細菌的研究自從十七世紀以來已經蓬勃發展至今。細菌在臨床分析、食品研究以及環境中扮演了非常重要的角色。因此，在於這些領域中去開發新的細菌偵測方法就顯得日益重要，並且也成為了目前的顯著指標。在過去已經有許多相當成功的方法用來偵測細菌種類，例如基因技術中的聚合酶連鎖反應(PCR)或是細菌培養等，但是缺點是相當耗時耗力，所需的實驗材料也較多。有鑑於此，本篇論文開發出新的偵測方法，利用離子溶液、離子液體以及奈米粒子等技術來提升細菌的萃取效率，並結合基質輔助雷射脫附游離質譜儀，樣品用量極少，快速、簡單地分析真實樣品中的細菌。
論文第一個部分，從傳統超商購得的新鮮優格做細菌的偵測實驗，由於市售優格大多數含有對人類腸道有助益的益生細菌，所以針對市售優格中的細菌做探討及研究是極其重要的。市售優格中都含有的特定的益生菌種，所以本論文的目標為分析優格中含有的已知細菌菌種。優格中含有豐富的奶製品成分，而使得利用質譜儀偵測優格中細菌會受到奶製品的訊號所干擾，所以本論文使用奈米粒子以及離子溶液去抓取細菌並提升了細菌偵測的訊號強度，由結果顯示在添加了離子溶液及銀奈米粒子後可有效地提升細菌偵測的訊號強度。此外，本論文也針對過期的優格分別儲存於冰箱以及室溫下做分析，比對新鮮優格中所含有的細菌數目和細菌菌落做定量分析，並將優格中的細菌置入乳酸桿菌培養基中做培養作為細菌比對的用途。另外則是連續七天針對存放在冰箱以及室溫下的兩種市售優格做偵測，並且也對優格細菌做培養以利日後細菌的比對。最後將細菌菌落數的數據做彙整，即可得知市售優格中添加的益生細菌在不同存放時間和存放條件下，也會有著不同的結果，進而對細菌消長做分析。
本論文第二個部分，利用基質輔助雷射脫附游離質譜儀於細菌感染的臨床分析研究上，利用基質輔助雷射脫附游離質譜儀偵測老鼠血液和尿液中的致病細菌-金黃色葡萄球菌。由於金黃色葡萄球菌是一種非常普及且常見的致病細菌，會導致人類產生一些皮膚和黏膜上的疾病，嚴重甚至導致死亡，所以在本論文的第二部份討論金黃色葡萄球菌在老鼠體內的消長和分析。另外我們也利用離子液體結合單滴微萃取技術在體外實驗中成功萃取純水中的金黃色葡萄球菌，並且大幅降低所能偵測到的偵測濃度限制至約105 cfu/mL。另外在老鼠的體內實驗中，首先，我們先將金黃色葡萄球菌注射到老鼠的內腹腔中，之後隨著固定時間點，從老鼠尾部取血，並且也採集尿液，最後分別置入營養瓊脂培養基中做細菌培養，培養後的細菌可做為實驗菌種的比對。同時也利用基質輔助雷射脫附游離質譜儀去分析血液和尿液中所含有的圖譜資訊，並且與金黃色葡萄球菌的標準品做質譜訊號比較，即可對血液和尿液中的細菌進行定性分析。然而由於老鼠的血液和尿液的背景干擾很大，尤其是血液，會大幅的降低我們所能偵測血液中的金黃色葡萄球菌的靈敏度。故我們開發了一些提升偵測靈敏度的方法，例如將被細菌感染的血液做前處理，或者是利用離子液體做萃取，成功地萃取出從被感染的老鼠其血液裡的金黃色葡萄球菌。

Bacterial research has been flourishing ever since 17th century. Bacteria play an
important role in clinical microbiology, food microbiology and environmental microbiology. Therefore, the rising need to detect bacteria in clinical, food and environmental samples is emerging onto a craving research frenzy. There are some established methods to study bacteria, such as genetic techniques like PCR, culturing and RNA based analysis, but these require time, resources and labor. This thesis concentrates on highlighting the importance of MALDI-MS in rapid, direct, and simple detection of bacteria from food and clinical samples. The method is to enable detection of bacteria in real samples.
