Design and Rapid Constructions of Microstructures in Optical Fibers Using CO2 Laser-Their Potentials for Biosensor Application

Rudi Irawan
Singapore-University of Washington Alliance, BMERC, Nanyang Technological University, Singapore. Department of Physics, University of Lampung, Bandar Lampung, Indonesia. erirawan@ntu.edu.sg
Abstract This paper reports on design and rapid constructions of microstructures, such as in-fiber microhole, Dshaped fiber, in-fiber microchannel, side-polished fiber and tapered fiber, in optical fibers using a CO2 laser system that help exposing the optical fiber core to the measurands. Exposing the optical fiber core to the measurands surrounding the fiber is often used to enhance the sensitivity of an optical fiber sensor. The microstructures in the fibers were examined using a SEM and an optical microscope. Testing on an in-fiber microchannel of 100m width, 210m depth and 10mm length for fluorescence sensor embedded in a microfluidic card shows the prospect of this method for use as a fluorescence biosensor in a microfluidic card. The microstructures were fabricated using a direct-write continuous wave (CW) CO2 laser system with maximum output power of 40 W. The direct-write CO2 laser machine is a flexible and fast machining tool for design and fabrication of microfluidic cards and microstructures in an optical fiber, and can possibly be a replacement of the time consuming chemical etching method that is usually used for microstructure fabrications in an optical fiber. Keywordsoptical fiber sensor, microfluidic microstructures in fibers, direct-write CO2 laser system. card,

Swee Chuan Tjin, Chia Meng Tay, Khay Ming Tan

Photonics Research Centre, School of Electrical and Electronics Engineering, Nanyang Technological University, Singapore.

generally immune to electromagnetic interference (EMI), light weight, small size, passive (all dielectric), chemically inert and can be manufactured cheaply using silica or polymeric fibers [1]. Fiber optic sensors can be divided into those relying on intensity modulation, polarisation modulation, wavelength/ frequency modulation, optical path modulation, time modulation and coherence length modulation. The simplest fiber optic sensor and the easiest to demodulate are the intensity based sensor, where the intensity of light in the guided mode of the fiber is modulated by changes in the external environment [2]. Refractive index sensing and fluorescence sensing commonly use intensity based sensors. The refractive index or fluorescence emission of a measurand affects the intensity of a fiber output at chosen wavelengths. A simple change of light intensity exiting the fiber can be detected easily, when a properly designed sensor reacts to changes in a physical quantity like refractive index or fluorescence intensity in the environment. This refractive index or fluorescence emission can possibly be correlated to the concentration of a measurand, which can be a biological or chemical analyte. As a result, this method is popularly used for chemical and biological analyte sensors. Fiber sensors can also be embedded in microfluidic cards conveniently due to their flexibility, high sensitivity and low cost [3-5]. Refractive index sensing has been used for detection of chemicals, or chemical concentrations in lab-on-chip or microfluidic applications [6, 7]. Fluorescence based sensing is more commonly employed for biological analyte sensing and for analyte sorting in lab-ona-chip applications due to its specificity and sensitivity [3, 8, 9]. When preparing fibers for chemical and biological sensing, the light in the fiber core must be able to be modulated by the measurands. Enhancing the interactions between the light guided in the fiber cores and the measurands by exposing the fiber cores to the external environment is popularly used to achieve this objective. In order to improve light coupling from the fiber core to the fiber exterior and then back again, microstructures are needed in the fiber to promote light

I. INTRODUCTION Optical sensors are ubiquitous in todays industry. Optical sensors perform best when measurements have to be taken in hazardous environments or when the sensor itself has to come into contact with the measurand or analyte [1]. Some examples of environments that degrade electronic sensors, but which optical sensors can survive in, are in environments with high humidity, high temperature, high electromagnetic flux, and in fluids. Fiber optic sensors are a class of optical sensors that are popular due to the boom in the fiber optic communications industry in the past twenty years. Fiber optic sensors are
__________________________________ This project is funded by the Biomedical Engineering Research Council of Singapore under the Singapore-University of Washington Alliance.

