Integrated microring resonator biosensors for monitoring cell growth and detection of toxic chemicals in water

Abstract

Integrated microring resonators fabricated on silicon wafers were used as signal transducers to detect alterations in physical traits of attached live mammalian cells. Cell adhesion and growth events could be monitored by the shift in resonance frequency of the microring resonator. Toxic chemical-induced changes in cell motility were rapidly detected based on variations in the fluctuation of resonance frequency. Microring resonators modified with an endothelial cell line (MS1) adhered onto its surface were used to detect the presence of two toxic chemicals, viz. sodium pentachlorophenate and Aldicarb at concentrations above the military exposure guideline levels within a duration of 1 h.

Introduction

Cell-based bioassays are widely used in biomedical, pharmaceutical and environmental applications for measurement of biomarkers, screening candidate drug molecules and sensing of toxic chemicals. A major advantage of using living cells is that the effect of complex biological interactions can be studied by monitoring changes in cellular physiology. Compared to other ecotoxicological assays, cell-based assays using in-vitro cultured animal or human cells have shown promise as a method for monitoring toxicity in environmental water (Ekwall et al., 1998). However, existing cell-based assays are limited by their slow response time and the need for specially trained technicians and dedicated lab equipment. They are not suitable for field deployment and automated detection (Ekwall et al., 1998, Shoji et al., 2000). A recent report by US army center for environmental health research concluded that none of the biosensors tested met the requirements for use in drinking water safety monitoring (van der Schalie et al., 2006).

Cell motility is highly correlated to its physiological functions, such as dynamic changes in cell volume, cell to cell contact and cell to matrix contacts. Cell motility could be affected by the presence of toxic reagents (Aschner et al., 1998, Allansson et al., 2001), excitability (Paoletti and Ascher, 1994, Casado and Ascher, 1998), changes in metabolism (Waldegger et al., 1997), apoptosis (Gomez-Angelats and Cidlowski, 2002, Bortner and Cidlowski, 2004), necrosis, neurotransmitters (Kimelberg, 2004, Sombers et al., 2004), cell division and growth (Grover and Woldringh, 2001, Remillard and Yuan, 2004). Cell swelling (volume change) is one of the earliest responses to stress and injury and can occur within minutes following exposure to toxic agents (Grover and Woldringh, 2001).

Methods to measure cell volume and motility have utilized dedicated lab equipment such as light or electron microscopy (Grover and Woldringh, 2001), fluorescence microscopy (Satoh et al., 1996, Crowe et al., 1995), electrophysiology (Dierkes et al., 2002, Kawahara et al., 1994), atomic force microscopy (Schneider et al., 2000), or electrical impedance (Oconnor et al., 1993; http://biophysics.com). These methods may however not be suitable for field or portable use. The development of a microfluidic cell volume sensor based on electrical cell-substrate impedance sensing (ECIS) was reported for use as a potential portable sensor system (Ateya et al., 2005). Several factors such as media conductivity and cell damage resulting from the applied potential can affect impedance measurements, thus the system must be operated under restricted conditions. Quartz crystal microbalance has also been recently suggested for use in probing cell motility (Sapper et al., 2006). While the sensitivity of this method was not specified, it does distinguish between cells in normal, dehydrated, and fixed status. This method was used only to monitor the averaged effect of thousands of cells attached to the sensor surface.

We recently reported the use of a microring resonator (MR) based optical refractive index sensor for biosensing applications (Ramachandran et al., 2008). The MR chips consisted of a closed-loop waveguide that was vertically coupled to input and output waveguides. Light at a frequency supporting the resonant modes of the microring can couple into the microring via the input waveguide. The coupled light continuously circulate in the high refractive index core of the microring giving an effective path length that is much longer than the physical size of the ring there by enhancing sensitivity. The output waveguide extracts the signal at resonance. An evanescent optical field extends outside the core to a range of a few hundred nanometers, and refractive index changes within this range will be detected as a change in resonance frequency.

The refractive index (RI) of cells is quite different from the surrounding media. Cell membranes consist of proteins, sugar, and lipids, which have a higher refractive index (∼1.46) than cytoplasm (∼1.36) and surrounding water (∼1.33) (Kleinfeld and LaPorta, 2003). Therefore, cell motility and volume change can be measured optically using a refractive index sensor. Surface plasmon resonance microscopy has been used to quantitatively measure the distance of adherent cells to the attached surface (Giebel et al., 1999). A grating-based refractive index sensor was recently demonstrated for monitoring dynamic mass redistribution of cellular content inside the cell (Fang et al., 2006).

Here we report the application of MR sensors for monitoring cell growth and for rapid detection of toxic chemicals in water. Although mammalian cells are a few microns thick, cell membranes attached to the surface are only 3–10 nm thick and are well within the MR sensing range. Due to the refractive index difference between cells and surrounding media, cell adhesion and growth can be monitored by the MR sensor. Changes in cell volume are one of the earliest responses to occur upon exposure to toxic chemicals (Grover and Woldringh, 2001). By attaching cells to the microring resonators, the volumetric and motility change of cells will cause refractive index shifts near the MR surfaces, which can be monitored with extreme sensitivity using the MR sensor.

Compared to existing cell monitoring methods MR sensors offer several advantages: (1) optical based refractive index sensing – enables truly label-free and non-invasive monitoring; (2) extremely small size – MR based sensors can be used detect the responses of only a few cells and possibly even a single cell; (3) Multiplexing – multiple MRs can be manufactured on the same chip and individually interrogated, which allows for multiplexed detection, self referencing, or redundancy sensing without sacrificing sensitivity; (4) interferences from the bulk solution is negligible as optical interrogation occurs mainly at the sensor surface; (5) Mass production – MR chips are manufactured by standard wafer technology therefore mass production at low cost is feasible.

The main limitations of cell-based sensing using MR sensors are (1) Changes occurring within the whole cell cannot be monitored due to the surface sensitive nature of MR sensors. Only the part of the cell attached on the sensor surface can be monitored. (2) MR based biosensor technology is still in the early stages of development. Additional research is needed to fully understand the relationship between the MR signal and cell behavior. (3) An important technical issue is the need of an automated or passive alignment mechanism to couple the MR chips with optic fibers, which will help in reducing the cost of sensor production and development of disposable sensors. (4) MRs are highly sensitive to environmental effects, like changes in temperature and pressure. It is highly important to maintain a steady environment or cancel out any such effects by accurate referencing.