First attempts were made to detect bacteria from yogurt sample, because as already known commercial yogurt samples contain bacteria. And yogurts were used in this study to demonstrate the capability of MALDI-MS technique to detect the bacteria directly by culture-free methods. Since the bacterial signals were quenched because of the strong milk peaks, we had to employ nanoparticles and ionic solution for affinity capture of the bacteria for bacterial detection. The results showed that addition of ionic solution and Ag nanoparticles lead to enhanced bacterial detection. The deterioration of the yogurt bacteria on improper storage ( at room temperature) and after extended storage in refrigerator beyond expiry date was studied using MALDI-MS. The day by day deterioration in the microbiological quality of the yogurt was also detected. A special selective media for growth of the yogurt bacteria, DifcoTM Lactobacilli MRS Broth (Agar) was used to quantify the bacteria in various experiments. MALDI-MS results were useful in understanding the shelf life and quality control on extended storage.
The second part of this thesis concentrates on the use of MALDI-MS in clinical analysis. The major goal in this work was to use MALDI-MS to detect bacteria in mousy blood and urine. Phase I of this study involved detection of the bacteria by direct MALDI-MS in blood and urine samples spiked with bacteria. We call these experiments as in vitro experiments. Analysis using MALDI was a challenge since the blood peaks interfered with the bacterial peaks and subdued them. Ionic liquid is a pretty excellent extraction solvent when combined with SDME (Single-drop Microextraction). Just one drop of 1-butyl-3methylimidazolium hexafluorophosphate can extract bacteria from spiked samples. What’s more, the LOD can be lower to ~105 cfu / mL using SDME with ionic liquid. It’s definitely showing that 1-butyl-3methylimidazolium hexafluorophosphate, that is to say, the ionic liquid has good ability to trap pathogenic bacteria.
This bacterium is injected into mouse and the growth kinetics of bacteria in vivo was studied as a function of time. Samples taken at specific time points were simultaneously analyzed using standard plate count method as well as MALDI-MS. Analysis using MALDI was a challenge since the blood peaks interfered with the bacterial peaks and subdued them. Therefore, we employ the use of silver and zinc oxide nanoparticles and ionic liquid.

Catalog
Acknowledgement………………………………………………………...……………...i
Chinese abstract…………………………………….……………………………...…..ii English abstract……………………………………………………….……………...…iv
Catalog………………….…………………………………………….…………..…….vii
Figure catalog………………..…………………………………………………………xii
Table catalog………………………………………………………………...………..xxiv

Chapter One Introduction...............................................................................................1
1-1 Prelogue……...…………………………………………………………………….1
1-2 The development of bacterial study…………………….…………………………2
1-2-1 Characteristics of bacteria- their classification………………..………………2
1-3 Identification of bacteria using molecular tools……….…………………………..5
1-3-1 PCR (Polymerase Chain Reaction)…………………………………...……….5
1-3-2 RAPD (Random Amplification of Polymorphic DNA)……………………….6
1-3-3 RFLP (Restriction Fragment Length Polymorphism)…………………………6
1-4 Matrix-Assisted Laser desorption/ionization mass spectrometry (MALDI-MS)…7
1-5 Bacterial analysis in MALDI-MS……………………………………………...….8
1-6 Nanoparticle (NPs) based MALDI-MS bacterial studies………………………….8
