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(373v) Biosensors Using Morpholinos

Authors: 
McConnell, J. T., University of Wyoming
Johnson, P. A., University of Wyoming
Zhang, H., University of Wyoming


In recent years, there has been a considerable amount of research devoted to developing the nucleic acid sensing capabilities of Surface Enhanced Raman Spectroscopy (SERS). Current methods of DNA detection, such as RT-PCR, can be costly and time consuming. DNA detection utilizing SERS provides a unique Raman spectrum which is specific for the nucleic acid sequence and can be performed within a matter of minutes. Furthermore, SERS-based detection has been shown to exhibit spectral band gaps significantly narrower than fluorescence spectroscopy. Applications of SERS-based DNA sensing include rapid identification of pathogens, disease prognosis and diagnosis and detection of biological agents. If practical use of such sensors is executed, many false diagnoses can be avoided and an efficient response to a biological attack would be ensured. Recent biosensing studies utilizing SERS employ one of two general techniques: adsorption of an analyte to a Raman-active surface or addition of an analyte to a noble metal colloid for subsequent SERS spectoscopy. Raman-active surfaces may be roughened or engineered to have a repeating geometric structure. Colloids can be composed of nanoparticles of a single metal or multi-layered structures of various topologies fabricated from two or more metals. In either of the previous circumstances the colloid may be analyzed in the liquid state or dried on inert glass, the former being the more rapid option. In many instances, a Raman dye is appended to the target DNA sequence as a result of the low specificity of DNA alone. Current DNA sensing research involves using thiol-fuctionalized DNA oligos tethered to colloidal Au nanoparticles coupled with methylene blue hybridized DNA oligos and has been shown to be a valid method of viral DNA detection. It has been of recent interest to utilize morpholinos in DNA sensing applications considering their attractive properties. Morpholinos have a morpholine ring in place of the deoxyribose ring present in DNA and have an uncharged phosphodiamidate backbone. Consequently, bond strengths of morpholino-DNA configurations are significantly higher than their DNA-DNA counterparts and morpholino oligos are resistant to nuclease degradation. The lack of a charged backbone is especially important in our application of morpholinos. We hypothesized that as a result of utilizing morpholinos in DNA biosensor applications, lower buffer concentrations could be used effectively. This is an important consideration, taking into account that the binding of two complementary DNA strands requires a high buffer concentration to effectively shield the negative charge possessed by each strand of DNA .The use of a low buffer concentration would eliminate much of the noise in SERS spectra caused by high concentration buffers which, in theory, would lower the detection threshold of DNA biosensors using SERS. In our experiments we compare the use of 60nm Au and Ag nanoparticles as Raman-active substrates for DNA detection using SERS spectroscopy, silica as a support for the nanoparticles and mehtylene blue as a Raman dye. The effect of buffer strength on DNA-morpholino binding was examined using quartz crystal microbalance with dissipation (QCM-D). We recorded 104-105 enhancements for both Au and Ag substrates. DNA-DNA hybridization experiments were successful on Au substrates but not of Ag substrates. Lack of a successful DNA-DNA hybridization on Ag substrates was a result of oxidation of Ag nanoparticles. QCM-D data show mass adsorption under 0.1M buffer conditions was similar to mass adsorption under 1.1M buffer conditions. These results indicate buffer strength does not have a substantial effect on morpholino-DNA binding.