Patterned and switchable surfaces for biomaterial applications

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Chapter 4 – Formation of a chemically patterned substrate for cell microarray applicationsHowever, the key advantage of this approach is the integration of the celladhesion regulating pattern with the subsequent formation of a small-molecule,protein, DNA or RNA microarray. By way of a demonstration, a TCM was preparedby first spotting an array of DNA vectors using pEGFP-N1, a plasmid encoding for aGFP, and pDsRed2, a plasmid encoding for a RFP, along with Effectene transfectionagent on top of the already prepared PLLc polymer array. Since surface patterningand microarray formation are carried out on the same instrument, alignment of thetwo surface patterns was easily achieved. At neutral pH, PLL is positively charged,thus stabilising electrostatically the deposited DNA. The interactions between DNAand PAA, polyallylamine (PALA), PEI, PLL and PVP surface coatings was assessedby atomic force microscopy force measurements (Appendix 2). This demonstratedthe electrostatic nature of interactions between DNA and PALA, PEI and PLL. ThePEG surface has previously been shown to restrict DNA adsorption, hencepreventing surface diffusion of the DNA spots on the surface [188, 207]. HEK-293cells were subsequently seeded onto the array and were visualised by fluorescencemicroscopy after 48 hr incubation at 37 °C to check for reverse transfection. Acheckerboard pattern of red and green cells resulted (Figure 4.7A), whereupon cellsfluorescing green were localised to regions where pEGFP-N1 had been spotted onPLLc (Figure 4.7B) whilst cells fluorescing red were localised to regions wherepDsRed2 had been deposited on PLLc (Figure 4.7C), confirming that reversetransfection had occurred. There were no fluorescent cells found outside the PLLcregions. Furthermore, green and red fluorescing cells were not observed on the samespot, demonstrating the absence of cross-contamination. Typically, transfectionefficiencies of 15-20% were observed, which is similar to previously reportedefficiencies [29, 35].4-150
Andrew Hook – Patterned and switchable surfaces for biomaterial applications(A)(B)(C)Figure 4.7. Fluorescence microscopy images of HEK-293 cells grown on a PLLcpattern on a PEG background with a checkerboard DNA vector arraypattern. (A) 4x magnification of entire checkerboard array with red andgreen fluorescence combined. Scale bar equals 500 m. (B) 20xmagnification of a single spot containing a plasmid encoding for GFP.Green fluorescence is due to the presence of GFP within cells. Scale barequals 200 m. (C) 20x magnification of a single spot containing a plasmidencoding for RFP. Red fluorescence is due to the presence of RFP withincells. Scale bar equals 200 m. GFP fluorescence taken through a 470-495nm excitation filter and a 510 nm suppression filter. RFP fluorescence takenthrough a 530-550 nm excitation filter and a 575 nm suppression filter.4-151
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Patterned andswitchable surfacesfor
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TABLE OF CONTENTSTABLE OF CONTENTS
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CHAPTER 1.INTRODUCTION1The content
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Chapter 1 - Introductionconsiderabl
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Chapter 1 - Introductioninteraction
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Chapter 1 - IntroductionIn general,
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Chapter 1 - IntroductionFor some ap
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Chapter 1 - Introductionangles rang
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Chapter 1 - Introductionof understa
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Chapter 1 - IntroductionSeveral str
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Chapter 1 - Introductioncues to con
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Chapter 1 - Introductionby controll
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Chapter 1 - Introduction1.2.2. Soft
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Chapter 1 - Introductionsubstrate.
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Chapter 1 - Introduction(A)(B)Figur
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Chapter 1 - Introduction(A)(B)(C)(C
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Chapter 1 - Introductioncomplex gen
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Chapter 1 - Introductionmorphology
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Chapter 1 - Introduction(A)(B)(C)(D
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Chapter 1 - Introductionthe LCST, a
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Chapter 1 - IntroductionHyun et al.
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Chapter 1 - Introductionmicelles. T
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Chapter 1 - Introduction1.4.1. DNA
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Chapter 1 - IntroductionA major pro
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Chapter 1 - Introductionattached to
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Chapter 1 - IntroductionTable 1.1.
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Chapter 1 - Introductionefficiencie
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Chapter 1 - Introductionachieved by
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Chapter 1 - IntroductionTable 1.2.
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Chapter 1 - IntroductionOnce releas
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Chapter 1 - Introductionimaging not
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Chapter 1 - IntroductionA number of
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CHAPTER 2.SPATIALLY CONTROLLED ELEC
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CHAPTER 3.COMPARISON OF THE BINDING
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CHAPTER 6.OVERALL CONCLUSIONS6.6An
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Chapter 6 - Overall conclusionsThes
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Force (nN)Force (nN)Force (nN)Force
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