1-6-1 TiO2 as a good bacterial trapping to the pathogenic bacteria…………...……11
1-6-2 Biofunctionalized Au nanoparticles as good bacterial trap for
pathogenic bacteria…………………………………………………………11
1-7 The motivation for this thesis…………………………………………………….12

Chapter Two Ionic solution and nanoparticles assisted MALDI-MS as bacterial biosensors for rapid yogurt analysis…………………………………...…………...…..13
2-1 Experiments……………………………………………………….……………...13
2-1-1 Instruments…………………………………………………..……………...13
2-1-2 Chemicals……………………………………………………………...……..15
2-2 Experimental steps and procedures………………………………………………16
2-2-1 Synthesis of Silver nanoparticles………………………………...…………..16
2-2-2 Description of real sample………………………………...…………...…….17
2-2-3 Culturing methods and quantification for yogurt bacteria……………...……18
2-2-3-1 Methodology for counting bacteria..………………………………..18
2-2-4 Sample preparation for MALDI-MS………………………………………...20
2-2-5 Microscope observation to see morphology of yogurt bacteria……………...21
2-3 Result and discussion…………………………………………………………….23
2-3-1 Yogurt bacteria cultured in different medium………………………………..25
2-3-2 Direct analysis of fresh yogurt sample using MALDI-MS…………...……..26
2-3-3 Direct analysis of expired yogurt sample using MALDI-MS…………...…..29
2-3-4 Bacteria growth of fresh yogurt and expired yogurt…………………………47
2-3-5 Quantification of bacteria in yogurt sample………………...……………….48
2-3-6 Siver nanoparticles effect on the yogurt bacteria…………………………….53
2-3-7 Ionic solution and ionic liquid effect on the yogurt bacteria………………...56
2-4 Conclusions………………………………………………………………………60

Chapter Three Using ionic liquid as a highly selective affinity probe to detect pathogenic bacteria in clinical samples such as blood and urine and also to study the in vivo growth kinetics of the pathogen Staphylococcus aureus………………...………..62
3-1 Experiments…………………………………………………………………......62
3-1-1 Instruments…………………………………………………………………...62
3-1-2 Chemicals…………………………………………………………………….64
3-1-3 Reasons for using Staphylococcus aureus as the test organism……………...65
3-1-4 Culturing methods of staphylococcus aureus………………………………..67
3-1-4-1 Opening a double-vial……………………………………………..67
3-1-4-2 Recovering culture from lyophilized specimen…………..………..67
3-1-4-3 Culturing of bacteria for experiment………………………………68
3-1-4-3-1 Standard protocol for counting bacteria……………….68
3-1-4-3-2 Standard protocol for counting bacteria………….…….69
3-1-4-3-3 Standard protocol for counting bacteria………….…….69
3-1-5 Experimental procedures followed…………………………...….…………..70
3-1-6 Use of Ionic liquid on bacterial research…………………………………….72
3-1-7 Single Drop Micro-extraction ( SDME )…………………………………….72
3-2 Result and discussion…………………………………………….………………74
3-2-1 Staphylococcus aureus appearance under TEM, and nutrient agar plate…….74
3-2-2 Fluorescence microscopic studies of Staphylococcus aureus………..............75
3-2-3 Direct analysis of standard Staphylococcus aureus using MALDI-MS using traditional SA matrix………………………………………………………….76
3-2-4 In-vitro detection of Staphylococcus aureus in blood and urine using traditional SA matrix…………………………………………………….…..78
3-2-5 Ionic liquid combined with SDME successfully trapping staphylococcus aureus in water…………………………………………………………….....82
3-2-6 Phase II- Tracing the In-vivo growth kinetics of S. aureus in blood and urine using Mice models…………………………………………………………...85
3-2-6-1 In vivo studies for tracing the pathogen by standard plate count method…………………………………………………………...…87
