식품 내 병원성 대장균 O157:H7의 검출을 위한 압타머 기반의 이중기능링커 개발 및 금 나노입자 응집 기반 비색 어세이 개발

학위논문(석사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부, 2023. 2. 최영진.In this study, we developed a colorimetric assay with bi-functional gold nanoparticle (AuNP) based on aptamer for sensitive detection of Escherichia coli O157:H7 using the aggregation of AuNP linked with a complementary DNA aptamer specific to E. coli O157:H7 aptamer. AuNP was used as a colorimetric linker and probe, respectively, and E. coli O157:H7 aptamer and its complementary DNA sequences were modified on each AuNP surface. AuNP labeled with E. coli O157:H7 aptamer was used as a bi-functional linker (BL) in a colorimetric competitive assay. Colorimetric assay with BL based on aptamer detected target bacteria by sequential competitive binding between colorimetric probes, which are AuNP having complementary sequences of E. coli O157:H7 (AA, Aggregation Assistant) and E. coli O157:H7 aptamer. In addition, under optimized conditions by main testing parameters, the assay exhibited a detection range of 101 to 106 CFU/400 µL and a detection limit of 57 ± 0.5 CFU/400 µL. In addition, the developed colorimetric assay with BL based on aptamer was excellently evaluated as an on/off detection system in coconut water, a real matrix. Thus, the proposed colorimetric competitive assay with BL based on aptamer can be a proper on-site detection method of foodborne pathogens. In addition, this study provides the basis for research into developing different colorimetric assay with aptamer for pathogen analysis in real matrices. This colorimetric assay with BL based on aptamer functions by driving the system between two competitive interaction dynamics: A) affinity between the aptamer sequence and the target, and B) affinity between the aptamer and the its complementary DNA aptamer.이 연구에서 우리는 E. coli O157에 특이적인 압타머와 상보적 DNA 압타머가 결합된 AuNP의 응집에 기초하여 E. coli O157:H7의 현장에서 신속하며 민감하게 검출할 수 있는, 압타머가 결합된 이중 기능링커 AuNP를 이용한 비색 검출법을 개발하였다. 이 시스템에서 AuNP는 이중기능링커 및 응집보조 프로브로 사용되었으며, 각 AuNP 표면은 E. coli O157:H7 대상 압타머와 상보적인 DNA 서열의 압타머로 결합되었으며, 이는 각각 이중기능링커 AuNP (BL)와 응집보조 프로브 AuNP (AA)로 명명하였다. BL은 대상균이 존재하지 않을 때에는 상보적인 서열에 의해 AA와 결합하였으며, 양적균형에 의해 특정영역(REVC, 120 – 220 μL)에서 응집 및 침전이 일어났다. 하지만, 대상균이 투입되었을 때에는 경쟁적 결합 우선순위에 의해 상보적 서열의 AA가 아니라 대상균인 E. coli O157:H7과 결합하게 된다. 이는 기존의 양적균형에서의 차이를 일으키게 되고 대조군과 유사한 응집 및 침전이 일어나기 위해서는 기존보다 더 많은 양의 BL이 반응에 참여하여야 한다. 이를 통해 대조군과 대상균 간에는 REVC 영역의 변화가 발생하게 되며, 이를 통해 대상균의 유무를 확인할 수 있었다. 검출은 가장 민감하며 구분되는 BL 영역인 100, 120, 140 μL에서 진행되었으며, 최적화된 조건에서 101 ~ 106 CFU/400 μL의 검출 범위와 57 ± 0.5 CFU/400 L의 검출 한계를 나타냈다. 또한 압타머 기반의 BL을 이용한 비색분석법은 실제 매트릭스인 코코넛워터에서도 on/off 감지 시스템으로 매우 우수하게 평가되었다. 따라서 제안된 압타머 기반의 BL을 이용한 비색분석법은 식중독을 일으키는 병원체를 현장에서 검출하는 데 적합한 방법이 될 수 있을 것으로 기대된다. 또한, 본 연구를 통해 실제 매트릭스에서 병원균 분석을 위한 앱타머 기반 비색 분석 개발에 대한 향후 연구의 기반을 제공할 수 있었다. 이 압타머 기반 비색 분석법은 A) 압타머 서열과 표적 사이의 친화성, B) 압타머와 그 상보적인 DNA 압타머 사이의 친화력이라는 두 가지 경쟁적인 상호 작용 역학에 의해 구동되었다.Ⅰ. INTRODUCTION １ Ⅱ. MATERIALS AND METHODS 5 2.1. Materials 5 2.2. Instrumentation 5 2.3. Culturing of Bacteria 6 2.4. Preparation of gold nanoparticles (AuNP) 6 2.5. Modification of AuNP with aptamer 7 2.6. Optimization of NaCl concentrations 8 2.7. Detection of E. coli O157:H7 in samples 8 2.8. Verification of the selectivity of the detection method 9 2.9. Statistical analysis 10 Ⅲ. RESULTS AND DISCUSSION 11 3.1. Overall detection procedures 11 3.2. Optimization of NaCl concentrations 17 3.3. Selectivity of the colorimetric assay 25 3.4. The range of REVC 27 3.5. Detection of E. coli O157:H7 in HEPES buffer 29 3.6. Detection of E. coli O157:H7 in coconut water 34 CONCLUSION 39 REFERENCES 41 국문초록 45석

