Design and Fabrication of Fiber-Optic Nanoprobes for Optical Sensing
© Zhang et al. 2010
Received: 7 July 2010
Accepted: 5 August 2010
Published: 31 August 2010
This paper describes the design and fabrication of fiber-optic nanoprobes developed for optical detection in single living cells. It is critical to fabricate probes with well-controlled nanoapertures for optimized spatial resolution and optical transmission. The detection sensitivity of fiber-optic nanoprobe depends mainly on the extremely small excitation volume that is determined by the aperture sizes and penetration depths. We investigate the angle dependence of the aperture in shadow evaporation of the metal coating onto the tip wall. It was found that nanoaperture diameters of approximately 50 nm can be achieved using a 25° tilt angle. On the other hand, the aperture size is sensitive to the subtle change of the metal evaporation angle and could be blocked by irregular metal grains. Through focused ion beam (FIB) milling, optical nanoprobes with well-defined aperture size as small as 200 nm can be obtained. Finally, we illustrate the use of the nanoprobes by detecting a fluorescent species, benzo[a]pyrene tetrol (BPT), in single living cells. A quantitative estimation of the numbers of BPT molecules detected using fiber-optic nanoprobes for BPT solutions shows that the limit of detection was approximately 100 molecules.
The emergence of nanotechnology opens new horizons for nanosensors and nanoprobes that are suitable for intracellular measurements. Nanosensors provide critical information for monitoring biomolecular processes within a single living cell, thus could provide great advances in biomedical research and clinical applications. Fiber-optic nanosensors with nanoscale dimensions are capable of sensing intracellular/intercellular physiological and biological parameters in submicron environments. Tapered fibers with distal diameters between 20 and 500 nm have been demonstrated for near-field scanning optical microscopy (NSOM) [1, 2]. Chemical nanosensors were developed for monitoring calcium and nitric oxide, among other physico-chemicals in single cells [3, 4]. Vo-Dinh and coworkers have developed nanobiosensors to detect biochemical targets inside living single cells [5–12]. Fiber-optic nanoprobe promises to be an area of growing research that could potentially provide an imaging tool for monitoring individual cells and even biological molecules. Single-molecule detection and imaging schemes using nanofibers could open new possibilities in the investigation of the complex biochemical reactions and pathways in biological and cellular systems leading to important applications in medicine and health effect studies.
Optical nanotips were first developed as scanning probes in near-field optical microscopes . Such nanoprobes can achieve resolution as high as λ/50, where λ is the wavelength of light . It is important to control aperture size, taper shape, and metal coating to achieve a better performance . The fiber-optic probes were fabricated by laser-heated pulling or chemical etching [14–16]. Laser-pulled fiber tips can achieve diameters smaller than 50 nm with small cone angles . Chemical etching tips have larger cone angles and similar apex sizes . However, it is often difficult to control the etching process. The side of the fiber was further coated with silver, aluminum, or gold films to confine the light [9, 14, 17]. Traditional manufacturing processes still limit the quality of metal-coated fiber probes. Optical throughput of pulled nanoprobes is limited by the sharp taper angle. Chemical-etched tips have higher throughput; however, they do not have a flat distal end as laser-pulled ones which are difficult to form well-defined nanoapertures in shadow evaporation. Moreover, shadow evaporation often leads to either complete or irregular coated tip. Grainy structures of metal thin film increase the distance between the aperture and the sample, which reduce the resolution and intensity. It is also easy to form pin holes at the tapered region that could cause light-leaking. The aperture deviates from ideal circular shape because of grains. A quantitative analysis of probe transmission efficiency becomes difficult. Focused ion beam (FIB) fabrication of nanostructures has been applied on optical fibers for chemical sensing . FIB milling for nanostructure formation allows precise control of size and shape in nanometer accuracy. This paper deals specifically with the metal coating on the formation of nanoaperture at the tip end. Coating materials and angles greatly affect the quality of the nanoprobe. By combining with focused ion beam milling, nanoprobes with well-defined aperture as small as 200 nm have been obtained. We investigate the capacity of the nanoprobes by detect benzo[a]pyrene tetrol in living cells.
The sidewall of the tapered end was then coated with a thin layer of metal, such as silver, aluminum, or gold to prevent light leakage of the excitation light on the tapered side of the fiber. An array of fiber probes was attached on a rotating motor inside a thermal evaporation chamber (Quorum Technologies E6700). The rotation rate was controlled by a microcontroller board (Parallax). While the probes were rotating, the metal was allowed to evaporate onto the tapered side of the fiber tip to form a thin coating. The nanoaperture was formed through shadowed evaporation as the fibers were tilting away from the source. The nanoprobes were characterized with scanning electron microscopy (FEI XL30).