3-2-6-2 In vivo studies for tracing the pathogen by MALDI-MS method....92
3-2-7 Improvement of bacterial detection using sample pretreatment……………..95
3-2-8 MALDI-MS direct rapid detection of the pathogen invivo in urine samples..97
3-2-9 Conclusions…………………………………………………………………100
Reference……………………………………………………………………………...100














Figure Catalog
Fig. 1. Synthesis of silver nanoparticles……………………………...………………...17
Fig. 2. The scheme of the yogurt experiment…………………………………………..22
Fig. 3. (a) TEM photo of the synthesized silver nanoparticles (b) UV-vis spectra of the synthesized silver nanoparticles………………………………………...……………...23
Fig. 4. (a) TEM photo of the fresh Lin Feng-Yin yogurt (b) TEM photo of the fresh AB yogurt (c) Fluorescence micrograph of AB yogurt bacteria with acridine orange staining (d) Fluorescence micrograph of Lin yogurt bacteria with acridine orange staining (e) Fluorescence micrograph of AB yogurt bacteria……………………………………….24
Fig. 5. (a) The image of AB yogurt cultured in the LB agar plate (b) The image of AB yogurt cultured in the nutrient agar plate (c) The image of AB yogurt cultured in the DifcoTM Lactobacilli MRS Broth agar plate……………………………...…………….26
Fig. 6. Direct analysis yogurt using 50mM of SA as matrix of (a) fresh AB yogurt (b) fresh Lin Feng-Yin yogurt……………………………………………………………...27
Fig. 7. Direct analysis of fresh AB yogurt using 50mM of SA as matrix. (a) without
any dilution. (b) is 101 dilution. (c) is 102 dilution. (d) is 103 dilution. (e) is 104 dilution………………………………………………………………………………….28
Fig. 8. Direct analysis of fresh Lin Feng-Yin yogurt using 50mM of SA as matrix. (a) is without any dilution. (b) is 101 dilution. (c) is 102 dilution. (d) is 103 dilution. (e) is 104 dilution.………………………………………………………………...……………….29
Fig. 9. AB yogurt in the refrigerator and analysis by MALDI-MS. SA matrix is 50
mM (a) is fresh yogurt after got from the convenience store. (b)~(h) is expired 1~7 days but also stored in the refrigerator. (b) 1 day expired. (c) 2 days expired. (d) 3 days expired. (e) 4 days expired. (f) 5 days expired. (g) 6 days expired. (h) 7 days expired.
…………………………………………………………...……………………………..31
Fig. 10. AB yogurt in the laminar flow at room temperature and analyzed by
MALDI-MS. SA matrix is 50 mM. (a) is fresh yogurt after got from the convenience store. (b)~(h) is expired 1~7 days but also stored in the laminar flow at room temperature. (b) 1 day expired. (c) 2 days expired. (d) 3 days expired. (e) 4 days expired. (f) 5 days expired. (g) 6 days expired. (h) 7 days expired……………………………...32
Fig. 11. Lin Feng-Yin yogurt in the refrigerator and analysis by MALDI-MS. SA
matrix is 50 mM. (a) is fresh yogurt after got from the convenience store. (b)~(h) is expired 1~7 days but also stored in the refrigerator. (b) 1 day expired. (c) 2 days expired. (d) 3 days expired. (e) 4 days expired. (f) 5 days expired. (g) 6 days expired. (h) 7 days expired…………………………………………………..……………………………...33
Fig. 12. Lin Feng-Yin yogurt in the laminar flow at room temperature and analyzed by MALDI-MS. SA matrix is 50 mM. (a) is fresh yogurt after got from the convenience store. (b)~(h) is expired 1~7 days but also stored in the laminar flow at room temperature. (b) 1 day expired. (c) 2 days expired. (d) 3 days expired. (e) 4 days expired. (f) 5 days expired. (g) 6 days expired. (h) 7 days expired………….…………………..34
Fig. 13. AB yogurt stored in the refrigerator and cultured by DifcoTM Lactobacilli
MRS Broth agar day by day. After cultured we scraped the bacteria and then put into 500μL of deionized water and incubated for five minutes. After that we used
