New principles of detecting specific DNA targets with liquid crystals

NEW PRINCIPLES OF DETECTING SPECIFIC DNA TARGETS WITH LIQUID CRYSTALS LAI SIOK LIAN (B. Eng. (Hons), Universiti Teknologi Malaysia) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2011 To my dearest parents, sisters, brother and Green Apple Fellowship ACKNOWLEDGEMENTS It is my pleasure to thank several persons who made this thesis possible. First and foremost, I am heartily thankful to my supervisor, Dr. Yang Kun-Lin, for his guidance and support from the beginning until the end of this research work which enable me to learn and develop an understanding of this work. His patience and encouragement have been my motivation when I hurdle the obstacles to complete the research work. I am indebted to him more than he knows. I would like to show my gratitude to all the members in the research lab for their insights, questions and suggestions highlighted during the progress of this work. They have given me a pleasant working environment and I am grateful to work with them. Appreciation is also to be given to lab officers, especially Mr Boey and Chai Keng, for all the invaluable lab assistances provided to ensure the smooth running of experiments. Thanks must be given to my family members for all their love and encouragement throughout my studies at University. Without their constant support and comfort, I might not be able to finish my PhD degree. Lastly, I would like to thank Almighty God for His blessings during the completion of my PhD works. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS iii TABLE OF CONTENTS iv SUMMARY ix LIST OF TABLES xi LIST OF FIGURES xii NOMENCLATURE xx CHAPTER INTRODUCTION 1.1 Background 1.2 Research Objectives LITERATURE REVIEW 2.1 Deoxyribonucleic Acids (DNA) 2.2 Solution Hybridization 12 2.3 DNA Hybridization on DNA Microarrays 16 2.3.1 Selection of Substrate 18 CHAPTER iv 2.3.2 DNA Immobilization Strategies 19 2.3.3 DNA Hybridization 24 2.4 Detection of DNA Targets on DNA Microarrays 26 2.5 Peptide Nucleic Acids (PNA) 27 2.6 Liquid Crystals (LCs) 30 2.6.1 Thermotropic Liquid Crystals 30 2.7 Interactions of LCs with Light 34 2.8 Orientations of LCs at Interface 35 2.9 Factors Affecting the Orientations of LCs at 37 Interface 2.9.1 Solid-LC Interfaces 37 2.9.2 Aqueous-LC Interfaces 41 Applications of LCs as Biological Assays 43 2.10.1 On Solid-LC Interfaces 43 2.10.2 On Aqueous-LC Interfaces 46 2.10 CHAPTER DEVELOPMENT OF PROTOCOL FOR DNA 49 IMMOBILIZATION AND HYBRIDIZATION ON SOLID SURFACES 3.1 Introduction 50 3.2 Experimental Section 51 3.3 Results and Discussions 54 3.3.1 Immobilization of Amine-Labeled DNA on Aldehyde 54 Decorated Surface v 3.3.2 Effect of the Salt Concentrations 55 3.3.3 Effect of the DNA Concentrations 56 3.3.4 Effect of the Immobilization Time 56 3.3.5 Effect of the Hybridization Buffer Concentration on 59 DNA Hybridization Efficiency 3.3.6 Effect of the DNA Target Concentrations 61 3.4 Conclusions 62 CHAPTER ENHANCING THE FLUORESCENCE 64 INTENSITY OF DNA MICROARRAYS BY USING CATIONIC SURFACTANTS 4.1 Introduction 64 4.2 Experimental Section 67 4.3 Results and Discussions 70 4.3.1 Emission Spectra of FAM-labeled DNA in CTAB 70 Solution 4.3.2 Role of DNA in Enhancing the Fluorescence Intensity 72 4.3.3 Influence of CTAB on FAM-labeled DNA on Solid 74 Surfaces 4.3.4 Influence of CTAB on Cy3-labeled DNA on Solid 78 Surfaces 4.3.5 Effect of CTAB on the Fluorescence Property of 79 Double-Stranded DNA 4.4 Conclusions 82 vi CHAPTER OPTICAL IMAGING OF SURFACE- 83 IMMOBILIZED OLIGONUCLEOTIDE PROBES ON DNA MICROARRAYS USING LIQUID CRYSTALS 5.1 Introduction 84 5.2 Experimental Section 86 5.3 Results and Discussions 91 5.3.1 Orientations of LCs on TEA-Decorated