Label-Free Biosensor Imaging on Photonic Crystal Surfaces

Abstract

We review the development and application of nanostructured photonic crystal surfaces and a hyperspectral reflectance imaging detection instrument which, when used together, represent a new form of optical microscopy that enables label-free, quantitative, and kinetic monitoring of biomaterial interaction with substrate surfaces. Photonic Crystal Enhanced Microscopy (PCEM) has been used to detect broad classes of materials which include dielectric nanoparticles, metal plasmonic nanoparticles, biomolecular layers, and live cells. Because PCEM does not require cytotoxic stains or photobleachable fluorescent dyes, it is especially useful for monitoring the long-term interactions of cells with extracellular matrix surfaces. PCEM is only sensitive to the attachment of cell components within ~200 nm of the photonic crystal surface, which may correspond to the region of most interest for adhesion processes that involve stem cell differentiation, chemotaxis, and metastasis. PCEM has also demonstrated sufficient sensitivity for sensing nanoparticle contrast agents that are roughly the same size as protein molecules, which may enable applications in "digital" diagnostics with single molecule sensing resolution. We will review PCEM's development history, operating principles, nanostructure design, and imaging modalities that enable tracking of optical scatterers, emitters, absorbers, and centers of dielectric permittivity.

Keywords: biomaterial detection; label-free bioimaging; live cell imaging; nanoparticle detection; nanophotonics; photonic crystal; photonic crystal biosensor; photonic crystal enhanced fluorescence (PCEF); photonic crystal enhanced microscopy (PCEM); photonic crystal surface; protein-protein binding detection.

Figure 1

Photonic Crystal (PC) Surface Biosensor. (A) Schematic of the PC surface on a substrate with structure parameters: grating period (Λ), grating depth (d), refractive index (RI) of low-RI grating material (n_low) and high-RI top layer (n_high), thickness of high-RI layer (t); (B) Band structure of a photonic crystal biosensor calculated by FDTD simulation; (C) Normalized reflection spectrum from the PC surface with resonant peak wavelength value (PWV) of λ₀; (D) Peak wavelength shift (PWS) of Δλ extracted from the normalized spectra with a background pixel (PWV of λ₀) and a pixel with surface-attached biomaterial (PWV of λ₁) on the PC biosensor.

Figure 2

Instrument 1: Label-free Biomolecular Interaction Detection (BIND) Scanner utilizing a PC surface biosensor. (A) Schematic of excitation/detection instrument where an imaging spectrometer gathers hundreds of reflected spectra simultaneously from one line across the sensor surface; (B) PWS images of Protein A (bright regions represent regions of greater PWS) gathered on a 6-mm diameter region of a PC biosensor, which is imaged at approximately 20 µm pixel resolution after writing the letters ‘NSG’ (Nano Sensors Group) with a microarray spotting tool (PerkinElmer, Inc. Piezoarray™) (Reprinted in part with permission from [50], © 2006 Future Drugs Ltd.); (C) PWS images with shift scale bars (ΔPWV) indicating the magnitude of wavelength shifts in nanometers. Pixels with higher PWS displayed in brighter colors indicate locations where Panc-1 cell attachment has occurred. The three columns of image sets represent the following: (a) untreated control, (b) extract that induced 100% cell death Petunia punctata Paxton (P. punctate), (c) extract that enhanced proliferation Anisoptera glabra Kurz (A. glabra). The top row of images was taken before exposure and the bottom row of images was taken after the 24 h exposure period with a plant extract at 100 μg/mL. Scale bar (white) = 300 μm (Reprinted in part with permission from [41], © 2010 BioMed Central Ltd.).

Figure 3

Instrument 2: Transmission acquisition mode of photonic crystal biosensor integrated with an upright imaging microscope and using laser as light source. (A) Schematic of combined label-free and enhanced-fluorescence imaging instrument; (B) Enhanced (a) fluorescence and (b) label-free images of 50 mg/mL SA-Cy5 spots on a PC biosensor. Inverted transmission versus angle response for a pixel inside and outside the SA-Cy5 spot in (c), and cross-section of the label-free image through two SA-Cy5 spots in (d). Rather than measuring the PWS, the label-free imaging system measures the angle of minimum transmission (AMT) by illuminating the PC sensor at a fixed wavelength while scanning the angle of illumination through computer-controlled rotation of the mirror (reprinted in part with permission from [76], © 2009 American Optical Society); (C) Label-free image of a DNA microarray measured with a PC biosensor. The white dashed box denotes the location of a set of 20 intentional blank spots. A line profile running through a row containing 4 blank spots followed by 12 probe spots is shown in (D) (Reprinted in part with permission from [37], © 2010 American Chemical Society).

Figure 4

Instrument 3: Reflection acquisition mode of photonic crystal biosensor integrated with an inverted microscope and using LED as light source. (A) Schematic of the structure of a photonic crystal (PC) surface biosensor with a surface-attached nanoparticle, inset: photo of a PC biosensor fabricated on a glass slide; (B) Instrument schematic of the modern Photonic Crystal Enhanced Microscopy (PCEM); (C) Scanning electron micrograph of the photonic crystal surface, inset: zoomed-in image on the edge of the PC biosensor; (D) Normalized spectrograph (surface plot) measured with PCEM. Inset: PCEM-acquired 3D spectrum data; (E) AFM images of a tDPN-printed 3 × 3 array of nano-dots (each with dimension of 540² × 40 nm³), inset: zoomed-in AFM image of one tDPN-printed dot; (F) PWV image of the tDPN-printed dots (displayed in a 3D surface plot) within a 20² µm² field of view, inset: 2D PWV image; (G) Normalized spectra of a representative tDPN-printed dot and a background pixel, inset: zoomed-in image of the spectra with 2D polynomial fitting (Reprinted in part with permission from [48], © 2014 RSC Publishing.).

Figure 5

Wavelength-sensitive live cell image from instrument 3–PCEM. (A) Brightfield and (B) PWV images of Panc-1 cells attached to the PC surface. Lamellipodial extensions are visible, especially from cell 2, demonstrating the ability of PCEM to resolve regional differences in single-cell attachment; (C) Representative spectra (normalized) from background regions and regions with cellular attachment. Selected areas of the PWV image from beneath a cell show the PWS of a typical Panc-1 cell is ~1.0 nm; (D) Time-lapse PWS images of cellular attachment of dental stem cells (mHAT9a) (Reprinted in part with permission from [45], © 2013 RSC Publishing).

Figure 6

PCEM detection of protein-protein binding. (A) Schematic illustration of the PCEM detection of protein-protein binding on the PC biosensor surface; (B) SEM images of AuNR-IgG (AuNR conjugated with SH-PEG-IgG) attached to the PC biosensor surface. Inset: zoomed-in image for one AuNR; (C) PCEM-detected peak intensity value (PIV) images (in grayscale) and the PIV-shift image indicating AuNR-IgG attached on the PC surface; (D) Two representative cross-section lines of the normalized intensity images with/without two AuNRs-IgG on the PC surface (Reprinted in part with permission from [48], © 2014 RSC Publishing.).

Figure 7

Photonic Crystal Enhanced Fluorescence (PCEF) portion on a PCEM imaging system. (A) Schematic of the PCEF portion on modern PCEM detection instrumentation. Inset (top left): angle reflection spectrum; (B) Brightfield and PCEF images of membrane dye-stained 3T3 fibroblast cells: (a) brightfield, (b) off-resonance PCEF, (c) on-resonance PCEF, (d) enhancement factor image, (e) 3D surface plot image of the enhancement factor (Reprinted in part with permission from [96], © 2014 RSC Publishing).