"Biosensors for the characterization of DNA molecules based on electronic-, thermal-, and photonic- sensing principles."
Deoxyribonucleic acid (DNA) recognition is an important tool in DNA biosensors. DNA biosensors are being developed with a rapid pace with the aim to achieve an inexpensive and rapid testing tool for genetic and infectious disease and to detect DNA damage and interactions. The study of single nucleotide polymorphisms (SNPs) and the analysis of gene sequences play a fundamental role in rapid detection of genetic mutations. It also opens up new opportunities for reliable diagnosis even before any symptoms of a disease appear. The aim of this thesis was to optimize and develop biosensors for the characterization of DNA molecules based on electronic-, thermal-, and photonic sensing principles.
In Chapter 1, a general description to DNA and DNA sensors was introduced. A brief explanation about the fundamentals of DNA as described by James D. Watson and Francis Crick was given. The concept and consequences of mutations in DNA sequences were explained briefly. The composition of a DNA sensor, the different sensing techniques used to sense DNA standards, and the sensor preparation with DNA were described briefly. Probe DNA, consisting of a 36-mer fragment was covalently immobilized on nanocrystalline chemical vapour deposition (CVD) diamond electrodes and hybridized with a 29-mer target DNA.
In Chapter 2, label-free real-time electronic monitoring of DNA denaturation upon exposure to NaOH solution at different flow rates and molarities, using electrochemical impedance spectroscopy as readout technology, was reported. The impedance response was separated into a denaturation time constant and a medium exchange time constant by means of a double exponential fit. It was observed that the denaturation time is dependent on the flow rate as well as on the molarity of the NaOH solution used. Surprisingly, it was observed that at low molarities (0.05 M) the DNA does not fully denature at low flow rates. Only after flushing the flow cell a second time with 0.05 M NaOH, complete denaturation was achieved. Confocal images were obtained and plotted in 3D graphs to confirm the results.
In Chapter 3, we showed that synthetic sapphire (Al2O3), an established implant material, can also serve as a platform material for biosensors comparable to nanocrystalline diamond. Sapphire chips, beads, and powder were first modified with (3-Aminopropyl) triethoxysilane (APTES), followed by succinic anhydride (SA), and, finally, single-stranded deoxyribonucleic acid (ss-DNA) probe was coupled using the zero-length cross-linker 1-ethyl-3-[3-dimethylaminopropyl]- carbodiimide (EDC) to the functionalized layer. The presence of ATPES-succinic anhydride layer on sapphire powder was confirmed by thermogravimetric analyis (TGA) and Fourier-transform infrared spectroscopy (FT-IR). The areal DNA density was quantified in X-ray photoelectron spectroscopy (XPS). Fluorescence microscopy was performed to demonstrate the successful coupling of fluorescently tagged target DNA to the pre-immobilized probe DNA. Synthetic sapphire is especially suitable for the heat-transfer method (HTM) due to its high thermal conductivity and chemical inertness. This measuring method analyzes the heat-transfer resistance at the solid-liquid interface when a target DNA molecule interacts with a ssDNA probe-functionalized surface. The heat transfer method was performed for the characterization of DNA on synthetic sapphire chips.
Within Chapter 4, a comparative theoretical study of an optical biosensor concept based on elastic light scattering from microspheres and the corresponding shift of whispering gallery modes (WGMs), after an add-on layer to the sphere, was performed. The study included sapphire, glass and diamond microspheres. The theoretical calculation of the expected resonant wavelength shifts were based on the generalized Lorenz–Mie theory (GLMT). The transverse electric (TE) and the transverse magnetic (TM) elastic light scattering intensity of electromagnetic waves at 600 and 1400 nm are numerically calculated for DNA and unspecific binding of proteins to the microsphere surface. The effect of changing the optical properties was studied for diamond (refractive index 2.34), glass (refractive index 1.50), and sapphire (refractive index 1.75) microspheres with a 50 µm radius. The mode spacing, the linewidth of WGMs, and the shift of resonant wavelength due to the change in radius and refractive index, were analyzed by numerical simulations. Preliminary results of unspecific binding of biomolecules showed that the calculated shift in WGMs can be used for biomolecules detection.
Therefore, an optical setup for DNA optical biosensor based on sapphire spherical microcavity was built as described in Chapter 5. Transmitted and elastic scattering intensity at 1510 nm were analyzed from a sapphire microsphere (radius 500 m, refractive index 1.77) on an optical fiber half coupler for the first time. The 0.43 nm angular mode spacing of the resonances correlated well with the optical size of the sapphire sphere. The spectral linewidths of the resonances were on the order of 0.01 nm, which corresponded to quality factors on the order of 105. As a proof for principle, polydopamine (PDA) layer has been used as a functionalizing agent on sapphire microspherical resonators in view of the implementation of biosensors. The various PDA layer thicknesses on the sapphire microsphere were characterized as a function of the resonances wavelength shift. It was shown that the polymeric functionalization does not affect the high quality factor (Q ≈ 104) of the sapphire microspheres. This functionalizing process of the microresonator constitutes a promising step towards the achievement of an ultrasensitive biosensor.
Then, the sapphire sphere was modified with DNA and an optical biosensor is demonstrated for the first time using an insulating implant material as illustrated in Chapter 6. Probe DNA, consisting of a 36-mer fragment was covalently immobilized on sapphire microsphere and hybridized with a 29-mer target DNA. Whispering gallery modes (WGMs) were monitored before the sapphire being functionalized with DNA and after it was functionalized with single stranded DNA (ssDNA) and double stranded DNA (dsDNA). The shift in resonances due to the surface modification with DNA was measured and correlated well with the estimated add-on DNA layer. It was shown that ssDNA are more uniformly oriented on the sapphire surface than the dsDNA. In addition, it was shown that functionalization of the sapphire spherical surface with DNA does not affect the high quality factor (Q ≈ 104) of the sapphire microspheres. Future work may focus on optimization of this method further and to perform measurements on mismatched dsDNA and in liquid medium.
All in all, we have taken the first step towards utilizing a structural, electrically insulating implant material as a heat-transfer based and optical microcavity based biosensor platform paving the way for future in vivo biosensing devices. Our efforts are expressed in details in the concluding Chapter 7.