Porous silicon Bloch surface and sub-surface wave structure for simultaneous detection of small and large molecules
© Rodriguez et al.; licensee Springer. 2014
Received: 1 May 2014
Accepted: 18 June 2014
Published: 7 August 2014
A porous silicon (PSi) Bloch surface wave (BSW) and Bloch sub-surface wave (BSSW) composite biosensor is designed and used for the size-selective detection of both small and large molecules. The BSW/BSSW structure consists of a periodic stack of high and low refractive index PSi layers and a reduced optical thickness surface layer that gives rise to a BSW with an evanescent tail that extends above the surface to enable the detection of large surface-bound molecules. Small molecules were detected in the sensor by the BSSW, which is a large electric field intensity spatially localized to a desired region of the Bragg mirror and is generated by the implementation of a step or gradient refractive index profile within the Bragg mirror. The step and gradient BSW/BSSW sensors are designed to maximize both resonance reflectance intensity and sensitivity to large molecules. Size-selective detection of large molecules including latex nanospheres and the M13KO7 bacteriophage as well as small chemical linker molecules is reported.
KeywordsBloch surface wave Bloch sub-surface wave Size selective Large molecule Biosensor M13KO7 bacteriophage Nanospheres
Porous silicon (PSi) has excelled as a biosensing platform due to its cost-effective and versatile fabrication, enhanced surface area, and chemical and biological compatibility. Well-established Si surface functionalization chemistry has led to specific binding of several relevant molecules including DNA , proteins , explosives , and illicit drugs  to PSi platforms. However, PSi refractometric sensing applications have generally been size limited to molecules that diffuse into the porous matrix to cause a measurable change in effective optical thickness. Pore sizes of 5 to 100 nm diameter have allowed for the detection of larger molecules such as bovine serum albumin (8 nm in width) and anti-MS2 antibodies (15 nm in width) [5, 6]. However, molecules approaching the average pore diameter clog the pore and hinder molecular infiltration, which significantly deteriorates the transduced signal. For example, 40-base DNA (~13 nm in length) cannot efficiently infiltrate 20-nm pores [7, 8]. Hence, there is a significant challenge in detecting biological entities such as viruses, bacteria, and blood cells that typically have sizes much larger than those of the pores. Alternative measurement techniques for the detection of surface-bound molecules on PSi include monitoring fluorescent labels and changes in reflectance intensity for the detection of MS2 bacteriophage  and Escherichia coli bacteria , respectively. However, emerging interest in lab-on-a-chip technologies has placed focus on label-free refractometric-based sensors in order to avoid the additional expense of fluorescent labels. In addition, refractometric sensing configurations are a popular choice due to the compact size, small active sensing region, ability to transduce molecular interaction with an electric field into a refractive index change, and ability to array and multiplex devices allowing several biosensors on a single chip. For example, silicon-on-insulator (SOI) waveguides (WGs) and surface plasmon devices utilize evanescent fields to detect surface-bound molecules of all sizes [10, 11]. PSi WGs have demonstrated sensitivities an order of magnitude greater than SOI WGs due to the direct interaction of small molecules with the guided field inside the porous layer; however, surface-bound large molecules present a detection challenge in PSi WGs due to the weak evanescent fields at the surface [8, 12, 13]. The PSi BSW/BSSW biosensor offers the possibility to detect both small molecules that infiltrate the pores and large molecules attached to the sensor's surface . The BSW mode is a surface state excited within the truncated defect layer at the surface of a multilayer Bragg mirror and has been previously reported in PSi sensing applications [14–17]. The novel BSSW mode is confined by a step or gradient refractive index within the multilayer and can selectively detect small molecules attached within the pores with an enhanced sensitivity (>2,000 nm/refractive index unit (RIU)) in comparison to band edge modes of the multilayer, microcavities, or traditional WG modes [8, 12, 16]. The BSW and BSSW modes are each manifested as a distinct resonance peak in the reflectance spectrum, and the angular shift of each peak can be used to quantify the number of molecules attached to the sensor. A thorough theoretical analysis of both the step and gradient BSW/BSSW configurations has been previously presented . In this report, the first fabricated step index and an optimized gradient index PSi BSW/BSSW biosensor are presented. Large M13KO7 bacterial viruses and 60 nm diameter latex nanospheres as well as small 3-aminopropyltriethoxysilane (APTES) and gluteraldehyde (GA) molecules are used as model systems to demonstrate the size-selective detection scheme.
PSi BSW/BSSW device fabrication
Latex nanosphere functionalization
M13KO7 bacteriophage functionalization
Viruses are infectious agents that can cause disease in humans, plants, and animals; antibodies are typically used in immunoassays to detect viruses in biological samples. The M13KO7 bacterial virus was used as a model system to determine if the large (approximately 2 μm in length; 16,400 kDa) M13KO7 could be directly bound to and detected on the PSi BSW/BSSW sensor surface. The M13KO7 bacteriophage is a low-cost, readily available, nonhazardous E. coli bacterial virus that can be readily detected using commercially available antibodies [18, 19]. The virus was covalently cross-linked to the PSi surface via APTES and GA linkers. APTES was attached as described above. GA is a homobifunctional cross-linker that can bind to and covalently link molecules through their free amines. A 2.5% GA in phosphate buffered saline (PBS) buffer solution was used to cross-link the APTES free amines on the sensor surface to the free amines on M13KO7 suspended in solution on the sensor surface. After a 30-min GA incubation step, a 1% sodium cyanoborohydride (Sigma-Aldrich, St. Louis, MO, USA) in PBS buffer solution was applied, followed by a 30-min incubation step to stabilize the Schiff base bonds formed during GA cross-linking . The M13KO7 (0.32 mg/ml carbonate/bicarbonate buffer, pH ~ 10) was diluted to a final concentration of 32 μg/ml in PBS buffer (final pH ~ 9.5) and applied to the sensor surface for 20 min at room temperature. The device was thoroughly rinsed with DI water. Figure 2b shows a top view SEM image of the M13KO7 bacteriophage immobilized on the PSi surface. Coulombic interactions prevent a uniform self-assembled monolayer due to the negatively charged nature of the virus.
Results and discussion
Resonance shifts illustrated in Figure 4
The fabrication and realization of step and gradient index BSW/BSSW sensors were demonstrated. The excitation of both BSW and BSSW modes within the same structure in both grating- and prism-coupled configurations allowed for simultaneous detection of APTES and GA with both modes and the detection of large 60-nm nanospheres and the large M13KO7 bacteriophage with the BSW. The strong confinement of the BSSW minimizes the overlap with surface immobilized analytes for high sensitivity, high selectivity applications. The evanescent field of the BSW allows for detection of very large molecules that could not be detected in typical PSi devices such as interferometers, microcavities, and waveguides. Size-selective detection using the same sensor platform is expected to be a significant advantage for future multianalyte detection schemes using a microfluidics approach.
Bloch surface wave
Bloch sub-surface wave
high refractive index
low refractive index
phosphate buffer saline
rigorous coupled wave analysis
scanning electron microscope
The authors acknowledge funding from the Army Research Office (W911NF-09-1-0101) and National Science Foundation (ECCS-0746296). A portion of this research was conducted at the Center for Nanophase Materials Sciences, which is sponsored at Oak Ridge National Laboratory by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. Additional fabrication was carried out at the Vanderbilt Institute of Nanoscale Science and Engineering (NSF ARI-R2 DMR-0963361). Lonai acknowledges the NSF-REU program at Vanderbilt (DMR-1005023).
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