Short term outcomes of topiramate monotherapy as a first-line treatment in newly diagnosed West syndrome

PurposeTo investigate the efficacy of topiramate monotherapy in West syndrome prospectively.MethodsThe study population included 28 patients (15 male and 13 female children aged 2 to 18 months) diagnosed with West syndrome. After a 2-week baseline period for documentation of the frequency of spasms, topiramate was initiated at 2 mg/kg/day. The dose was increased by 2 mg/kg every week to a maximum of 12 mg/kg/day. Clinical assessment was based on the parents' report and a neurological examination every 2 weeks for the first 2 months of treatment. The baseline electroencephalograms (EEGs) were compared with the post-treatment EEGs at 2 weeks and 1 month.ResultsWest syndrome was considered to be cryptogenic in 7 of the 28 patients and symptomatic in 21 patients. After treatment, 11 patients (39%) became spasm-free, 6 (21%) had more than 50% spasmsreduction, 3 (11%) showed less than 50% reduction, and 8 (29%) did not respond. The effective daily dose for achieving more than 50% reduction in spasm frequency, including becoming spasm-free, was found to be 5.8±1.1 mg/kg/day. Nine patients (32%) showed complete disappearance of spasms and hypsarrhythmia, and 11 (39%) showed improved EEG results. Despite adverse events (4 instances of irritability, 3 of drowsiness, and 1 of decreased feeding), no patients discontinued the medication.ConclusionTopiramate monotherapy seems to be effective and well tolerated as a first line therapy for West syndrome and is not associated with serious adverse effects

Characterizations of realized metal-insulator-silicon-insulator-metal waveguides and nanochannel fabrication via insulator removal

We investigate experimentally metal-insulator-silicon-insulator-metal (MISIM) waveguides that are fabricated by using fully standard CMOS technology. They are hybrid plasmonic waveguides, and they have a feature that their insulator is replaceable with functional material. We explain a fabrication process for them and discuss fabrication results based on 8-inch silicon-on-insulator wafers. We measured the propagation characteristics of the MISIM waveguides that were actually fabricated to be connected to Si photonic waveguides through symmetric and asymmetric couplers. When incident light from an optical source has transverse electric (TE) polarization and its wavelength is 1318 or 1554 nm, their propagation losses are between 0.2 and 0.3 dB/mu m. Excess losses due to the symmetric couplers are around 0.5 dB, which are smaller than those due to the asymmetric couplers. Additional measurement results indicate that the MISIM waveguide supports a TE-polarized hybrid plasmonic mode. Finally, we explain a process of removing the insulator without affecting the remaining MISIM structure to fabricate similar to 30-nm-wide nanochannels which may be filled with functional material.open8

Characterization of the Lytic Bacteriophage phiEaP-8 Effective against Both Erwinia amylovora and Erwinia pyrifoliae Causing Severe Diseases in Apple and Pear

Bacteriophages, bacteria-infecting viruses, have been recently reconsidered as a biological control tool for preventing bacterial pathogens. Erwinia amylovora and E. pyrifoliae cause fire blight and black shoot blight disease in apple and pear, respectively. In this study, the bacteriophage phiEaP-8 was isolated from apple orchard soil and could efficiently and specifically kill both E amylovora and E. pyrifoliae. This bacteriophage belongs to the Podoviridae family. Whole genome analysis revealed that phiEaP-8 carries a 75,929 bp genomic DNA with 78 coding sequences and 5 tRNA genes. Genome comparison showed that phiEaP-8 has only 85% identity to known bacteriophages at the DNA level. PhiEaP-8 retained lytic activity up to 50 degrees C, within a pH range from 5 to 10, and under 365 nm UV light. Based on these characteristics, the bacteriophage phiEaP-8 is novel and carries potential to control both E. amylovora and E. pyrifoliae in apple and pear

Comparative analysis of FBS containing media and serum free chemically defined media, CellCor for adipose derived stem cells production

Background: As a result of the aging society, the average OECD life expectancy has grown to about 80 years, yet the average health life still remains at only 65 years, leaving more than 15 years of life in an uncertain health state. Regenerative medicine is a new concept of medicine that combines cells and biomaterials to restore the functions of aged or damaged tissues or organs. It is also a good treatment for chronic diseases and incurable diseases, receiving attention as a new paradigm for treating diseases. Problems: As the market for regenerative medicine grows, mass production of consistent quality cells is required. Media is the most important thing in mass production of consistent quality cells. However, the fetal bovine serum (FBS) containing media that is currently wide used has many problems, such as unidentified viral infection, immunogenicity, lot variations, unstable supply, and ethical issues. To solve these problems and make rapid progress in regenerative medicine, a high-performance serum free chemically defined media (CDM) is needed. Solution: CellCor is a serum free CDM that provides excellent performance, safety, economy and consistency in stem cell production. CellCor allows higher-speed cell production rate than current FBS containing culture media (Figure 1). Compared to the FBS containing media, CellCor is able to maintain stem cell markers, higher population homogeneity, genetic stability, and excellent differentiation potency even at later passage. Please click Additional Files below to see the full abstract