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scattering as well as evanescent field coupling. Commonly used methods to obtain the microstructures in the fiber are side polishing the fiber [6], etching the fiber [5, 7], tapering the fiber tip [3] or D-shaped fiber [10, 11]. They are time consuming, use expensive reagents, are less flexible to turn new optical fiber sensor designs and are therefore not practical for making large quantities or fast prototypes of sensors. Hence, a simple and effective fabrication technique is needed for the fabrication of microstructures in optical fibers. In this paper, we report a direct write method for the microfabrication of microstructures in optical fibers using a continuous wave (CW) CO2 laser system. It is believed that this technique can replace some of the conventional techniques, such as using the time-consuming photomask generation, photolithography and etching processes which hamper the rapid turnaround of new fiber sensor designs. The proposed method uses a computer aided design software for drawing different patterns and designs which are transferred to the computer-controlled CO2 laser cutting machine which will then imprint the patterns directly onto the fiber. Changing and improving designs are extremely rapid and simple and making large quantities of sensors or prototyping new designs is fast as well. Different structures like in-fiber microhole, D-shaped fiber, in-fiber microchannel, side-polished fiber and tapered fiber have been fabricated using the CO2 laser. Different types of fibers, single mode fibers, large core silica fibers and Polymethyl Methacrylate (PMMA) fibers were used. Hence, complicated microstructures have been fabricated onto silica or PMMA fibers which can be used for sensing applications like fluorescence biosensing in microfluidic devices. A fluorescence sensing application with fluorescein solutions as samples was tested in our experiment. The in-fiber microchannel structure, with a feature resolution of about 100m, provides a novel geometry where biological or chemical fluids can flow, biological analytes can be immobilized within the in-fiber channel and the fluorescence sensing can take place. If the analytes of interest are fluorophore tagged biological analytes, this structure allows for efficient collection of fluorescence emission. This fluorescence sensor will be more sensitive than conventional fluorescence sensors as the immobilization of analytes is achieved in the structures within the fiber and the fluorescence emission is contained inside the structures. II. METHODOLOGY

40 W, and consists of computer-controlled two-dimensional robotic arms to move across the entire machining area 61 cm x 30.5 cm. Fibers with microstructures, particularly PCS and single mode fibers, need to be handled gently and carefully, since they are very fragile. The sizes and the patterns of microstructures were drawn using a computer aided design program, and transferred to the computer-controlled direct-write CO2 laser machine. Careful control of laser intensity and number of laser passes determine the structure depth, but a balance must be adjusted between the laser power, number of laser passes and speed of laser scan to create well defined structures. B. Test for Sensor Application in a Microfluidic Card In-fiber microchannel structure made from PCS fiber was chosen for fluorescence sensor application test in this experiment. The width, depth and the length of the fabricated microchannel tested were 100 m, 210 m and 10 mm, respectively. The scanning electron microscope (SEM) images of the in-fiber channel structures after fabrication are shown in Figs. 6. Fluorescein was used as the fluorophore for testing the optical fluorescence sensor. Fluorescein is used because it is widely available, inexpensive, and commonly used as a biomolecular fluorescence tag. Fluorescein powder was dissolved in phosphate-buffer saline (PBS) to stabilize its pH at 7.4, as fluorescence spectrum is affected by pH of solution. The prepared fluorescein solutions were stored and enclosed properly to protect the solutions from excessive exposure to light before any measurement was conducted. This is because an overexposure of the fluorescein solution to light can lead to photo-bleaching. Different concentrations of fluorescein solutions from 0.001 g/L to 1g/L were prepared to characterize the response of the sensor. Fig. 1 illustrates the experimental setup used in the testing of the fiber fluorescence sensor. The light source used was a Nichia blue LED with an excitation wavelength centered at 470nm. A bandpass filter, 470nm5nm, was attached to the excitation source to limit the excitation spectrum, so that the excitation spectrum does not overlap with the fluorescence emission spectrum. The optical fiber with microfluidic channel (the sensor section) under the test was installed inside a microfluidic card made from polymeric sheets, as illustrated in Fig. 2. The fiber was extended to the exterior of the microfluidic card, so that it can be accessed easily by the light source and detection systems. The output of fiber sensor is a mixture of fluorescence emission and excitation light, so that a high pass filter, 550nm, was installed in front of the detector to filter out the excitation wavelength entering the detector. Finally, the emission light was focused into a mini-compact module of photon multiplier tube (PMT, Hamamatsu H578402) and the output voltage was measured using a volt meter. The experiments were started from the lowest concentration of prepared sample solutions, and then progressed to consecutively higher concentrations. During the measurements, the fluorescein solutions were pumped continuously into the channel using a micropump. After each measurement, the channel was cleaned thoroughly by flushing with buffer.