Surfaces 91 5.3.2 Imaging Immobilized Oligonucleotides with LCs 93 5.3.3 Effect of Oligonucleotides Length 94 5.3.4 Orientations of LCs on mixed TEA/ DMOAP 95 Surfaces 5.3.5 Imaging Immobilized Oligonucleotides on mixed 97 TEA/ DMOAP Surfaces 5.3.6 Effect of Droplet Size on Detection Limit 98 5.3.7 Assessing the Quality of a DNA Microarray Using 100 LCs 5.4 Conclusions CHAPTER DETECTING DNA TARGETS THROUGH THE 105 106 FORMATION OF DNA/CTAB COMPLEX AND ITS INTERACTIONS WITH LIQUID CRYSTALS vii 6.1 Introduction 107 6.2 Experimental Section 110 6.3 Results and Discussions 115 6.3.1 Optical Image of LC Supported on DNA/ Surfactant 115 Complexes 6.3.2 Reversible Formation of DNA/ CTAB Complexes 117 6.3.3 Effect of CTAB Concentrations 118 6.3.4 Optical Image of LC with PNA/ DNA Targets on 120 Solid Surface 6.3.5 Specificity of Detection 123 6.4 Conclusions 124 CHAPTER SELF-ASSEMBLY OF CHOLESTEROL DNA 125 AT LIQUID CRYSTAL/AQUEOUS INTERFACE AND ITS APPLICATION FOR DNA DETECTION 7.1 Introduction 125 7.2 Experimental Section 127 7.3 Results and Discussions 129 7.4 Conclusions 136 CHAPTER CONCLUSIONS AND RECOMMENDATIONS 137 BIBLIOGRAPHY 145 APPENDIX A: LIST OF PUBLICATIONS 157 viii SUMMARY Detecting DNA targets with specific sequence is important in the identification and detection of single nucleotide polymorphisms and gene expression profile analysis. Traditionally, fluorescence is used to report the presence of DNA targets hybridized to surface-immobilized DNA probes. However, this method requires fluorescent labeling of DNA targets, and the sensitivity remains a challenge. In the first part of this thesis, we used cetyl trimethylammonium bromide (CTAB) for enhancing the fluorescence intensity of 6-carboxy-fluorescene (FAM)-labeled DNA targets hybridized to the immobilized DNA probes. The fluorescence intensity shows a 26fold increase for perfect-match DNA targets. The contrast ratio between perfectmatch and 1-mismatch DNA is increased from 1.3-fold to 15-fold. This method offers a simple and efficient technique to enhance the fluorescence detection limit on solid surface. In the second part, we used liquid crystal (LCs) as an imaging tool to detect DNA targets. The imaging principle is based on the disruption of the orientations of LCs by single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). Because LCs are birefringent materials, disruption of their orientations can manifest as optical signals visible to the naked eye. Firstly, LCs was used to image ssDNA immobilized on solid surfaces. Interestingly, a clear transition of the optical appearance of LCs ix from dark to bright at a threshold ssDNA concentration was observed. This enables us to correlate the LCs interference colors with ssDNA concentrations, and it also serves as a basis for the quantification of immobilized ssDNA on solid surface. Later on, we hybridized the ssDNA with complementary targets and used LCs to image the dsDNA. However, because the color contrast between ssDNA and dsDNA is not significant, we added the surfactants, CTAB, to the DNA targets to aid the re-organization of LCs because the hydrocarbon tail of surfactants has strong orientational effect on LCs molecules. This approach is further explored to show the ability to discriminate the complementary strands from the non-complementary strands even at low hybridization efficiency (33%). Even though the detection of DNA targets can be carried out by using the LCbased method above on a solid surface, the long duration of DNA hybridization makes this method not feasible for real-time detection. Thus, in our final part of this thesis, we developed an LC-based method which can detect DNA targets in a realtime manner. We self-assembled cholesterol-labeled DNA