Enhancing the surface sensitivity of colorimetric resonant optical biosensors

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Abstract

Through alteration of the structure of a colorimetric resonant optical biosensor, the relative contribution of surface-binding effects to bulk refractive index effects can be influenced to favor detection of material in direct proximity to the sensor surface structure. The sequential buildup of polyelectrolyte monolayers has been used as a characterization method for determining the sensitivity of the device as a function of deposited thickness in an aqueous environment. A subtle change in the sensor structure is found to enhance surface sensitivity by ∼4.5×.

Introduction

Several optical biosensor approaches have been demonstrated which rely upon the detection of increased optical density caused by binding of biological material (for example: proteins, DNA, cells, bacteria) on a surface that has been activated with a receptor molecule [1], [2], [3], [4], [5], [6], [7]. Such sensors register a change in signal for any event that changes the optical density of the medium in contact with the sensor surface, whether it is from a biochemical-binding event or from a change in the “bulk” refractive index of the analyte solution. Most typically, an optical biosensor detects a combination of surface and bulk effects simultaneously. The bulk effect is subtracted from the measurement by referencing to a negative control surface that is known not to have adsorbed material present. The relative contribution of bulk and surface effects is determined by the spatial profile of the optical mode as it extends from the sensor surface into the test solution. Typically, an evanescent field extends from a planar sensor surface into the test sample with an exponentially decaying profile with a sampling distance on the order of hundreds of nanometers [8]. Only events occurring within the evanescent zone, whether surface or bulk effects, have the opportunity to influence the sensor measurement. Because biochemical interactions generally occur very close to the sensor surface (with dimensions of typical proteins ranging from 5 to 50 Å), it is of primary importance to have the highest sensitivity in the 500 Å region nearest to the sensor surface. Biochemical interaction with immobilized receptors generally is not occurring beyond this region, where bulk effects predominate.

In previous work, we have described a novel technology based upon a narrow bandwidth guided mode resonant filter structure that has been optimized to perform as a biosensor [9]. The sensor utilizes a sub-wavelength grating waveguide structure to provide a surface that, when illuminated with white light at normal incidence, reflects only a very narrow (resonant) band of wavelengths. The resonantly reflected wavelength is modified by the attachment of biomolecules to the waveguide, so that small changes in surface optical density can be quantified without attachment of a label to the detected biomolecule. Unlike optical detection approaches that rely upon interaction of detected molecules with an evanescent wave, the detection phenomenon in this work actually occurs within the waveguide, and thus provides for a strong interaction between surface-binding events and the transduced signal. Further advantages of the sensor approach are that the resonant reflected signal is measurable with the sensor either dry or immersed in liquid, and the simplicity of the non-contact excitation/detection instrumentation. Equivalent sensor structures have been fabricated onto glass substrates and incorporated into sheets of plastic film. Previous results demonstrate the ability to produce the biosensor in plastic over large surface areas and the incorporation of the sensor into large area disposable assay formats such as microtiter plates and microarray slides [10].

Often, the sensitivities of optical biosensor detection technologies are compared by determining the minimum change in liquid bulk refractive index that can be resolved. This comparison technique is only useful when the surface and bulk sensitivities scale together, and does not reflect the fact that such sensors must be optimized to measure primarily surface effects. However, thorough characterization of sensor response as a function of distance from the sensor surface is difficult to perform, particularly within liquid media or under conditions that mimic protein binding that occurs during a biochemical assay.

In order to study the spatial-dependent sensitivity of the colorimetric resonant optical biosensor apart from the context of a biomolecular assay, experiments were performed in which a series of polyelectrolyte layers with defined thickness and refractive index are built on the surface. Previous researchers have identified this method as a reliable means for characterizing the surface sensitivity of optical biosensors without performing assays using immobilized protein receptor molecules. Because protein analytes are subject to the effects of surface capacity, binding conditions, and molecular orientation, polyelectrolyte deposition has been shown to be a more reliable means for obtaining a known amount of material on the sensor surface. A procedure for deposition of a sequence of positively and negatively charged polyelectrolyte films has been demonstrated as a means for reliably calibrating and comparing the response of various optical biosensors [11]. The polyelectrolyte multilayers behave as homogeneous and isotropic monolayers, while multilayers with proteins are expected to have more complex refractive index profiles.

In this work, we will use deposition of polyelectrolyte multilayers to evaluate and compare the bulk and surface contributions of two slightly different sensor designs. The results will show that a small change in sensor design can have a profound effect on the relative surface and bulk sensitivities, and that surface sensitivity of the biosensor has been improved substantially beyond values that have been reported previously. The results show that the surface and bulk sensitivity do not scale in the same proportion, and that it is possible to substantially reduce the relative contribution of bulk refractive index effects.

Section snippets

Sensor design and fabrication

The sensor structure requires a grating with a period lower than the wavelength of the resonantly reflected light [12], [13]. Structures reported in this work utilized a linear grating with a period of 500 nm and a depth of ∼170 nm. As shown in Fig. 1, the grating structure is fabricated from a low refractive index material that is overcoated with a thin film of higher refractive index material. The grating structure was micro-replicated within a layer of cured epoxy on the surface of a polyester

Bulk refractive index sensitivity

The dependence of PWV on bulk refractive index was determined by placing droplets of different solvents on the sensor that span a wide range of refractive index (methanol, water, acetone, isopropyl alcohol, and glycerol) and recording the PWV. The PWV as a function of solution refractive index for the “tie layer” and “no tie layer” sensors is shown in Fig. 2 along with the bulk refractive index sensitivity of our first published sensor, which utilized an etched silicon nitride grating structure

Discussion

If deposition of polymer layers were to continue, one would expect that eventually δPWV/δd would approach zero for both sensors, as the polymer thickness extends beyond the region where the optical field travels along the sensor surface. The curves in Fig. 5 were numerically integrated in order to compare the total integrated sensitivity of both sensor structures over their optical sampling depth. (The integral is computed by taking the sum of the δPWV/δd values of Fig. 5 multiplied by 88 Å, so

Conclusion

A polyelectrolyte multilayer characterization method has been used to study the relative contributions of surface and bulk effects on the PWV shifts measured on a colorimetric resonant optical biosensor. We have shown that it is possible to optimize the sensor structure through the design of the dielectric coating to substantially bias the sensitivity toward the sensor surface, thereby reducing the relative importance of bulk refractive index effects. We have also shown that sensor

Acknowledgements

The authors gratefully acknowledge John Gerstenmaier for performing the polyelectrolyte multilayer deposition experiments, and Brenda Hugh for fabrication of the sensors used in this work.

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