A. In-fiber Microstructure Fabrications In this experiment, three types of optical fibers were tested for microstructure fabrications. They are the PMMA optical fiber (PMMA fiber)- 750 m core diameter with 10 m fluorinated polymer cladding from Industrial Fiber Optics, Inc., the large core silica fiber (PCS fiber)- 600m/750m (core/clad diameter) from Ceram Optec Industries Inc. and the single mode optical fiber. Various patterns of microstructures, in-fiber microhole, D-shaped fiber, in-fiber microchannel, side polished fiber and tapered fiber, were machined on the optical fibers using the ablation process in which the laser beam from the CO2 laser was focused onto the optical fibers. The CO2 laser was operated at a wavelength of = 10.6 m with full power of

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Lens system to focus light and 470nm bandpass filter 470nm LED

Microfluidic card with in-fiber channel inside

550nm high pass filter PMT

5.0V

microstructures in fibers may also be used for other lab-on-chip applications. To mention a few of them, in-fiber microchannel in Figs. 6 can be used not only for sensing purposes, but also as substrates of disposable microfluidic channels. In addition, the in-fiber microhole shown in Fig. 4 can be used both as a reaction chamber and sensing area. These applications are useful for lab-on-chip applications.

Lens system to couple light from fiber

V V
A

A A
V C OM

OFF

Figure 1. Experimental setup for testing of the fiber optic fluorescence sensor. PMT photon multiplier tube.

Microchannel A

In-fiber channel

Optical fiber and channel A

Figure 3. D-shaped fiber constructed on 600 m silica core fiber.

Microfluidic card (a) (b)

Figure 2. (a) Schematic side view of the microfluidic card; (b) Crosssectional view (A-A) of the device

III.

RESULTS AND DISCUSSION

A. Microstructures Constructed in Fibers Various microstructures, D-shaped fiber, in-fiber microhole, side-polished fiber, in-fiber microchannel and tapered fiber, have been fabricated in large cores of PMMA and silica fibers, and a single mode optical fiber using a CW CO2 laser machine, which in turn can be used as optical sensors. The SEM images of these microstructures are shown in Figs. 3, 4, 5, 6, and 7. The CO2 laser machine used was originally designed for engraving polymeric materials. However, in this experiment this laser set-up was tested to fabricate microstructures in optical fibers, both polymeric core optical fiber and silica core optical fiber, in a few seconds. This method does not only reduce the fabrication time, but also eases the complexity of development of an optical fiber sensor based on enhancing the fiber core exposure to measurand surrounding the fiber which is widely used for chemical and biological sensors. The patterns of microstructures can be designed easily using a computer aided design software, and then the files are transferred to the computer controlled CO2 laser machine. The laser beam directed by mirrors mounted at the computer-controlled two-dimensional robotic arms writes the patterns onto the fibers. This fabrication method has the potential for both fast flexible prototyping and reproducible mass production. To the best of our knowledge no publications so far have been published about the use of a direct-write CO2 laser machine to write microstructures in optical fibers. The conventional method used for fabrication of microstructures in fibers is etching the silica fibers using chemical wet etching process or polishing the fibers that are time consuming processes that hamper the rapid turnaround of new fiber sensor designs. Together with sensing applications, the

Figure 4. A microhole fabricated in a silica core fiber.

Figure 5. Side polished single mode fiber.

Figure 6. In-fiber microchannels written in (a) 600 m silica core fiber, (b) PMMA fiber.