probes at the LC-aqueous interface. When the system is exposed to perfect match DNA targets, the optical appearance of LC shows a continuous change from dark to bright under the crossed polars within 15 min. No obvious change can be observed when the system is exposed to or base-pair mismatch DNA targets. x Chapter 8.2 Recommendations Based on the experimental results and discussions from this thesis, we make some recommendations for future investigation. First, the current hybridization procedure gives relatively low hybridization efficiency, i.e. 28%, as compared to nearly 100% as reported by (Levicky et al. 1998). The improvement on the hybridization efficiency is needed to increase the sensitivity in the fluorescence signals detection and to discriminate the perfect match and 1-mismatch DNA probes more effectively. Besides, by increasing the hybridization efficiency, the surface density of dsDNA can be increased and the disruption of the orientations of LCs can also be increased. If the hybridization efficiency is high enough, then perhaps strategies of using surfactants to enhance the LC signal will no longer be needed. To improve the hybridization efficiency, several parameters such as the types of hybridization buffer used, the temperature of hybridization and the correlation between the DNA probes density and DNA targets concentration can be adopted and studied to understand their effects on the hybridization performance (Levicky et al. 1998). In term of using LCs to detect DNA targets, several factors can be tuned in order to improve the detection signal. For instance, the property of the boundary surfaces can affect the orientations of LCs (Price and Schwartz 2006). By using a more hydrophilic or lower anchoring energy surfaces, LCs will tend to orientate in tilted or planar orientations. DNA on surface with lower anchoring energy can disrupt the orientations of LCs more easily. Besides, the thickness of the fabricated LC cell also affects the orientations of LCs (Price and Schwartz 2006). Thinner cell will orientate LCs homeotropically as compared to thicker cell. 142 Chapter Because the detection of DNA targets on LC-solid interface poses some limitations such as low sensitivity and affected by the property of the surfaces used, LC-aqueous interface system gives a better option to detect DNA targets. We have shown the used of the LC-aqueous interface system in Chapter but the study is preliminary and further study is required to improve the system. Our current system is able to detect perfect match DNA targets and discriminate them from the 1-mismatch DNA targets but the detection limit is not very high. One of the possible reasons is the low DNA hybridization efficiency. Even though solution hybridization efficiency is normally higher than that on the surface, the stringency of hybridization buffer which are affected by the concentration of salt and DNA targets and the addition of formamide (Price and Schwartz 2008) worth a detailed study in order to increase the amount of DNA targets hybridized to their perfect match DNA probes. The second reason is because of the remaining cholesterol-DNA probes in the bulk solution which can self-assemble at the LC-aqueous interface and restrict the orientational change of LCs. The cholesterol-DNA probes are not removed after self-assembling at the LCaqueous interface because the cholesterol-DNA probes are not stable at the interface if we change the bulk solution condition, i.e. they will desorb from the interface after the bulk solution is changed to a fresh solution. So, more stable DNA probes need to be used such that the DNA probes can anchor at the LC-aqueous interface even after the bulk solution is changed. This can be