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研究生：	葉曜緯 Yeh, Yao-Wei
論文名稱：	發展速效多重光源滅菌技術與多功能水凝膠敷料在防止感染之應用 Development of Rapid Multi-wavelength LED Sterilization Technology and Multifunctional Hydrogel Dressings for Infection Prevention Applications
指導教授：	吳炳慶 Wu, Ping-Ching
學位類別：	博士 Doctor
系所名稱：	工學院 - 生物醫學工程學系 Department of BioMedical Engineering
論文出版年：	2023
畢業學年度：	111
語文別：	英文
論文頁數：	120
中文關鍵詞：	多波長、發光二極體、光動力失活、活性氧類、核苷酸切除修復、環丁烷嘧啶二聚體、光裂合酶、抗生素、水凝膠、超音波影像
外文關鍵詞：	Multi-wavelength, Light-emitting Diodes, Photodynamic Inactivation, Reactive Oxygen Species, Nucleotide Excision Repair, Cyclobutane Pyrimidine Dimer, Photolyases, Antibiotics, Hydrogels, Ultrasound Image
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感染控制的議題在流行傳染病肆虐的近年來扮演著相當重要的角色。濫用抗生素所產生的抗藥性菌種不僅造成臨床照護的難度，更是大幅地增加感染的可能性。本研究將針對接觸器械及傷口照護所造成的感染問題進行探討。
首先，結合紫外光及藍光兩種波長的LED光源與核黃素搭配，可以有效降低紫外光的照射劑量。由於汞的使用受到法規的限制，傳統的紫外汞燈已無法使用。並且在低紫外線波段的發光二極體製程存在著技術上的瓶頸，因此發展滅菌的替代方案勢在必行。結果顯示，利用藍光與核黃素對微生物造成的光動力失活效應與紫外光搭配，不同的照射序列會有不同的殺滅效果。透過活性氧對DNA的鹼基對結構造成破壞，接著輔以紫外光造成DNA損傷產生環丁烷嘧啶二聚體和6-4光產物。原先受到藍光照射產生的光裂合酶暴露於紫外光會降低酶活性並誘導過氧化氫的積累，進而提高殺菌的效果。
另一方面，在傷口照護造成感染問題的部分，開發結合桿菌肽、硫酸新黴素和多黏菌素B三種抗生素的水凝膠敷料。有別於市售敷料僅有隔絕接觸的作用，此多功能水凝膠敷料具有多孔結構可增加吸收組織液的能力，並在再水化之後延展性及柔軟度也大幅提升。經過分析，抗生素包覆率>88%，並且可以在48小時內持續地釋放。同時此款水凝膠具有高生物相容性及良好的抑菌效果。更可以在人體及模擬皮膚於微生物生長的情況下透過30 MHz的頻率在B模式獲取清晰的即時超音波影像。本研究針對可能造成接觸感染的手術器械與衛生用品提供一種快速滅菌的方法，以及可同時保護、治療及監控傷口是否感染的新型敷料。由造成感染的源頭到後續的保護監控提供一套完整的解決方案。

The issue of infection control has played a very important role in recent years as infectious disease epidemics have raged. Drug-resistant bacteria produced by the misuse of antibiotics not only complicate clinical care, but also greatly increase the possibility of infection. This study will address the issue of infections due to exposure to instruments and wound care.
First, the combination of the dual-wavelength ultraviolet (UV) and blue light emitting diode (LED) source with riboflavin 5'-phosphate can effectively reduce the exposure dose of UV light. Conventional mercury UV lamps are no longer available due to regulatory restrictions on the use of mercury. In addition, there are technical bottlenecks in the process of LED in the low UV band, so it is imperative to develop alternative solutions for sterilization. The results show that the photodynamic inactivation effect of blue light and riboflavin-5'-phosphate on microorganisms is combined with UV light, and different irradiation sequences have different sterilization effects. The base pair structure of DNA is damaged by reactive oxygen species (ROS) and then supported by UV light to cause DNA damage to produce cyclobutane pyrimidine dimer (CPD) and 6-4 photoproduct (6-4PP). Exposure of CPD photolyases, originally produced by blue light, to UV light reduces enzyme activity and induces the accumulation of hydrogen peroxide, thereby enhancing the bactericidal effect.
On the other hand, in the part of the wound where there is a problem of infection, a hydrogel dressing that combines three antibiotics - bacitracin, neomycin sulfate and polymyxin B - has been developed. Unlike commercially available dressings that function only to isolate contact, this multifunctional hydrogel dressing has a porous structure that can increase the ability to absorb interstitial fluid, and its extensibility and softness are also greatly improved after rehydration. After analysis, the antibiotic coverage rate is >88%, and it can be released continuously within 48 hours. At the same time, this hydrogel has high biocompatibility and good antibacterial effect. It can also obtain clear real-time B-mode ultrasound images through the frequency of 30 MHz in the case of microbial growth in the human body and simulated skin. This study provides a rapid sterilization method for surgical instruments and hygiene products that can cause contact infection, as well as a new type of dressing that can simultaneously protect, treat and monitor wound infection. Providing a complete solution from the source of infection to subsequent protection monitoring.

