Silk-Encapsulated Plasmonic Biochips with Enhanced Thermal Stability

Abstract

Because of their high sensitivity, cost-efficiency, and great potential as point-of-care biodiagnostic devices, plasmonic biosensors based on localized surface plasmon resonance have gained immense attention. However, most plasmonic biosensors and conventional bioassays rely on natural antibodies, which are susceptible to elevated temperatures and nonaqueous media. Hence, an expensive and cumbersome "cold chain" system is necessary to preserve the labile antibodies by maintaining optimal cold temperatures during transport, storage, and handling. Herein, we introduce a facile approach to preserve the antibody activity on a biosensor surface even at elevated temperatures. We show that silk fibroin film could be used as a protective layer to preserve the activity of a model antibody (Rabbit IgG) and cardiac troponin antibody at both room temperature and 40 °C over several days. Furthermore, a simple aqueous rinsing process restores the biofunctionality of the biosensor. This energy-efficient and environmentally friendly method represents a novel approach to eliminate the cold chain and temperature-controlled packing of diagnostic reagents and materials, thereby extending the capability of antibody-based biosensors to different resource-limited circumstances such as developing countries, an ambulance, an intensive care unit emergency room, and battlefield.

Keywords: biopreservation; gold nanorods; localized surface plasmon resonance; plasmonic biosensor; silk.

Figure 1

Schematic illustration depicting the concept of silk-encapsulated plasmonic biochips with enhanced thermal stability obviating the need for refrigerated transportation, handling, and storage of the plasmonic biochips.

Figure 2

(A) TEM image of AuNRs used as plasmonic nanotransducers. The dimensions of the AuNRs were found to be 50 × 18 nm. (B) Extinction spectra showing the LSPR shift after conjugation of AuNR with IgG in solution. (C) LSPR shift of AuNR-IgG conjugates on glass substrates exposed to different concentrations of anti-IgG (mean ± standard deviation, N = 3) (D) Extinction spectra of AuNR-IgG conjugates on the glass substrate before (red) and after exposure to 24 μg/mL of anti-IgG (blue) showing a shift of ∼16 nm.

Figure 3

(A) Representative extinction spectra of AuNR-IgG conjugates on the glass substrate before (red) and after silk film coating (green), after rinsing silk film (purple) and after exposure to 24 μg/mL of anti-IgG (blue). (B) LSPR shift corresponding to each step in (A). (C) FTIR spectra of silk film before (black) and after rinsing (red) with water. (D) Peak deconvolution of amide I band of silk film before rinsing. (E) Peak deconvolution of amide I band of silk residue after rinsing. The black lines are the original FTIR spectra and the dotted lines are the fitted curves. (F) Histogram showing the relative content of different secondary structures of the silk before (black) and after rinsing (red). (G) AFM image showing uniformly adsorbed AuNR-IgG on glass substrate before silk film coating. (H) AFM image showing silk film deposited on AuNR-IgG conjugates adsorbed on glass substrate. (I) AFM image of the AuNR-IgG conjugates after rinsing the silk film with water evidencing the removal of most of the silk film.

Figure 4

(A) Retained sensitivity of AuNR-IgG conjugates over 7 days at room temperature and 40 °C. (B) Retained sensitivity of AuNR-anti cTnI conjugates over 7 days at room temperature and 40 °C. Control experiments (without silk coating) show antibody on AuNR lost 90% of sensitivity in 2 days at room temperature and 40 °C (mean ± standard deviation of three measurements obtained from samples from three different batches).

Figure 5

(A) Extinction spectra of AuNR-IgG conjugates on the glass substrate before (red) and after hyaluronic acid film coating (green), after hyaluronic acid film rinsing (purple) and after binding exposure to 24 μg/mL of anti-IgG (blue). (B) Histogram showing the comparison of retained sensitivity of IgG-AuNR conjugates underneath silk, BSA, and HA films after 1 day storage at room temperature (mean ± standard deviation of three measurements obtained from samples from three different batches).