MALDI-MS to do the analysis. SA matrix is 50 mM. (a) is fresh yogurt after got from the convenience store. (b)~(h) is expired 1~7 days but also stored in the refrigerator. (b) 1 day expired. (c) 2 days expired. (d) 3 days expired. (e) 4 days expired. (f) 5 days expired. (g) 6 days expired. (h) 7 days expired………………………………………...35
Fig. 14. AB yogurt stored in the laminar flow at room temperature and cultured by
DifcoTM Lactobacilli MRS Broth agar day by day. After cultured we scraped the
bacteria and then put into 500μL of deionized water and incubated for five
minutes. After that we used MALDI-MS to do the analysis. SA matrix is 50 mM. (a) is fresh yogurt after got from the convenience store. (b)~(h) is expired 1~7 days but also stored in the laminar flow at room temperature. (b) 1 day expired. (c) 2 days expired. (d) 3 days expired. (e) 4 days expired. (f) 5 days expired. (g) 6 days expired. (h) 7 days expired………………………………………………………………………………….36
Fig. 15. Lin Feng-Yin yogurt stored in the refrigerator and cultured by DifcoTM
Lactobacilli MRS Broth agar day by day. After cultured we scraped the bacteria
then put into 500μL of deionized water and incubated for five minutes. After that
we used MALDI-MS to do the analysis. SA matrix is 50 mM. (a) is fresh yogurt after got from the convenience store. (b)~(h) is expired 1~7 days but also stored in the refrigerator. (b) 1 day expired. (c) 2 days expired. (d) 3 days expired. (e) 4 days expired. (f) 5 days expired. (g) 6 days expired. (h) 7 days expired…………….………………..37
Fig. 16. Lin Feng-Yin yogurt stored in the laminar flow at room temperature and
cultured by DifcoTM Lactobacilli MRS Broth agar day by day. After cultured we
scraped the bacteria then put into 500μL of deionized water and incubated for five
minutes. After that we used MALDI-MS to do the analysis. SA matrix is 50 mM. (a) is fresh yogurt after got from the convenience store. (b)~(h) is expired 1~7 days but also stored in the laminar flow at room temperature. (b) 1 day expired. (c) 2 days expired. (d) 3 days expired. (e) 4 days expired. (f) 5 days expired. (g) 6 days expired. (h) 7 days expired………………………………………………………………………………….38
Fig. 17. Direct analysis of (a) fresh AB and (b) one-week spoilt AB yogurt using 50mM of SA as matrix…………………………………………………………………………40
Fig. 18. Direct analysis of (a) fresh Lin Feng-Yin and (b) one-week spoilt Lin Feng-Yin yogurt using 50mM of SA as matrix………………………………………………….41
Fig. 19. AB yogurt stored in the refrigerator and detected by date. SA matrix is 50 mM. (a) Fresh beyond the expired day. (b) One-week expired but stored in the refrigerator (c) Two-week expired but store in the refrigerator…………………...……………………42
Fig. 20. Lin Feng-Yin yogurt stored in the refrigerator and detected by date. SA matrix is 50 mM. (a) Fresh beyond the expired day (b) One-week expired but stored in the refrigerator (c) Two-week expired but store in the refrigerator………………………...43
Fig. 21. (a) Fresh AB yogurt direct analysis using MALDI-MS, we could see both milk protein and bacterial peaks. (b) Fresh AB yogurt cultured in the DifcoTM Lactobacilli MRS Broth agar and also scraped the bacteria from the plate and analyzed. SA matrix is 50 mM………………………………………….……………………………………….44
Fig. 22. (a) Two-week expired AB yogurt direct analysis using MALDI-MS, we could see only milk protein but no bacterial peaks. (b) Expired AB yogurt cultured in the DifcoTM Lactobacilli MRS Broth agar and also scraped the bacteria from the plate and analyzed. SA matrix is 50 mM……………………………………………..…………..45
