|Home | About | Journals | Submit | Contact Us | Français|
Optical label-free detection avoids the cost and complexity of fluorescence and radio labeling while providing accurate quantitative and kinetic results. We have developed a new optical label-free sensor called the liquid core optical ring resonator (LCORR). The LCORR integrates optical ring resonator sensors into the microfluidic delivery system by using glass capillaries with a thin wall. The LCORR is capable of performing refractive index detection on liquid samples, as well as bio/chemical analyte detection down to detection limits on the scale of pg/mm2.
The liquid core optical ring resonator (LCORR) sensing platform (1–8) integrates micro-capillary fluidics with label-free optical ring resonator sensing technology. Optical ring resonators have been studied for sensing applications for a decade (9–24). However, due to the use of the capillary as the ring resonator, the LCORR inherently integrates the sensor head with the sample fluidics, which can increase optical performance while simplifying the system design.
The LCORR sensing platform is illustrated in Figure 1. The ring resonator is formed in the circular cross section of the capillary. The capillary wall acts as a waveguide; light travels repeatedly in a circle around the circumference of the capillary as the ring guides the light through total internal reflection. Light is evanescently coupled into the ring resonator using a fiber taper or a pedestal waveguide. Light that forms an integer number of wavelengths around the circumference of the ring is resonant; the resonating modes are called whispering gallery modes (WGMs). As shown in Figure 1(B), the WGM has an evanescent field that extends beyond the inner wall of the LCORR capillary, where it interacts with the sample as it moves through the inside of the capillary. Analytes are detected by immobilizing biorecognition molecules (e.g. antibodies) that capture the analytes at the inner LCORR surface, where they interact with the evanescent field of the WGM. The presence of the analytes in the optical field changes the effective refractive index experienced by the WGM. Thus the effective optical path length around the ring changes, which causes the resonating wavelength to shift spectrally. This change in WGM spectral location over time as the sample passes through the LCORR capillary is the sensor signal, as illustrated in Figure 1(C).
In this chapter, the method for creating LCORRs is described, the preparation of the LCORR sensor setup is presented, and the steps for performing sensing measurements are given in detail. Protocols for some sensing applications of the LCORR are presented. These include a bulk refractive index measurement, an assay for detecting protein adsorption and protease activity, and an assay for detecting the presence of a specific DNA sequence in a sample.
This chapter describes in detail the process for first setting up an LCORR measurement and then the process for performing a sample analysis with the LCORR. At this stage of the development of the LCORR for use in bio/chemical analysis, it is necessary to assemble many of the components in the lab, and to produce LCORRs on site. Figure 2 presents a flow chart of all of the steps necessary to conduct a single sample analysis, from producing the LCORR to analyzing the data. This chapter further explains how to create the experimental setup, as well as how to assemble on-site LCORR and fiber taper production systems.
The primary component of the LCORR sensing system is a glass capillary that acts simultaneously as a microfluidic channel for sample delivery and as a ring resonator for sample detection. This capillary is unlike typical capillaries utilized for liquid or gas sample movement because the wall of the LCORR must be very thin. In fact, for sensing purposes, the LCORR capillary wall must be less than around 5 μm thick. As capillaries with this dimension are not available today, it is required to produce LCORR capillaries in the lab before conducting any detection assays.
To produce a capillary with a thin wall, we stretch a commercially available glass capillary to thin its dimensions (see Note 1). This is analogous to the process of drawing fiber optic cable from a preform. The preform glass capillary is heated to the softening point while one end is pulled. As wall thickness is critical, attention must be given to the temperature and pulling speed, as these parameters dictate the change in aspect ratio (diameter to wall thickness) during pulling. Higher speed and lower temperature will combat the effects of surface tension, which is pulling the softened glass radially inward and decreasing the aspect ratio. Preservation of nearly 100% of the aspect ratio during capillary drawing has been exhibited with our LCORR drawing technique, and has been demonstrated in similar work (25).