In order to fabricate well-defined fiber-optic nanoprobe tips, we employed focused ion beam (FIB) milling of nanoapertures in the metallic films deposited on tapered tips of optical fibers. Before carrying out FIB milling, the optical fibers were coated with metallic films (aluminum, silver or gold) using electron beam evaporation (CHA Industries Solution E-Beam). During the evaporation process, the fiber-optic tips faced the metal source to ensure that the fiber side walls and the tips were completely covered with a thin metallic layer (100–150 nm). The sample mount was rotated to improve uniformity and the thickness of the metallic film was monitored by a quartz crystal monitor. The deposition rate was varied between 0.05 and 0.2 nm s-1 at a chamber pressure of ~3 × 10-6 Torr for the electron beam evaporated films.
A Hitachi FB2100 focused ion beam milling machine with a gallium ion source was used to fabricate the nanoapertures on the fiber tips. Beam currents and accelerating voltages of 0.01 nA and 40 keV energy were typically used. The desired nanostructures were milled by rastering the ion beam and employing a beam blanker. The beam blanker shuts on and off according to a 8-bit grayscale, 512 by 512 pixel image file. Tapered optical fiber tips with nanoapertures were fabricated by employing FIB milling at magnifications varying between 3000× and 18000× depending on the desired minimum aperture size. To form metallic nanostructures on the tips of optical fibers, a special fiber holder that could fit in the FIB stage was fabricated.
Nanoprobes were also used to investigate BPT in single cells. PC3 human prostate cancer cells were incubated with 1 μM BPT in PBS for 2 h. Control cells are treated with PBS only. All dishes were rinsed with PBS prior to measurement. Nanoprobes were controlled by the micromanipulator to puncture the cell and keep inside while taking the measurement.
Results and Discussion
Effect of the Metal Evaporation Angle
Effect of the Different Metal Coatings on the Nanoprobes
The metallic coating process is a critical step in nanoprobe fabrication. A thin film of an optically opaque metal such as aluminum, silver, or gold is coated along the outside walls of the tapered optical fiber tip to form an optical light pipe free of defects, which would permit photons to escape from the tapered sides of the optical fiber. An optical aperture to allow evanescent wave excitation is formed at the tip's apex by angled evaporation. Silver has been used in nanoprobe fabrication . It has high reflectivity in the visible and IR range and very stable in aqueous solutions as long as oxidizing agents or complexing agents are not present. But a silver layer will oxidize rapidly under ordinary atmospheric conditions and will not exhibit a high reflectance below 400 nm. Therefore, it is desirable to use the nanoprobe right after metal evaporation. Otherwise, the light shielding will deteriorate or even the coating will peel off after a few days in air.
Gold thin film was demonstrated to be a very stable coating under environmental conditions although it does not have high reflectance in visible range. An interface layer such as Cr is required to increase adhesion between gold and the fiber silica surface. Gold has a high melting temperature (660°C for Al, 960°C for Ag, and 1,060°C for Au) and a good thermal resistance. The thermal stress generated during metallic film deposition damages the aperture due to very different thermal expansion coefficients of metal and quartz. Gold coating has the lowest thermal expansion coefficient (23.1 × 10-6/°C for Al, 18.9 × 10-6/°C for Ag, 14.2 × 10-6/°C for Au, 0.55 × 10-6/°C for SiO2), which will reduce the thermal destruction of the fiber tip.
Nanoprobe Fabrication using Focus Ion Beam (FIB) Milling
Fluorescence Measurement of Benzo[a]Pyrene Tetrol (BPT) Using Nanoprobes
Fiber-optic nanoprobes have opened up new applications in molecular biology and medical diagnostics. Due to their small sizes, nanosensor provides important tools for minimal invasive analysis at single cellular or sub-cellular level. Because transmission efficiency is highly related to the aperture size, control the nanoaperture size is essential in the fabrication of high-quality nanoprobes. Subtle changes in the tilt angle during metal evaporation can greatly affect the size or even the existence of the aperture. A much more rational fabrication process would involve a nanofabrication technique such as FIB, in which aperture size could be independently controlled from evaporation. The detection sensitivity of fiber-optic nanoprobes depends mainly on the extremely small excitation or detection volume set by the aperture sizes and penetration depths. This effectively reduces background fluorescence, thereby enhance detection sensitivity. Nanofabrication would also greatly improve the reproducibility of aperture shapes and hence the optical performance of near-field probes.
The author acknowledges the contribution of G.D. Griffin, J.P. Alarie, B.M. Cullum, and P. Kasili. This research is sponsored by the National Institutes of Health (1R01ES014774 and R01-EB006201) and US Army Medical Research and Material Command (W81XWH-09-1-0064).
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