Fig. 23. (a) Fresh Lin Feng-Yin yogurt direct analysis using MALDI-MS, we could see both milk protein and bacterial peaks. (b) Fresh Lin Feng-Yin yogurt cultured in the DifcoTM Lactobacilli MRS Broth agar and also scraped the bacteria from the plate and analyzed. SA matrix is 50 mM……………………………………………...………….46
Fig. 24. (a) Two-week expired Lin Feng-Yin yogurt direct analysis using MALDI-MS, we could see only milk protein but no bacterial peaks. (b) Expired Lin Feng-Yin yogurt cultured in the DifcoTM Lactobacilli MRS Broth agar and also scraped the bacteria from the plate and analyzed. SA matrix is 50 mM…………………………….……….47
Fig. 25. Two brands of yogurt cultured in the DifcoTM Lactobacilli MRS Broth agar plate and the temperature was set at 37oC. SA matrix is 50 mM. (a) is bacteria colonies cultured by fresh AB yogurt. (b) is bacteria colonies cultured by two-week expired but stored in the refrigerator AB yogurt. (c) is bacteria colonies cultured by fresh Lin Feng-Yin yogurt. (d) is bacteria colonies cultured by two-week expired but stored in the refrigerator Lin Feng-Yin yogurt……………………………………………………….48
Fig. 26. Both two brands of yogurt samples cultured in the DifcoTM Lactobacilli MRS Broth agar plate and then calculated the bacterial concentration in the yogurt
sample………………………………………………………….……………………….50
Fig. 27. (a) Bacterial growth curves showed bacterial growth of AB yogurt from
0-7 days. (b) Bacterial growth curves showed bacterial growth of Lin yogurt from 0-7 days. Bacteria were grown on the DifcoTM Lactobacilli MRS Broth agar plate…….....51
Fig. 28. Fresh AB yogurt adding to different concentration of silver nanoparticles and analyzed by MALDI-MS. SA matrix is 50 mM. (a) is without adding to any silver nanoparticles. (b) is adding 0.035 g AgNPs to the fresh AB yogurt. (c) is adding 0.35 g AgNPs to the fresh AB yogurt…………………………………………………….……55
Fig. 29. Fresh Lin Feng-Yin yogurt adding to different concentration of silver
nanoparticles and analyzed by MALDI-MS. SA matrix is 50 mM. (a) is without adding to any silver nanoparticles. (b) is adding 0.035 g AgNPs to the fresh AB yogurt. (c) is adding 0.35 g AgNPs to the fresh AB yogurt……………..……………………………56
Fig. 30. Fresh AB yogurt adding to kinds of ionic liquid and ionic solutions. And analyzed by MALDI-MS. SA matrix is 50 mM. (a) is without adding to any ionic liquid and ionic solutions. (b) is adding 1-butyl-3methylimidazolium hexafluorophosphate to the fresh AB yogurt. (c) is adding CrO42- to the fresh AB yogurt. (d) is adding PO43- to the fresh AB yogurt…………………………………………………….……………….58
Fig. 31. Fresh Lin Feng-Yin yogurt adding to kinds of ionic liquid and ionic solutions. And analyzed by MALDI-MS. SA matrix is 50 mM. (a) is without adding to any ionic liquid and ionic solutions. (b) is adding 1-butyl-3methylimidazolium hexafluorophosphate to the fresh AB yogurt. (c) is adding CrO42-- to the fresh AB yogurt.(d) is adding PO43- to the fresh AB yogurt……………………………………...59
Fig. 32. Possible mechanism for the affinity capture of the yogurt bacteria from yogurt samples by Cr+6………………………………………………………………………..60
Fig. 33. Scheme of the S. aureus experiment methodology……………………..……..71
Fig. 34. The device figure of SDME ( Single-Drop Microextraction ). In the small vial placed two kinds of solvent. The round drop marked with green circle line was the ionic liquid. And the aqueous solvent was the bacterial solution. The magnetic stir bar was 0.2 cm in diameter. Take use of the magnetic rotation, the ionic is going to trap bacteria from aqueous solution………………………………….………………………………73
