The combination of electrochemistry with microfluidic sample processing is a viable option to reduce the size, logistics load and power consumption of biosensors. Modern microfluidics technology makes it possible to perform sample clean-up, PCR, sample concentration and transduction on the same disposable chip. This presentation will discuss two novel electrochemical techniques which do not require a sandwich assay and can be employed on a disposable microfluidic chip, reducing logistics load and microfluidic complexity. Transduction is achieved via an electrochemical DNA hybridization sensor similar to a molecular beacon removing the need for a sandwich assay also referred to as E-DNA. The sensor is designed where a DNA stem-loop structure is immobilized on a gold electrode with a redox label held close to the surface. Upon hybridization the stem-loop opens and the label pulls away from the surface so that current cannot flow to the electrode under positive bias. This paper will primarily discuss experiments trying to understand the hybridization event and effect of surface morphology on electrochemical signal transduction.
Interfacing organisms and hardware is a promising and challenging research frontier. The threat of biological weapons and the rising cost of health care have pushed detection of pathogens and their toxins to the forefront of that frontier. Both the military and the civilian sectors require that this detection be fast, accurate, sensitive, and inexpensive. We describe an electrochemical detection method that relies upon "molecular-scale gates" capable of being activated by a biological agent. We discuss our most recent experimental and modeling results, which take into account DNA folding and introduce the concept of tethering to boost the detection signal. Preliminary results show dramatic and specific recognition of target molecules.
There is a great need for high throughput and sensitive sensors for genetic analysis. These sensors can be used for varied purposes from monitoring gene expression in organims to speciation of possible pathogens. Consequently, an instrument capable of these tasks would be a great benefit for food and water safety, medical diagnostics and defense of military and civilian populations from biological threats. This work examines the development of a hybridization-based biosensor using a novel tapered fiber optic rpobe. The immobilization of single-stranded, synthetic ologinucleotides utilizing aminoproplytriethoxysilane and glutaraldehyde was implemented on the fiber optic sensor. Hybridization takes place with a complementary analyte sequence followed by a fluorescent, labeled signaling probe to form a sandwich assay. Following hybridization, the fiber is interrogated with a diode laser source and the resulting fluorescence signal is detected using a miniature spectrometer.
In this study we show that steady-state fluorescence anisotorpy within PEBBLEs can be used for the optochemical sensing of analytes such as Zn2+, O2, and Ca2+. Steady-state fluorescence anisotropy is a non- time resolved method that measures a combination of rotational and fluorescence lifetimes. This eliminates the need for reference dyes and ratiometic techniques to obtain quantitative results, even when using intensity-based sensor dyes. An advantage to working with PEBBLE nanosensors is that the encapsulated dye is localized in a constant rotational environment. This is in contrast to the use of free dyes, which can be affected by interferents such as protein binding.
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