Biotechnology, Mechatronics Engineering
  6. Anahtar Kelimeler: Biyoaerosol, Biyoçip, Elektrokimyasal analiz, LAMP, Mikroçipler, Bioaerosol, Biochip, Electrochemical analysis, LAMP, Microchip
  7. Yıl: 2025
  8. Dil: Türkçe
  9. Üniversite: İstanbul Teknik Üniversitesi
  10. Enstitü: Lisansüstü Eğitim Enstitüsü
  11. Ana Bilim Dalı: Mekatronik Mühendisliği Ana Bilim Dalı
  12. Bilim Dalı: Mekatronik Mühendisliği Bilim Dalı
  13. Sayfa Sayısı: Belirtilmemiş.

Özet

Günümüzde hava kalitesi, özellikle kapalı ortamlarda insan sağlığını doğrudan etkileyen önemli bir çevresel faktördür. Havadaki biyolojik partiküller olarak tanımlanan biyoaerosoller, patojen mikroorganizmalar içerebildikleri için solunum yoluyla bulaşan enfeksiyonların yayılımında kritik rol oynamaktadır. Bu doğrultuda, biyoaerosollerin hızlı, güvenilir ve taşınabilir sistemlerle tespit edilmesi hem halk sağlığının korunması hem de bulaşıcı hastalıkların erken teşhisi açısından büyük önem arz etmektedir. Bu tez çalışmasında, havadaki Escherichia coli (E. coli) biyoaerosollerinin, izotermal koşullarda çalışan bir moleküler tanı yöntemi olan LAMP (Loop Mediated Isothermal Amplification) tekniği ile tespit edilmesi amaçlanmış; ayrıca bu yöntemin kültürleme, jel elektroforez ve elektrokimyasal ölçüm teknikleriyle karşılaştırmalı analizi gerçekleştirilmiştir. Tezin literatür bölümünde, biyoaerosollerin yapısı, çevresel etkileri, örnekleme yöntemleri (pasif ve aktif), moleküler tespit teknikleri ve biyosensör teknolojileri detaylı şekilde incelenmiştir. Özellikle sıvıya çarptırma (impingement) yöntemi, biyoaerosol toplama verimliliği açısından ön plana çıkmıştır. Ardından, hava örnekleme sistemine entegre edilebilecek bir çip tasarlanmış, PDMS (polidimetilsiloksan) kullanılarak üretimi gerçekleştirilmiştir. Bu çipin üretiminde kullanılan kalıp için PCB (baskı devre kartı) prototipleme cihazı kullanılmıştır. LAMP amplifikasyonlarında ısı kontrolü için özgün bir ısıtıcı sistem ve hava örnekleme numunesi çekme işlemlerinde kullanılmak üzere şırınga pompası tasarlanıp üretilmiştir. Deneysel süreçte, farklı konsantrasyonlarda hazırlanmış E. coli çözeltileri bir nebülizatör aracılığıyla kapalı bir ortama aerosolize edilmiştir. Özel olarak tasarlanan hava örnekleme şişesinde, bu bakteriler LB besiyerinde çoğaltılmış ve belirli zaman aralıklarında (10, 30, 60, 90 ve 120 dakika) peristaltik pompa ya da şırınga pompası ile sıvı numune çekme işlemi yapılmıştır. Elde edilen örnekler öncelikle kültürleme yöntemiyle agar plaklar üzerinde inkübe edilerek değerlendirilmiş, ardından LAMP yöntemi uygulanmıştır. Sonuçlar jel elektroforez yöntemi ve elektrokimyasal ölçümlerle doğrulanmıştır. Bu çalışmada geliştirilen sistem, klasik kültürleme ve PCR (Polimeraz Zincir Reaksiyonu) tabanlı analiz yöntemlerine kıyasla çok daha kısa sürede, yüksek özgüllük ve hassasiyette sonuç vermesi bakımından, acil durumlarda ve sahada kullanılabilir tanı sistemleri için umut vadetmektedir. Ayrıca çip tasarımı entegrasyonu ile sistemin taşınabilirliği artırılmış, düşük maliyetli ve kompakt bir biyosensör platformunun temelleri atılmıştır. Bu tez, çevresel sağlık alanına, enfeksiyon kontrolüne, mikrobiyal tespit ve mobil tanı alanlarına önemli katkılar sunan, yenilikçi ve çok disiplinli bir yaklaşımı yansıtmaktadır.

Özet (Çeviri)