Fig. 35. (a) TEM micrograph of Staphylococcus aureus used in the study (b) TEM micrograph of Staphylococcus aureus spiking into mouse blood. (c) Staphylococcus aureus injected into mice and the infected blood cultured in nutrient agar plates showing distinct golden yellow colonies characteristic of S. aureus…………………………….74
Fig. 36. (a) Fluorescent Stained cells of Staphylococcus aureus used in the study. (b) Fluorescent stain called DAPI used, but not successful in selectively staining the bacteria alone, blood cells and bacterial cells got stained. (c) Fluorescent stain Acridine orange successfully distinguished the bacterial cells by fluorescing orange from the blood cells which took up green fluorescence………………………………………….76
Fig. 37. MALDI-MS spectra of BCRC 10451 of Standard Staphylococcus aureus 4.43x1010 cfu/mL. Staphylococcus aureus was dispersed in the autoclaved deionized water……………………………………………………………………………………77
Fig. 38. MALDI-MS spectra of Standard Staphylococcus aureus in autoclaved sterile water direct analysis using 50 mM sinapic acid, (a) 1.9x109 cfu/mL of bacteria, (b) 1.9x108 cfu/mL of bacteria, (c) 1.9x107 cfu/mL of bacteria, and (d) 1.9x106 cfu/mL of bacteria………………………………………………………………………………….78
Fig. 39. Staphylococcus aureus spiking experiment on rabbit blood. 50mM SA as matrix. (a) is the rabbit blood using MALD-MS direct analysis. (b) is the BCRC 10451 Standard Staphylococcus aureus 4.43x1010 cfu/mL direct analysis. (c) is spiking 100μL of Standard Staphylococcus aureus 4.43x1010 cfu/mL into rabbit blood and analyzed by MALDI-MS…………………………………………………………………………….80
Fig. 40. Staphylococcus aureus spiking experiment on human urine. 50mM SA as matrix. (a) is the human urine using MALD-MS direct analysis. (b) is the BCRC 10451 Standard Staphylococcus aureus 2.1x109 cfu/mL direct analysis. (c) is spiking standard staphylococcus aureus 2.1x109 cfu/mL into human urine and analyzed by MALDI-MS. (d) is spiking of standard staphylococcus aureus 2.1x108 cfu/mL into human urine and analyzed by MALDI-MS. (e) is spiking of standard staphylococcus aureus 2.1x107 cfu/mL into human urine and analyzed by MALDI-MS……………………………….81
Fig. 41. MALDI-MS spectra of SDME of staphylococcus aureus combined with ionic liquid ( 1-butyl-3methylimidazolium hexafluorophosphate ). Standard Staphylococcus aureus was spiked in the autoclaved sterile water using SDME of ionic liquid. 50 mM sinapic acid as matrix, (a) 2.22x108 cfu/mL of bacteria, (b) 2.22x107 cfu/mL of bacteria, (c) 2.22x106 cfu/mL of bacteria, (d) 2.22x105 cfu/mL of bacteria and (e) 2.22x104 cfu/mL of bacteria. We could know that Staphylococcus aereus in sterile water using traditional SA matrix, the lowest detectible concentration of staphylococcus aureus in water was ~105 cfu/mL…………………………………………………………………83
Fig. 42. MALDI-MS spectra of SDME of Staphylococcus aureus combined AgNPs with ionic liquid ( 1-butyl-3methylimidazolium hexafluorophosphate ). Standard Staphylococcus aureus was spiked in the autoclaved sterile water using SDME of ionic liquid. 50 mM sinapic acid as matrix, (a) 1.9x109 cfu/mL BCRC 10451 of Standard Staphylococcus aureus (b) the lowest detectible concentration of staphylococcus aureus in water was 1.9x108 cfu/mL. (c) SDME with AgNPs in ionic liquid of staphylococcus aureus in water was 1.9x106 cfu/mL. (d) SDME with AgNPs in ionic liquid of staphylococcus aureus in water was 1.9x105 cfu/mL. (e) SDME with AgNPs in ionic liquid of staphylococcus aureus in water was 1.9x104 cfu/mL………………………...84