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the laser system shifts from the channel surface as the channel gets deeper. B. Fluorescence Sensor Application in a Microfluidic Card In-fiber microchannel structure was selected to study the prospects for optical sensor application, fluorescence sensor. The in-fiber microchannel structure is interesting not only because it can be used for sensor applications, but also because it can be used as substrates of disposable microfluidic channels for lab-on-chip systems in which the fluorescence sensors are popularly used [3, 8, 9, 13 - 17]. Its potential to be used both as the substrate of a disposable microfluidic channel and also as the fluorescence sensor will make a lab-on-chip system more compact and practical. The conventional methods to measure fluorescence emission from the sample in microchannels involve optically exciting the sample and detecting the fluorescence emission from the surface of the channels. The light source is focused onto the sample and the fluorescence emission is collected and focused into a detector which can be at the same side as or the opposite side (with reference to the microchannel) from the excitation source. This type of setup usually hinders the compactness and simplicity of the system. Focusing light beams onto a microchannel is challenging, particularly if the light beams diverge, such as light from LEDs. Poorly focused beam may cause that the light is scattered by the walls of microchannels which in turn can source of noise and cross-talk [18]. Collection efficiency of fluorescence emission using this method is also low, usually less than 1% [19]. Moreover, this type of set-up is sensitive to the alignment issues with regards to the focusing system, microchannels, and collection system. Any minor misalignment can cause severe problems to the consistency of the results. In the method using in-fiber microchannel tested in this experiment, the excitation light can be focused more easily into the optical fiber, transmitted through the optical fiber and will interact with the fluorescence sample situated in the microchannels through evanescent wave and/or scattered light mechanisms. The evanescent and/or scattered light excites the fluorophores. In this experiment, fluorescein solutions were used as the samples that absorb the excitation light, blue light, and emit fluorescence, green light. The fluorescence emission is isotropic in nature, and since the sample is contained inside the microfluidic channel in the fiber, most of the fluorescence emission is coupled back into the same fiber efficiently. This emission light is then transmitted through the same fiber and can be collected at the ends of the fiber. Unlike conventional methods which usually take point measurements of fluorescence emission, the technique reported here collects fluorescence emission from along the channel. It may be necessary to have an averaged measurement over the whole channel, since immobilization processes of real biological samples in a microchannel are usually statistical processes. Hence, the fluorescence sensor based on the in-fiber microchannel adds at least a two-fold advantage to the sensor; namely providing the channel for sample detection, and exposure of the fiber core to the sample for light-matter interaction, through evanescent wave and light scattering, which in turn increases the sensor sensitivity. The

Figure 7. Tapered single mode fiber.

Unlike UV-lasers, a CO2 laser, operated at a wavelength of = 10.6 m, creates the microstructures mainly due to the photothermal ablation mechanism. This far-infrared laser beam is absorbed by fiber materials, then heats, melts, and vaporizes the materials. This process leaves a void in the work piece. If the laser beam is scanned along an optical fiber, a microstructure will be created in the fiber. The patterns of microstructures are predetermined by the drawings and the depth of microstructures are dictated by several factors, such as the characteristic of work pieces, power of laser beam, number of laser passes, and moving speed of laser scan. The microstructure depth increases linearly with the laser power set [12]. However, the laser power needs to be optimized depending on the characteristics of material. If too much power is used, most of the surrounding material can be burned, and the microstructures will not be well formed. The structure depth also increases linearly with the number of laser passes [12]. It was found that ideally lower power should be used, but the laser beam should be often multiply scanned over the same area of the optical fiber to create well defined structures. Because the glass transition temperature of silica is higher than PMMA, to obtain the same depth of structures the silica core fibers require higher laser power, or a greater number of laser passes, or both than PMMA fibers do. It was also found that the ablation process on the PMMA fibers caused a thin layer of white residue to be formed on the surface of microstructures, such as microchannels. On the other hand, the ablation process on the silica core fibers still maintains the microstructures with clear and relatively smooth surfaces. It is believed that if the fibers are used for sensor applications, thin layers of white residues on the surfaces of microstructures fabricated in PMMA fibers may affect the sensor performance. If the CO2 laser beam is used to write microchannels or microholes, the cross sections of the channels or holes depend on the thermal diffusivities of the materials and on the intensity distribution within the CO2 laser beam. Since the thermal diffusivities of optical fiber materials used here, silica and PMMA core fibers, are very low, it is believed that the intensity distribution in the laser beam dictates the channel cross section [12]. The intensity distribution of the CO2 laser beam used here has a Gaussian-like profile, so that the cross sections of microchannels and microholes obtained have Gaussian-like profiles as shown in Figs. 4 and 6. The width of the microchannel surface is slightly bigger than 100 m, the size we specified, that might be due to that the focal point of

