Label-free detection of DNA hybridization using gold-coated tapered fiber optic biosensors (TFOBS) in a flow cell at 1310 nm and 1550 nm
Introduction
Detection of DNA interactions at femtomolar concentrations has many applications, such as biotechnology [1], genetics [2], medical diagnostics [3], [4], pathogen detection [5], [6], and drug screening [7]. To date the most popular methods of detection use ssDNA probes as recognition molecules [2], [4], [5], [8], [9]. Most of these methods depend on a fluorescent-labeled probe to provide and amplify sensing signal [2], [5], [10], [11]. Although the use of fluorescent labels improves sensitivity and selectivity, such a step requires laborious and time-consuming sample preparation steps. Furthermore, nucleic acids are often present in such small amounts that they need to be amplified using polymerase chain reactions (PCR) such that enough DNA exists to cause a change in sensor signal. Thus, it is highly desirable to reduce or eliminate the need for labeling and amplification, in order to develop a rapid, reliable, and user-friendly sensor.
For the past few years, tapered fiber optic biosensors (TFOBS) have been investigated in our laboratory as an alternative detection device for biomolecules. TFOBS have been widely investigated for the monitoring of physical properties [12], [13], [14], [15], [16], chemicals [17], [18], [19], [20], [21], [22], and biological molecules [23], [24], [25], [26], [27], [28]. TFOBS has several applications in environmental monitoring, drug screening, clinical diagnostics, and national security. From a detection standpoint, some advantages of TFOBS include the exposure of evanescent field beyond the surface of the sensing region, fast and real-time response, and the ability to respond without labels. Based on the femtogram to pictogram per mL sensitivity we obtained for protein bovine serum albumin (BSA) at near-IR wavelengths and flow conditions [29], we postulate that DNA can be detected using modification to our current system, resulting in label-free and continuous detection in the flow setting, at near-IR wavelengths.
In this study, TFOBS are coated with gold and housed in a flow cell. Thiolated 15-mer single stranded (ssDNA) probes were immobilized on the TFOBS gold surface. Complementary 10-mer ssDNA target strands were then detected while they hybridized with the immobilized probes at concentrations as low as 750 fM. The sensor also showed selectivity against a single nucleotide mismatch.
Section snippets
Physics of sensing
Optical fibers are light transmission waveguides made out of silica. They consist of a cylindrical core surrounded by a cladding. The core is doped with Ge such that its refractive index (RI) becomes slightly higher than that of the cladding. Since the core has a higher RI than the cladding, light is reflected along the fiber axis when it reaches the core-cladding interface, and therefore propagates through the optical fiber by total internal reflection (TIR). Aside from the large portion of
Flow cell apparatus
The flow cell assembly was described in our previous work [29] and is shown in Fig. 1. The chamber consists of a cylindrical well 4 mm in diameter and 3 mm in depth, and is centered on a 6 mm thick Plexiglas (∼3 cm × 5 cm). The approximate volume of the sample chamber is 40 μL. A sample inlet and outlet were drilled from the side edges of the Plexiglas to the sample chamber wall, to allow fluid flow through the sample chamber. The tapered region was positioned within the sample chamber. To complete the
Effect of gold thickness
Prior to performing DNA experiments, the ideal gold thickness was obtained by determining its effect on the TFOBS response due to Protein G (PG) immobilization. PG was chosen as the test analyte because its mechanism of attachment is similar to that of ssDNA, but can be used at higher concentrations and is of lower cost. Since the Denton Desk II System was capable of sputtering 100–1000 nm of gold, the experimental thicknesses were set within that range. In order to limit the number of
Conclusions
Through the use of a model system, we have shown that DNA hybridization can be detected using the TFOBS. The notable aspects of our detection system are intensity-based sensing, the use of near-IR wavelengths, and the ability to detect label-free DNA. The detection limit is comparable to many detection systems, and there should be some effort dedicated to lowering the limit. After the optimization of detection limit, the next step would be the detection of DNA from live cells.
Angela Leung received her BASc in biomedical engineering from University of Toronto in 2003. She is currently a final year doctoral student in Drexel University, where she is anticipating a PhD in chemical and biological engineering in June 2007. Her research area is in biosensors, particularly low concentration protein and DNA detection and design and fabrication of tapered fiber optic sensors and devices. Her interest also includes development of validation methods for bioprocesses and
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Angela Leung received her BASc in biomedical engineering from University of Toronto in 2003. She is currently a final year doctoral student in Drexel University, where she is anticipating a PhD in chemical and biological engineering in June 2007. Her research area is in biosensors, particularly low concentration protein and DNA detection and design and fabrication of tapered fiber optic sensors and devices. Her interest also includes development of validation methods for bioprocesses and biomedical devices.
P.M. Shankar received his MSc (1972) in physics from Kerala University, India, MTech (1975) in applied optics and PhD in electrical engineering (1980) from Indian Institute of Technology, Delhi, India. He was a visiting scholar at the School of Electrical Engineering, University of Sydney, Australia, from 1981 to 1982. He joined Drexel University in 1982 and is currently the Allen Rothwarf Professor and interim department head of Electrical and Computer Engineering. He is the author of the textbook ‘Introduction to Wireless Systems’, published by John Wiley & Sons, 2002. His research interests are in wireless communications, fiber sensors, biosensors, and statistical signal processing for medical applications.
Raj Mutharasan received his BS degree (1969) in chemical engineering from IIT Madras (India) and a PhD (1973)in chemical engineering from Drexel University. He joined the faculty ranks at Drexel University in 1974 after a post doctoral year at University of Toronto, Canada. He was appointed to the position of Frank A. Fletcher Professor of Chemical and Biological Engineering in 1995. His research interests are in biophysics, biophotonics and cantilever for sensor development, and process biotechnology. He has published extensively and is the author of several patents. He is a Fellow of American Institute of Chemical Engineers and American Institute for Medical and Biological Engineering.