The configuration utilized for drawing LCORR capillaries and a photo of the setup are shown in Figure 3. The entire apparatus is contained within a clear acrylic enclosure to reduce air currents, which can cause fluctuations in the temperature of the heating zone. CO2 lasers are used as the heat source to soften the glass capillary in the heating zone (see Note 2). Two CO2 lasers are used on opposite sides to provide more evenly distributed heating (see Note 3). The power of the lasers, which is computer-controlled using a data acquisition card, is dependent upon which type of glass is used (see Notes 4, 5). While the lasers soften the glass tube in one spot, the two stages holding each end of the glass tube are moved to stretch the glass. The tube can be taped onto each stage, although any temporary clamp will suffice. The stages are mechanical slides with stepper motors for movement. Movement of the stages is controlled with a PCI-based motion controller. One stage is pulled quickly away from the heating zone while the other is slowly pushed toward the heating zone, keeping constant the mass of glass in the heating zone. The ratio of pulling speed to feed-in speed controls the diameter of the pulled capillary (see Note 6).
In this prototype implementation, computer code controlling the stage movements and laser power is developed using LabView software (National Instruments). User inputs to the program include the desired laser power, the pulling speed, and the feed-in speed. The program operates through the DAQ card and the PCI-based motion controller to generate the appropriate voltage-based outputs to control the laser power and the pull/feed stages.
The steps to be performed for pulling an LCORR capillary are listed below.
For prototyping and pre-clinical applications of the LCORR sensing system, it is sometimes practical to utilize a tapered fiber optic cable to excite WGMs in an LCORR see Note 8). The operating principle of the fiber taper is that a fiber optic cable that is thinned to a few micrometers in diameter will have an evanescent field outside the cable. This evanescent field is capable of coupling light into the LCORR. In general, fiber tapers must be produced in the lab in which they will be used because of their fragility. Thus, taper production is presented here as a necessary component in the LCORR assay development.
An illustration of the setup for pulling fiber tapers and a photo of the setup are presented in Figure 5. The taper is produced by stretching a fiber optic cable under heat. Typical single mode fibers are utilized as the tapering fiber. The heat source is a clean flame provided by gas torch (see Note 9) (26). The fiber optic cable is stretched slowly from both sides of the heat zone while the heat zone is scanned back and forth by about 1 cm in order to provide a region of constant diameter. Just as with the LCORR pulling apparatus, the stages are mechanical slides controlled by a computer via code developed in LabView and a PCI-based motion controller. The LabView program is very similar to the one used for pulling LCORR capillaries. It may be desirable to pass laser light through the fiber and to measure the loss during taper pulling to monitor the health of the taper while it is produced.
The steps to be performed for pulling fiber taper are listed below.
The purpose of the experimental setup is to pass the sample through the LCORR while monitoring the sample's effect on the spectral position of the WGMs. The experimental setup is diagrammed in Figure 7. The LCORR is connected to tubing so that the sample can be passed through using a pump. The taper is connected on one end to a tunable laser (see Note 13) and on the other to a photodetector. The tunable laser scans across a spectral range wide enough to detect a WGM and to track its movement. Typically, 10–15 GHz (i.e., about 100 pm for a center wavelength of 1550 nm) of tuning range is sufficient for this. When the scanning laser passes through a resonant wavelength, destructive interference occurs on the fiber taper at the coupling point with the LCORR, resulting in an observable decrease in the output power. Thus, scanning the laser across one WGM will produce a measured waveform like the one shown in Figure 7 (see Note 14).
Tunable laser control and data collection is performed by a data acquisition (DAQ) card, under the direction of code written in LabView. The DAQ voltage output scans the output wavelength of the laser while the input samples the voltage at the output of the photodetector. After each scan, the sample values are stored in a respective file on the computer. Also, the LabView program displays the measured voltage in real time. Following the experiment, a simple program written in Matlab (Mathworks) is used to scan each file for the spectral location of the voltage minimum (indicative of the WGM), and then a data set is created to represent the WGM spectral position over time. This data set is the sensorgram. An exemplary sensorgram is shown in Figure 1(C).
As with many label-free optical sensors, the signal from the LCORR sensor is temperature dependent (4). Therefore, a temperature control system is required in the experimental setup to suppress temperature fluctuations in the LCORR sensing region that would be translated to noise in the sensing signal. Our design is based on a thermo-electric cooler (TEC) and TEC controller. The setup is illustrated in Figure 8.