摘要　I
ABSTRACT　II
ACKNOWLEDGEMENT　IV
CONTENTS　V
LIST OF FIGURES　VIII
LIST OF TABLES　XI
NOMENCLATURE　XII
CHAPTER 1 INTRODUCTION　1
1	The Importance of Medical Device Sterilization　1
1.1	The Risk of Surgical Site Infection (SSIs)　1
1.2	Sterilization Validation of Medical Devices　2
1.3	Difficulties and Problems in the Sterilization of Medical Devices　4
1.4	Other Infectious Diseases　5
2	Photoinactivation　6
2.1	Ultraviolet Sterilization　6
2.2	Restrictions on the Use of UV Light Source Types　8
2.3	Technical Barriers to LED Manufacturing　9
2.4	Near-infrared Sterilization　11
2.5	Photodynamic Inactivation (PDI)　12
3	The Importance of Preventing Wound Infection　15
3.1	Extrinsic Factors and Types of Wound Infection　15
3.2	Severity of Wound Infection　18
3.3	The Importance of Early Prevention of Wound Infection and Healing　20
4	Development of Antibacterial Dressings to Prevent Wound Infection　22
4.1	Development of Multifunctional Antibacterial Dressings　22
4.2	Restrictions and Technical Limitations of Antimicrobial Dressings　24
5	Technology for Early Detection of Bacteria to Prevent Infection　24
5.1	Traditional Clinical Visual Inspection, Pathological Examination　24
5.2	Application of High Frequency Ultrasound in Infection Detection　26
6	Specific Aim　28
CHAPTER 2 MATERIAL & METHODS　30
1	Materials and Agents　30
2	Experimental Instrument　31
3	Multi-wavelength Light-emitting Diodes Module　32
4	Spectrometry of Multi-wavelength LED　34
5	Microbial Culture　34
6	Microbial Survival Fraction Calculation　35
6	Temperature Measurement　36
7	Measurement of ROS　37
8	Integrity Assay of Genomic DNA in E. coli　37
9	Cyclobutane Pyrimidine Dimer Quantitation　37
10	Simulated Sterilization on a Toothbrush　38
11	Fabrication of the Multiple Antibiotics Hydrogels　38
12	Determination of Antibiotic-containing Hydrogels　38
13	In Vitro Drug Release Study　40
14	Morphological Analysis　40
15	Tensile Testing of the Hydrogels　41
16	Water Absorption Efficacy Measurements　41
17	Antimicrobial Analysis　42
18	Cell Cytotoxicity Assay　42
19	Ultrasound Attenuation Coefficient　43
20	Wire Phantom on Material　43
21	B-mode Imaging of Human Skin　44
22	Detection of Bacterial Colonies in Ultrasound B-mode Images　44
CHAPTER 3 INFECTION PREVENTION IN MEDICAL DEVICES　45
1	The 1st Gen of Dual-wavelength LED Sterilization System　45
1.1 Output Current and Corresponding Power　45
1.2 Monochromatic LED Sterilization Effect　47
1.3 The Sterilization Effect of Synchronous Irradiation with UV and NIR Light　48
1.4 The Sterilization Effect of Asynchronous Irradiation with UV and NIR Light　49
1.5 The Sterilization Effect of NIR Monochromatic LED Intermittently of Microorganisms　50
1.6 Evaluation of the Bactericidal Effect of Dual-wavelength LED Combinations on Microorganisms　52
2	The 2nd Gen of Dual-wavelength LED Sterilization System　54
2.1 Output Current and Corresponding Power　54
2.2 Temperature Effect on Samples After LED Irradiation　56
2.3 The Sterilization Effect of 270 nm UV Monochromatic Light　59
2.4 The Amount of ROS Produced by 400 nm Blue Light Combined with FMN as the Photosensitizer　60
2.5 The Sterilization Effect of 400 nm Blue Monochromatic Light　61
2.6 The Sterilization Effect of 270 nm UV Monochromatic Light with FMN　62
2.7 The Sterilization Effect of Synchronous Irradiation with UV and Blue Light　63
2.8 Comparison of Inactivation Effects of UV Monochromatic LED and Dual-Wavelength LED Asynchronous Irradiation　64
3	The 3rd Gen of Dual-wavelength LED Sterilization System　65
3.1 Output Current and Corresponding Power　65
3.2 Effect of 255 nm LED on the Temperature of the Sample After Irradiation　67
3.3 The Sterilization Effect of 255 nm Monochromatic Light without FMN　69
3.4 The Sterilization Effect of 255 nm Monochromatic Light with FMN　70
4	Synergistic Mechanism of Dual-wavelength LED Sterilization　71
4.1 Integrity assay of genomic DNA　71
4.2 Effect of Dual-wavelength LED on CPD　74
5	Simulate the Actual Microorganism Sterilization Scenario　75
5.1 Effect of Temperature on the Toothbrush After LED Irradiation　75
5.2 Simulated Bactericidal Effect on Toothbrushes　76
6	Discussion　77
CHAPTER 4 INFECTION PREVENTION IN WOUNDS　88
1	Multifunctional Hydrogel Dressing Properties　88
1.1 Hydrogel Loading Capacity of Antibiotics　88
1.2 Surface Structure　90
1.3 Tensile Mechanical Properties　92
1.4 Hydrogel Swelling Capacity　93
2	Antibacterial Properties of Multifunctional Hydrogel Dressing　93
2.1 Hydrogel Antibiotic Release Rate　93
2.2 Biocompatibility　94
2.3 Zone of Inhibition　95
3	Real-time Ultrasonic Image Detection Function　96
3.1 Attenuation Coefficients of Hydrogels and Commercial Synthetic Skin　96
3.2 Ultrasound B-mode Imaging Quality　98
3.3 Ultrasound B-mode Scans of Human Skin　99
3.4 Ultrasound B-mode Scans of Colonies on LB Agar　100
3.5 Ultrasound B-mode Scans of Simulated Wound　101
4	Discussion　103
CHAPTER 5 CONCLUSION　107
REFERENCES　108
                                    

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