Fluorescent sensors using DNA-functionalized graphene oxide

The final publication is available at Springer via http://dx.doi.org/10.1007/s00216-014-7888-3In the past few years, graphene oxide (GO) has emerged as a unique platform for developing DNA-based biosensors, given the DNA adsorption and fluorescence-quenching properties of GO. Adsorbed DNA probes can be desorbed from the GO surface in the presence of target analytes, producing a fluorescence signal. In addition to this initial design, many other strategies have been reported, including the use of aptamers, molecular beacons, and DNAzymes as probes, label-free detection, utilization of the intrinsic fluorescence of GO, and the application of covalently linked DNA probes. The potential applications of DNA-functionalized GO range from environmental monitoring and cell imaging to biomedical diagnosis. In this review, we first summarize the fundamental surface interactions between DNA and GO and the related fluorescence-quenching mechanism. Following that, the various sensor design strategies are critically compared. Problems that must be overcome before this technology can reach its full potential are described, and a few future directions are also discussed.University of Waterloo || Natural Sciences and Engineering Research Council || Ontario Ministry of Research and Innovation || Foundation for Shenghua Scholar || National Natural Science Foundation of China || Grant No. 81301258, 21301195 Postdoctoral Science Foundation of Central South University and Hunan province ||Grant No. 124896 China Postdoctoral Science Foundation || Grant No. 2013M540644 Hunan Provincial Natural Science Foundation of China || Grant No. 13JJ4029 Specialized Research Fund for the Doctoral Program of Higher Education of China || Grant No. 2013016212007

Fluorescence Sensing Using DNA Aptamers in Cancer Research and Clinical Diagnostics

Among the various advantages of aptamers over antibodies, remarkable is their ability to tolerate a large number of chemical modifications within their backbone or at the termini without losing significant activity. Indeed, aptamers can be easily equipped with a wide variety of reporter groups or coupled to different carriers, nanoparticles, or other biomolecules, thus producing valuable molecular recognition tools effective for diagnostic and therapeutic purposes. This review reports an updated overview on fluorescent DNA aptamers, designed to recognize significant cancer biomarkers both in soluble or membrane-bound form. In many examples, the aptamer secondary structure switches induced by target recognition are suitably translated in a detectable fluorescent signal using either fluorescently-labelled or label-free aptamers. The fluorescence emission changes, producing an enhancement ("signal-on") or a quenching ("signal-off") effect, directly reflect the extent of the binding, thereby allowing for quantitative determination of the target in bioanalytical assays. Furthermore, several aptamers conjugated to fluorescent probes proved to be effective for applications in tumour diagnosis and intraoperative surgery, producing tumour-type specific, non-invasive in vivo imaging tools for cancer pre- and post-treatment assessment

DNA-stabilized fluorescent silver nanoclusters: A versatile nanomaterial for the specific detection of DNA

DNA-templated silver nanoclusters (DNA-AgNCs) are fluorescent molecules containing few-atom clusters of silver stabilized by a short strand of DNA. They fluoresce at wavelengths in the visible spectrum with emission colors tuned by varying the sequence of the DNA used for their synthesis. The combination of their small size, tunable spectra and biocompatibility opens exciting, new possibilities for their use in chemical sensing, biological labeling and imaging, genetic mutation detection and the development of self-assembled DNA nanotechnologies. A detailed understanding of the structure of DNA-AgNCs is vital for precisely engineering their properties for future applications. Specifically, changes in the arrangement and composition of bases surrounding a AgNC can strongly influence its fluorescence. Although this sensitivity has already been leveraged for the development of novel chemical sensors, a better understanding of the mechanism is needed to improve performance and broaden the applicability of DNA stabilized AgNCs. In this work, we use high-resolution microfluidic capillary electrophoresis to show that, although AgNC depend on single-stranded DNA for their stabilization, changes made to bases in the double-stranded stem region of a DNA hairpin can perturb their structure, leading to differences in fluorescence emission. We also explore ways in which AgNC can be robust to DNA sequence changes. We document one DNA-AgNC in which the DNA adopts different conformations, or shapes, that yield clusters with the same emission color. We also show that poly-thymidine regions, which are know to bind silver poorly,can act as convenient handles to adjust the electrophoretic mobility of DNA-AgNCs without affecting their fluorescence.Using poly-thymidine appendages, we demonstrate a way to tune the electrophoretic mobility of a AgNC-based DNA probe for use in a microfluidic assay. These label-free probes are composed of a DNA hairpin that generates a fluorescent silver nanocluster only after binding to a specific target DNA sequence. By tuning the mobility of probes designed to bind different targets, we demonstrate a rapid microfluidic separation assay for the multiplexed fluorescent detection of nucleic acid targets for Hepatitis A, B and C. The probe design initially used for these studies suffered from several shortcomings. Most significantly, the probe DNA failed to generate a fluorescent AgNC for some binding domain sequences. To overcome this, as well as provide added functionality, we engineer a new DNA-AgNC based sensor that supports ratiometric fluorescence measurements for the sensitive, specific and low-cost detection of DNA. Probes based on our new design generate a green emitting AgNC in its hairpin state, and a red emitting AgNC after binding their target. The ratiometric fluorescence provides a stable signal and rapid quantification of DNA concentration regardless of the choice of target, a dramatic improvement over similar turn-on fluorescent probes