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microchannel also enhances the coupling efficiency of fluorescence emission back into the fiber. Besides the drawbacks of LEDs, such as diverging beams and broad band spectra, the use of a blue LED as an excitation source in this test makes the system compact, cheap, simple and stable. An LED of 470 nm wavelength is a broad band source, so a 470 nm interference band-pass filter needs to be installed at the light source. Recently, a compact laser diode at 470 nm wavelength is becoming commercially available, but the cost is still very expensive and the size is bulky. Otherwise, a laser diode at 470 nm wavelength is a good excitation source, as it has a very small line-width with other advantages like a low divergence beam and high intensity. The output of optical fiber sensor can be collected easily and focused into the detector, a mini-compact module of photon multiplier tube, as depicted in Fig. 1. This PMT module is very small in size (2cm x 2cm x 6cm) which includes a high gain built-in amplifier and a built-in DC-DC high-voltage converter which make the system is compact and simple, and requires only 15V DC power supply. The sensitivity of the optical fiber sensor was evaluated by filling the microchannels with known concentrations of fluorescein solutions. To minimize the photobleaching effect, the fluorescein solutions were continuously pumped into the microfluidic channels during the measurements. The measurement was started from the lowest concentration following with higher concentrations sequentially. The reason for progressing from low to high concentrations was to eliminate any possible errors due to some residue from previous measurements, although extra effort was made to clean the channel thoroughly between the measurements. The fluorescence intensities of various fluorescein concentrations were corrected with respect to the background fluorescence, which was the fluorescence of the channel loaded by a buffer only. Fig. 8 shows the promising result, the plot of the fluorescein concentrations against the fluorescence intensities. The plot shows that the sensor has very good sensitivity, able to detect 0.005 g/L fluorescein solution, and has linear response. This result shows the prospect of this fiber sensor as a fluorescence sensor, such as a fluorescence immunosensor.
15000 14000

In addition to the sensitivity test, repeatability was tested as well. Fig. 9 indicates that the sensor performance is reproducible. It implies that the fluorescence sensor is stable, and it may be reusable provided the sensor can be cleaned properly.
3.5x10
6

3.0x10

6
-1

Fluorescence intensity (a.u)

2.5x10 2.0x10

250 g l -1 100 g l -1 50 g l

1.5x10

1.0x10

5.0x10

0.0 1

Experiment sequences

Figure 9. Three reproducible results of fluorescence measurements

Fluorescence intensity (a.u.)

13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

IV. CONCLUSIONS This paper has described the prospect to design and construct microstructures in optical fibers for sensor applications using a CO2 laser direct writing machine. It is believed that this technique can replace standard techniques, such as chemical etching and polishing fibers, to construct an optical fiber sensor. Various microstructures in optical fibers, in-fiber microhole, D-shaped fiber, in-fiber microchannel, sidepolished fiber and tapered fiber, have been constructed easily, quickly and reproducibly. The CO2 laser direct writing machine which is originally designed for engraving polymer materials enables rapid prototyping and iterative design cycles of microstructures in both PMMA fiber and silica core fiber. That designing and changing microstructure patterns are easy and fabrication process is rapid makes this technique suitable for fast prototyping new fiber sensor designs. The microfabrication using CO2 laser machine may leave particles or debris in the structures that can be cleaned easily by sonicating the microstructures in water. The optical fibers are inexpensive and the microconstruction fabrication using a CO2 laser machine is easy and rapid, so that the sensors may also be treated as a disposable and consumable component in mass production. The fluorescence sensor using an in-fiber microchannel has also been tested, and the result shows the sensor is sensitive; it is able to detect fluorescein concentrations 0.005 g/L. Besides as the sensor area, the in-fiber channel can function as the channel for the flowing chemical or biological samples as well. The fluorescence sensor based on in-fiber channel construction also avoids complex geometrical optical systems to focus the excitation light into the microchannels and to collect the fluorescence emission from the sample inside the microchannels. These advantages make the in-fiber channel sensor is suitable for compact lab-on-chip applications, particularly for biosensors. The results provide a platform to develop a compact, robust, sensitive and affordable fiber based optical biosensors.

R2 =0.999

Fluorescein concentration (g/l)

Figure 8. Concentrations of fluorescein solutions vs. fluorescence intensities if in-fiber channel made from PCS fiber is used as an optical fiber sensor.

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