achieved by using two head cholesterolDNA probes which exhibit stronger coupling strength to lipid membranes (Pfeiffer and Höök 2004). Another type of DNA probes which behave as amphiphiles, such as a hydrocarbon chain tagged DNA probes (Xu et al. 2005), can be used to self- 143 Chapter assemble at the LC-aqueous interface and the anchoring stability is compared with the cholesterol-DNA probes. So far, we have only used synthetic DNA targets in our experiments. Therefore, in the next phase of this study, biological samples should be used to test the feasibility of the developed LC-based system. From our previous sections, we discussed that the orientations of LCs are greatly affected by the materials or solution at the interfaces of LCs. Thus, the effect of buffer solution used in the preparation of biological samples on the orientations of LCs (i.e. whether the buffer solution will cause homeotropic orientations or planar orientations) needs to be known beforehand. 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Interfacial immobilisation of DNA molecules. Annu. Rep. Prog. Chem. 103, 261–286. 156 LIST OF PUBLICATIONS 1. S. L. Lai, S. Huang, X. Bi, and K.-L. Yang, “Optical Imaging of SurfaceImmobilized Oligonucleotide Probes on DNA Microarrays Using Liquid Crystals”, Langmuir, 2009, 25 (1), 311-316. 2. S. L. Lai, D. Hartono, and K.-L. Yang, “Self-assembly of cholesterol DNA at liquid crystal/aqueous interface and its application for DNA detection”, Appl. Phys. Lett., 2009, 95 (15), 153702. 3. S. L. Lai, C.-H. Chen, K.-L. Yang, “Enhancing Fluorescence Intensity of DNA Microarrays by Using Cationic Surfactants”, Langmuir, 2011, 27 (9), 5659-5664. 4. S. L. Lai, K.-L. Yang, “Detecting DNA targets through the formation of DNA/ CTAB complex and its interactions with liquid crystals”, Analyst, 2011, 136, 33293334. 5. S. L. Lai, W. L. Tan, K.-L. Yang, “Detection of DNA Targets Hybridized to Solid Surfaces by Using Optical Images of Liquid Crystals”, ACS Applied Materials & Interfaces, 2011, (9), 3389-3395. 6. X. Bi, S. L. Lai, and K.-L. Yang, “Liquid Crystal Multiplexed Protease Assays Reporting Enzymatic Activities as Optical Bar Charts”, Anal. Chem., 2009, 81 (13), 5503-5509. 7. D. Hartono, S. L. Lai, K.-L. Yang, and L.-Y. Lanry Yung, “A liquid crystal-based sensor for real-time and label-free identification of phospholipase-like toxins and their inhibitors”, Biosens. Bioelectron., 2009, 24 (7), 2289-2293. 8. X. Bi, H. Xu, S. L. Lai, and K.-L. Yang, “Bifunctional oligo(ethylene glycol) decorated surfaces which permit covalent protein immobilization and resist protein adsorption”, Biofouling, 2009, 25 (5), 435-444. 9. S. L. Lai, K.-L. Yang, “Super Bright DNA Microarrays with High Fluorescence Intensity”, US Provisional Patent, Application No. 61/165,555, 1st Apr, 2009. 157 [...]... optical images of LC show 10 and 5 µM of PNA spots were hybridized to (A) 5, and (B) 0 µM of DNA targets (T1) before treating with 0.1 mM CTAB To study the effect of DNA target concentrations on the optical images of LCs, glass slides with same concentration of PNA spots were hybridized with (C) 1 and (D) 0.1 µM of DNA targets (T1) before treating with 0.1 mM CTAB 121 Figure 6.6 Effect of CTAB on the... PNA spots hybridized to 5 µM DNA targets Figure 6.7 Specificity of DNA targets detection The optical images of LC for 5 µM PNA spots hybridized to 5 µM of (A) DNA targets (T1), (B) single-base mismatch DNA targets (T3), and (C) noncomplementary DNA target (T2) All surfaces were immersed in 0.1 mM of CTAB solution after DNA hybridization 124 Figure 7.1 Self-assembly of Chol -DNA at the 5CB/ aqueous interface... properties DNA microarray has thousands of DNA probe with specific sequence immobilized at specific location on solid surface which allows the simultaneous detection of thousands of DNA targets at one time The miniaturized scale of DNA microarray means that the required volume of targets sample is reduced DNA microarray can be fabricated either by using in situ DNA probe synthesis or transferring synthetic DNA. .. 