Fig. 43. Scheme of the outline methodology used for this phase of study……………..86
Fig. 44. The scheme of preparation of standard staphylococcus aureus and injection into mousy mousy intraperitoneal organs. Before injection of bacteria, we took its 10 μL of blood and urine first for the background. Then after infection, at each time point, we took blood and urine for culturing………………………………………..…………….89
Fig. 45 Mouse injected Staphylococcus aureus 4.43x1010 cfu/mL 200uL and blood detection. (a) background of mouse blood, (b) 1h of blood after infection, (c) 3h of blood after infection, (d) 6h of blood after infection, (e) 18h of blood after infection, and (f) 30h of blood after infection…………………………………………………………90
Fig. 46 Invivo growth kinetics of S. aureus in murine model showing three phases in the infection pattern…………………………………………………………….…………..91
Fig. 47. Show photograph of the colonies obtained from blood samples at (a) 2 h, (b) 24 h, (c) 48 h and (d) 96 h. infection periods………………………………..…………….92
Fig. 48. MALDI-MS spectra when 500µL of bacteria was injected still leading to observation of only few bacterial peaks………………………………………………..93
Fig. 49. MALDI-MS of (a) Blood background. (b) Blood of injection after 6 h of Staphylococcus aureus 2.5x1011 cfu/mL. (c) Blood of injection after 6 h of Staphylococcus aureus 2.5x1011 cfu/mL and adding to Ag NPs. (d) Blood of injection after 6 h of Staphylococcus aureus 2.5x1011 cfu/mL and adding to ZnO NPs. (e) Blood of injection after 6 h of Staphylococcus aureus 2.5x1011 cfu/mL and adding to1-butyl-3methylimidazolium hexafluorophosphate. (f) Blood of injection after 6 h of Staphylococcus aureus 2.5x1011 cfu/mL and adding to 1-Butyl-3-methylimidazolium tetrafluoroborate……………………………………………………..…………………94
Fig. 50. MALDI-MS of (a) 2.5x1011 cfu/mL BCRC 10451 of Standard Staphylococcus aureus. (b) Blood of injection after 6 h of Staphylococcus aureus 2.5x1011 cfu/mL. (c) Using 1-Butyl-3-methylimidazolium tetrafluoroborate enabled successful detection of bacteria in the blood sample…………………………………..………………………..95
Fig. 51. MALDI-MS of (a) 4.43x1010 cfu/mL BCRC 10451 of Standard Staphylococcus aureus. (b) Blood background. (c) Blood of injection after 3 h of Staphylococcus aureus 4.43x1010 cfu/mL. (d) Blood sample centrifuged at 500 rpm twice and the supernatant was analyzed. (e) Blood sample centrifuged at 5000 rpm twice and the precipitate was analyzed…………………………………….…………………………………………..96
Fig. 52. Mouse injected Staphylococcus aureus 4.43x1010 cfu/mL 200uL and urine detection. (a) background of mousy urine, (b) 1h of urine after infection, (c) 3h of urine after infection, (d) 6h of urine after infection, (e) 18h of urine after infection, and (f) 30h of urine after infection………………………………………………………………….98
Fig. 53. Spectra of the bacterial growth in mouse urine from 1 h to 96 h culture in the nutrient agar plates after injected 200 μL of 4.43x1010 cfu/mL Staphylococcus aureus and scanning in the cut-off mode < 9000Da . This made more bacterial peaks evident………………………………………………………………………………….99









Table Catalog
Table. 1. The list of bacterial peaks obtained from AB and Lin Feng-Yin yogurt using the MALDI-MS. Bacterial colonies were grown from DifcoTM Lactobacilli MRS Broth agar plate………………………………………………………………………53

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