Improving multiplexed RNA detection assays by interfacing enzymatic amplification strategies with silicon photonic microring resonators

The ability to make multiplexed measurements has significantly improved our understanding of disease onset and progression. This newfound understanding has the potential to transform clinical diagnostics. Also known as personalized medicine, diagnostic decisions are improved by relying on a detailed knowledge of an individual’s biochemical signature. While routine clinical tests detect one biomarker at a time, new technologies are needed that enable the analysis of multiple targets per clinical sample. This doctoral dissertation presents a platform that can complete these goals by developing assays that combing enzymatic processing steps with silicon photonic microring resonators, a technology pioneered by the Bailey Research Laboratory. While other efforts in lab have been geared to other classes of biomolecules, the developed assays discussed in this dissertation are designed to profile nucleic acid biomarkers in a host of clinically relevant samples. The results from these studies are confirmed using clinical gold standard techniques and compared with findings in the literature to validate the platform. Chapter 1 discusses how silicon photonic microring resonators fit into the landscape of next-generation multiplexed biomolecular detection platforms while also developing the motivation to use enzymatic processing of nucleic acids to produce ultra-sensitive detection platforms. Chapter 2 gives an exhaustive review of current microRNA (miRNA) detection platforms, both clinical gold standards and emerging technologies. Given the unique detection challenges of microRNAs, this class of RNA molecule was used to develop a detection platform which could then be translated to other RNA molecules. Chapter 3 describes the use of enzymatic processing of miRNA sequences and subsequent on-chip enzymatic signal enhancement strategy to lower the required input of RNA material to a clinically relevant amount. Chapter 4 outlines further improvements to enzymatic pre-processing of miRNA molecules by interfacing an adapted polymerase chain reaction process with the microring platform to study miRNA expression in glioblastoma patients. It also eliminates the need for on-chip signal amplification. Chapter 5 adapts this workflow and uses it for the detection of long-noncoding RNA (lncRNA) molecules in a previously uncharacterized glioblastoma cell line. Chapter 6 outlines additional research efforts and future directions, which include efforts to build a platform combining enzymatic pre-processing with microring resonator detection and efforts to push into an expanded set of clinical and research applications where low sample inputs and short analysis times are needed

Rolling circle amplification as an efficient analytical tool for rapid detection of contaminants in aqueous environments

Environmental contaminants are a global concern, and an effective strategy for remediation is to develop a rapid, on-site, and affordable monitoring method. However, this remains challenging, especially with regard to the detection of various contaminants in complex water environments. The application of molecular methods has recently attracted increasing attention; for example, rolling circle amplification (RCA) is an isothermal enzymatic process in which a short nucleic acid primer is amplified to form a long single-stranded nucleic acid using a circular template and special nucleic acid polymerases. Furthermore, this approach can be further engineered into a device for point-of-need monitoring of environmental pollutants. In this paper, we describe the fundamental principles of RCA and the advantages and disadvantages of RCA assays. Then, we discuss the recently developed RCA-based tools for environmental analysis to determine various targets, including heavy metals, organic small molecules, nucleic acids, peptides, proteins, and even microorganisms in aqueous environments. Finally, we summarize the challenges and outline strategies for the advancement of this technique for application in contaminant monitoring