A comparative study of in-flow and micro-patterning biofunctionalization protocols for nanophotonic silicon-based biosensors

Abstract

Reliable immobilization of bioreceptors over any sensor surface is the most crucial step for achieving high performance, selective and sensitive biosensor devices able to analyze human samples without the need of previous processing. With this aim, we have implemented an optimized scheme to covalently biofunctionalize the sensor area of a novel nanophotonic interferometric biosensor. The proposed method is based on the ex-situ silanization of the silicon nitride transducer surface by the use of a carboxyl water soluble silane, the carboxyethylsilanetriol sodium salt (CTES). The use of an organosilane stable in water entails advantages in comparison with usual trialkoxysilanes such as avoiding the generation of organic waste and leading to the assembly of compact monolayers due to the high dielectric constant of water. Additionally, cross-linking is prevented when the conditions (e.g. immersion time, concentration of silane) are optimized. This covalent strategy is followed by the bioreceptor linkage on the sensor area surface using two different approaches: an in-flow patterning and a microcontact printing using a biodeposition system. The performance of the different bioreceptor layers assembled is compared by the real-time and label-free immunosensing of the proteins BSA/mAb BSA, employed as a model molecular pair. Although the results demonstrated that both strategies provide the biosensor with a stable biological interface, the performance of the bioreceptor layer assembled by microcontact printing slightly improves the biosensing capabilities of the photonic biosensor.

Introduction

The past few years have witnessed a significant progress in the development of biosensing devices based on silicon photonics. These devices can offer several advantages over traditional methodologies, such as ELISA or RIA test, which are time-consuming, expensive, and need from bulky instrumentation usually located in laboratory environments. Photonic biosensors based on evanescent wave detection are able to perform highly sensitive detections in a label-free scheme providing rapid, affordable, and simple analysis. Among evanescent wave optical biosensors, interferometric ones are recognized to be one of the most sensitive devices for label-free analysis [1]. Moreover, they can be fabricated with standard silicon technology affording mass production and miniaturization which make interferometric devices suitable candidates for providing a multiplexed and portable analytical tool.

However, the sensitivity of a biosensing platform is a complex parameter, directly related to the transducer sensing principle, but strongly dependent of other parameters such as the material, working wavelength, surface cleanness, protein surface coverage, and biorecognition method. A reliable immobilization of bioreceptors over any sensor surface is the most crucial step for achieving high performance, selective, and sensitive biosensors able to analyze human samples directly, without the need of previous processing. Integrated nanophotonic devices commonly use silicon nitride (Si₃N₄) as core layer due to its high refractive index that allows the confinement of light. Additionally, Si₃N₄ has excellent properties to be an optimal sensing area surface such as high density and chemical inertness that make it resistant to ion species, oxygen, and moisture permeation [2]. Covalent attachment of molecules to this surface is preferred due to its long-term stability in fluid systems allowing the biosensor to be reused several times [3]. However, the covalent strategy is more complex than physical adsorption [4] implying the modification of the Si₃N₄ surface by incorporating functional groups able to react with the biomolecules. Among the available functional groups, carboxyl groups are the most suitable candidates in order to conjugate proteins to surfaces [5], [6], [7]. Several strategies to provide silicon-based surfaces with carboxyl groups have been reported: via the attachment of a trifluoro ethanol ester and subsequent thermal acid hydrolysis, or through the attachment of a photocleavable ester and subsequent photochemical cleavage [5], besides via the attachment of long-chain carboxylic acid terminated monolayers [6], [7]. However, these strategies involve a large number of steps, require of additional instrumentation, and are time-consuming.

Silanization methods are the simplest and most common way to covalently modify silicon-based surfaces and have been extensively reviewed in the literature [8]. Organofunctional trialkoxysilanes (such as 3-aminopropyltriethoxy (APTES) silane [9] and the 3-trimethoxysilyil propyl methacrylate (MPTS) silane [10]) are the most employed for liquid-phase silanization. An undesirable effect regarding the use of trialkoxysilanes is the polymerization that can occur at the free silanol groups on the surface or in solution, leading to highly heterogeneous surfaces, a potential disadvantage when dealing with biosensors. To avoid this negative effect, anhydrous solvents have been used to limit the amount of water reacting during the monolayer formation [11]. However, small variations in the amount of water during the silanization reaction can dramatically alter the thickness of the final film giving place to highly irreproducible surfaces due to the formation of silane multilayers [9].

To achieve a carboxylic acid terminated layer on the Si₃N₄ surface by a simple silanization method, avoiding the derived problems of using anhydrous solvents, we chose to study the use of a carboxyethylsilanetriol sodium salt (CTES). This aqueous soluble silane is extensively used for the functionalization of silica particles [12], [13] and as a co-structure directing agent [14]. In spite of its wide use for the synthesis of particles, few works can be found about functionalization of silicon surfaces with CTES silane [15]. Due to its short alkyl chain of approximately 6 Å and to the hydrophilicity of the functional carboxyl group that contains, CTES is an organosilane stable front the cross-linking in water. This is an unusual property for a silane molecule that makes the use of CTES especially attractive to avoid concerns regarding the use of organic solvents and the generation of organic waste by the rinsing steps. Another attractive aspect of the use of water as solvent for the reaction is its larger dielectric constant, which favors the formation of packaged monolayers [16]. Moreover, the solvent compatibility with the polymer materials employed in our fluidic systems allows the use of CTES to silanize the sensor area inside the fluidic cell (in-flow) when necessary for specific applications.

Another important issue related with the formation of a bioreceptor layer is the method employed to place the biomolecules on the sensor surface. The most explored methods for the immobilization of the bioreceptor layer in biosensors are the in-flow strategy [17] and the patterning of surfaces [18]. The in-flow strategy uses smalls channels with low Reynolds number that generates a laminar flow [19], and allows the real-time monitoring of the layer formation. On the other hand, the surface patterning is based on the selective deposition of small volumes of samples under static conditions, which avoids the formation of the typical geometrical patterns due to the laminar flow [17].

In this work, we report the functionalization of the Si₃N₄ surface of a nanophotonic biosensor with carboxyl groups by an organosilane molecule stable in water. We found out the optimum reaction parameters (time and concentration) and the resulting film morphology of the carboxylic acid terminated CTES silane monolayer on Si₃N₄ test surfaces by using standard techniques such as contact angle characterization, atomic force microscopy (AFM), and fluorescent analysis. After that, the optimized silanization protocol was applied to the sensor area surface of a Bimodal Waveguide device (BiMW) to evaluate its biosensing capabilities by using the proteins BSA/mAb BSA as a model molecular pair. Both immobilization strategies, in-flow and micropatterning of surfaces, were applied to the BiMW sensors for comparison. The main goal of this work was not only to find out the optimized parameters for a new silanization method based on an organosilane stable in water but also to assess its application with two different protein immobilization methods for nanophotonic biosensing applications.