In recent years, the issue of air quality has attracted significant attention due to its direct impact on public health, environmental sustainability and socioeconomic development. Among the various factors contributing to both indoor and outdoor air pollution, biological airborne particles, referred to as bioaerosols, pose a critical threat. Bioaerosols are defined as airborne microscopic particles of biological origin, including bacteria, viruses, fungal spores, pollen and fragments of plant or animal matter. These particles have the potential to cause a range of adverse health effects, from allergic reactions to serious respiratory infections. They are particularly dangerous in enclosed environments with limited ventilation. It is imperative to recognise the importance of the ability to detect, identify and quantify bioaerosols in real time, as this is instrumental in ensuring the protection of public health and the implementation of timely response strategies, particularly during disease outbreaks or in high-risk environments such as hospitals, laboratories and industrial workplaces. This thesis presents the designs and experimental studies developed for an integrated, portable, and rapid detection system for airborne Escherichia coli (E. coli) bioaerosols using Loop Mediated Isothermal Amplification (LAMP), a molecular technique offering high specificity and sensitivity under isothermal conditions. The primary objective of this study is to establish a novel methodology that combines a custom-designed air sampling system with a chip for use with the LAMP technique. The performance of the proposed method is compared with conventional techniques, including culture-based methods, gel electrophoresis, and electrochemical impedance spectroscopy (EIS), in terms of speed, accuracy, and field applicability. The integration of automatic sampling from the air sampling system to the reaction chip and detection by the LAMP method aims to overcome the limitations of traditional bioaerosol detection techniques, which are often time-consuming and require complex equipment and trained personnel. The literature review presented in this study offers a comprehensive examination of bioaerosols, including their sources, physical and biological characteristics, and health-related effects. Particular emphasis is placed on the disadvantages of existing detection methods. Despite their simplicity and cost-effectiveness, passive sampling techniques frequently exhibit deficiencies in terms of accuracy and temporal resolution. In contrast, active sampling methods, such as impaction, filtration, and impingement, are more effective for collecting viable microorganisms in real time. Among these, impingement is especially advantageous due to its ability to preserve microbial viability and to facilitate immediate molecular analysis in liquid media. Furthermore, molecular detection techniques are explained in detail, with particular emphasis on polymerase chain reaction (PCR), quantitative PCR (qPCR), and loop-mediated amplification (LAMP) methods. These techniques are evaluated in terms of their advantages and selection criteria. The findings highlight that, unlike PCR, LAMP does not require thermal cycling and can be conducted at a constant temperature, which makes it highly suitable for point-of-care applications and field deployment. In the experimental phase of the study, an air sampling bottle was designed to collect airborne E. coli particles directly into a liquid medium. The system was optimized to maximize collection efficiency and to support bacterial enrichment in a controlled, closed environment. E. coli bacteria were aerosolized into the air sampling chamber via a nebulizer were effectively captured using air sampling bottle pre-filled with Luria-Bertani (LB) broth, which acted both as a collection and enrichment medium. Thereby simulating a contaminated indoor environment. The integration of the collected aerosol with LB medium facilitated the cultivation of E. coli and enabled subsequent microbiological and molecular analyses. Air sampling was performed at predefined intervals (10, 30, 60, 90 and 120 minutes) by withdrawing bacterial LB fluid via hoses extended into the closed system from a peristaltic or syringe pump. This enabled the analysis of bacterial concentration as a function of sampling time. The LAMP technique was used to detect E. coli in the collected samples. Amplification reactions were performed at 65°C for 30 to 60 minutes using standard LAMP reagents. Furthermore, reaction chips were designed and moulds were produced using the prototype processing device (LPKF). These moulds were then used to produce chip assemblies using polydimethylsiloxane (PDMS). These chips were developed to facilitate the integration of the method into a compact, portable platform. In order to validate the LAMP results, conventional analytical techniques were applied. Air samples were then subjected to an incubation process at a temperature of 37°C for a duration of between 24 and 72 hours, utilising agar plates as a medium for bacterial cultivation. This procedure was employed to ascertain whether the samples were capable of capturing bacteria. The gel electrophoresis technique was utilised to evaluate the specificity of LAMP products. Moreover, an alternative verification method was implemented by conducting electrochemical measurements with a produced E. coli detection sensor. The findings of this study demonstrate that the developed detection system is capable of identifying airborne E. coli bioaerosols with high sensitivity and in significantly shorter time frames compared to conventional methods. In contrast to culture-based techniques, which necessitate protracted incubation periods, the LAMP method yielded swift and dependable results. Furthermore, LAMP is particularly advantageous for detecting bacteria that have entered a viable but non-culturable (VBNC) state or have sustained cellular damage, conditions under which culture-based methods may fail to yield positive results. As a result of the experiments, the limit of detection (LOD) was determined as 65 CFU/L. The LOD is quite low compared to the literature for the early identification of bacterial contamination. Furthermore, the special primer design minimized the risk of cross-reactivity. The proposed detection system offers numerous practical advantages. The technology enables real-time monitoring of microbial air quality in diverse settings, including hospitals, public transportation systems, industrial facilities, and crowded indoor spaces. It is imperative to note that rapid detection is of particular significance during outbreaks of infectious diseases or in the context of bioterrorism threats. In such circumstances, timely intervention is of the essence to prevent widespread transmission. Moreover, the system's cost-effectiveness, portability, and ease of use render it especially beneficial in settings with limited resources and restricted access to laboratory facilities. In conclusion, this thesis demonstrates that the combination of a modified air sampling system, chip design, and LAMP technology provides an effective and practical approach for the rapid detection of airborne bacterial pathogens. The system addresses the limitations of conventional methods and presents a scalable and adaptable platform for further development. Future research could focus on expanding the detection range to include additional pathogens, developing automated sample flows incorporating the microfluidic chip, and incorporating wireless communication capabilities for remote monitoring and data sharing. The multidisciplinary approach adopted in this study, combining microbiology, chip geometry design, molecular diagnostics, and engineering, has contributed significantly to the advancement of next-generation biosensor technologies that can operate outside traditional laboratory settings. In summary, this study proposes a novel and robust methodology for detecting airborne bioaerosols. This approach integrates a new air sampling system, automatic sample collection, chip design, and a rapid and easily implementable isothermal nucleic acid amplification technique (LAMP) for detection. The results of the study provide a valuable contribution to the fields of environmental microbiology, public health monitoring, and biosensor development. In view of the persistent threat posed by airborne pathogens, there is an urgent need for the development of rapid and portable diagnostic systems. This thesis provides a solid foundation for future innovations in the real-time detection of bioaerosols and the protection of human health.

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