132 Figure 7.3 Optical responses of 5CB to (A) 51 µg/ mL of noncomplementary DNA targets after 15 min, and (B-D) 51 µg/ mL of complementary DNA targets after 5, 10 and 15 min (Scale bar, 200 μm) 134 Figure 7.4 Optical responses of 5CB to (A) 51 µg/ mL of 1MM DNA targets, and (B) 51 µg/ mL of 2MM DNA targets Images were taken at 0, 5, and 15 min after the addition of the DNA target solution (Scale bar,... responses of 5CB after 24 h The aqueous solution is Tris buffer (20 mM, pH 8.5) containing 5 mM of MgCl2 and (A) 69 µg/ mL of Chol -DNA, (B) 77 µg/ mL of cholesterol-free DNA, and (C) 34 µg/ mL of Chol -DNA (Scale bar, 200 μm) 131 Figure 7.2 Schematic illustration of two different orientations of 5CB (A) Before and (B) after the hybridization of self-assembled of Chol -DNA probes with complementary DNA targets. .. intensity value of 60 (B) Fluorescence-labeled DNA targets were hybridized to DNA microarray after the microarray was contacted with LCs and the LCs were removed The intensity plot across five fluorescence spots showed the average intensity value of 60, which was comparable with (A) Figure 6.1 (Top) Effect of DNA/ surfactant complexes on the orientations of LC The surface was decorated with 10 µM of DNA and...LIST OF TABLES Table 3.1 Fluorescence Intensity Analysis of DNA Spots in Figure 3.6 59 Table 3.2 Fluorescence Intensity Analysis of DNA Spots Hybridized to Different Concentration of DNA Targets 61 Table 4.1 Comparison of the Fluorescence Intensity for P2M, P1M, and PP Spots in Figure 4.9 80 xi LIST OF FIGURES Figure 2.1 Structure of DNA molecule which composes of a sugar ring, a phosphate... fabricate the DNA microarray The performance of the developed DNA microarray, in terms of sensitivity in detecting DNA targets and specificity in discriminating single-base mismatch targets, will also be tested by using the DNA microarray 5 Chapter 1 2) Enhance the Fluorescence Intensity of DNA Microarray by using Cationic Surfactants Fluorescence detection used in most conventional DNA microarray... Polymorphisms ssDNA Single-stranded DNA TEA Triethoxysilyl butyl aldehyde xx Chapter 1 CHAPTER 1 INTRODUCTION 1.1 Background Detecting DNA targets with specific sequence is important in the identification and detection of single nucleotide polymorphisms (SNPs), the analysis of gene expression profile and the identification of pathogens (Heller 2002) SNPs analysis is important in the identification of alleles... Emission spectra of solutions containing 1 µM of FAM-labeled DNA (FAM-P25) in aqueous solution without CTAB (dashed line) and in aqueous solution with various concentrations of CTAB: 0.1, 0.5, 1, and 2.5 mM (solid line) 70 Figure 4.3 Effect of DNA chain length on the fluorescence enhancement Emission spectra of solutions containing 1 µM of 40 mer, 25 mer, and 10 mer FAM-labeled DNA without CTAB solution . NEW PRINCIPLES OF DETECTING SPECIFIC DNA TARGETS WITH LIQUID CRYSTALS LAI SIOK LIAN (B. Eng. (Hons), Universiti Teknologi Malaysia) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR. Detection of DNA Targets on DNA Microarrays 26 2.5 Peptide Nucleic Acids (PNA) 27 2.6 Liquid Crystals (LCs) 30 2.6.1 Thermotropic Liquid Crystals 30 2.7 Interactions of LCs with Light. 0.1 µM of DNA targets (T 1 ) before treating with 0.1 mM CTAB. 121 Figure 6.6 Effect of CTAB on the binding stability of DNA targets on PNA probes. The fluorescence images of PNA/ DNA duplexes