The steps to be performed for creating the experimental setup are listed below.
After pulling the LCORR capillary, the wall thickness may not be as thin as desired for sensing purposes. Additionally, the sensitivity must be characterized before use. Sub-micron differences in wall thickness result in significant differences in the sensitivity of the LCORR, and thus it must be well-characterized. Therefore, once an LCORR is pulled, it is placed in the experimental setup for sensitivity optimization and characterization.
To set the sensitivity of the LCORR, hydrofluoric acid (HF) is used to slowly etch away the inner capillary surface. The amount of glass to be removed depends on the preform and the capillary pulling process. For example, if the preform had an outer diameter of 1 mm and an inner diameter of 0.9 mm, and the LCORR outer diameter is 100 μm, then the thinnest that the wall can be is 10 μm. To achieve a good sensitivity, around 7 μm may need to be removed with HF. This is done by passing diluted concentrations of HF through the LCORR while intermittently characterizing the sensitivity.
The sensitivity is characterized using solutions of known refractive indices, as the sensing mechanism of the LCORR is based on refractive index detection. We use solutions of ethanol in water (1), as the difference in refractive index of the varying concentrations is known (27). Determining the WGM spectral shift for a particular change in refractive index of the sample leads to the refractive index sensitivity of the LCORR sensor.
The steps for optimizing and characterizing the sensitivity of the LCORR are listed here.
The characterization procedure outlined in Section 3.4 illustrates that the LCORR sensing platform can be used to identify the refractive index of sample liquids. Once the sensitivity has been characterized, liquids of unknown refractive index can be passed through the LCORR while the WGM spectral position is monitored. This can be used, for example, to identify if a sample has a small amount of contaminants.
The following procedure is used to measure the bulk refractive index.
The LCORR can utilize its refractive index sensing capabilities to detect biomolecule analytes that are captured at the inner surface of the capillary. Typically, biomolecules have refractive indices around 1.45 to 1.55, while buffers are typically close to the range of 1.3 – 1.35. Thus, when biomolecules bind to the surface, the local refractive index in the region of the WGM evanescent field increases. This RI increase is reflected in the sensor signal by a red shift of the WGM spectral position. Figure 12 illustrates an exemplary sensor signal reflecting the binding of BSA molecules at the inner surface of the capillary. Because the total WGM spectral shift is proportional to the number of molecules that bind to the surface, the concentration of analytes in the sample can be determined when using the LCORR as a sensor.
Through the same refractive index sensing mechanism that causes a red-shift for binding analytes, a blue shift in the WGM spectral position occurs when analytes are removed from the capillary surface. Thus, the LCORR can also be used to detect proteolytic activity. Figure 13 shows the sensor signal when trypsin is introduced into the same LCORR following the binding of BSA (shown in Figure 12). Trypsin is known to cleave BSA molecules at a number of residues, which will cause a significant amount of the BSA mass to be removed from the LCORR.
As an example of protein detection, the steps for detecting BSA molecules in a sample are listed below.
One important application of the label-free biomolecule detection capability of the LCORR sensing platform is the identification of a specific DNA sequence in a sample. Similar to DNA microarray technology, an oligonucleotide probe that is designed to be the complement of the single-stranded target is immobilized on the sensor surface. If the target sequence exists in the sample, it will bind with an immobilized oligo probe at the LCORR inner surface. As shown in Subsection 3.6, the LCORR quantitatively detects biomolecules that bind to at the surface.
Surface chemistry for successful immobilization of the oligo probes is critical. First, the surface of the LCORR and the 5' end of the oligo probe are functionalized with amine groups. Then, dimethyl adipimidate (DMA) is used to crosslink the oligo probes to the amino-functionalized LCORR surface. This surface chemistry is illustrated in Figure 14. Upon immobilizing the probes, the LCORR is prepared to detect the presence of the complementary probe sequence in the sample. Figure 15 presents an exemplary sensorgram, which shows the WGM spectral shift during the functionalization, oligo probe immobilization, and sample analysis processes.
The steps for conducting the DNA sequence detection measurement are outlined below.