Photonic crystal enhanced fluorescence immunoassay on diatom biosilica

Abstract

Fluorescence biosensing is one of the most established biosensing methods, particularly fluorescence spectroscopy and microscopy. These are two highly sensitive techniques but require high-grade electronics and optics to achieve the desired sensitivity. Efforts have been made to implement these methods using consumer grade electronics and simple optical setups for applications such as point-of-care diagnostics, but the sensitivity inherently suffers. Sensing substrates, capable of enhancing fluorescence are thus needed to achieve high sensitivity for such applications. In this paper, we demonstrate a photonic crystal-enhanced fluorescence immunoassay biosensor using diatom biosilica, which consists of silica frustules with sub-100 nm periodic pores. Utilizing the enhanced local optical field, the Purcell effect and increased surface area from the diatom photonic crystals, we create ultrasensitive immunoassay biosensors that can significantly enhance fluorescence spectroscopy as well as fluorescence imaging. Using standard antibody-antigen-labeled antibody immunoassay protocol, we experimentally achieved 100× and 10× better detection limit with fluorescence spectroscopy and fluorescence imaging respectively. The limit of detection of the mouse IgG goes down to 10 M (14 fg/mL) and 10 M (140 fg/mL) for the two respective detection modalities, virtually sensing a single mouse IgG molecule on each diatom frustule. The effectively enhanced fluorescence imaging in conjunction with the simple hot-spot counting analysis method used in this paper proves the great potential of diatom fluorescence immunoassay for point-of-care biosensing.

Photonic crystal-enhanced fluorescence imaging immunoassay for cardiovascular disease biomarker screening with machine learning analysis

“…The fabrication follows our earlier work with minor modifications (Squire et al 2018). Diatom culturing and isolation techniques as well as populating a coverslip are described in the Supporting Information.…”

“…The details about the fluorescence microscope, light source, and image capture parameters are described in the Supporting Information. The acquired optical and fluorescence images were saved as .tiff files and imported into Matlab to be prepared for processing, following the similar approach outlined in our previous work (Squire et al 2018), which is described in Fig. 2.…”

“…The periodic, nanostructured pore arrays of diatom frustules can enable natural photonic crystal behavior. Our group has proven that diatom biosilica is capable of enhancing Raman and fluorescence signals due to their photonic crystal structure as well as their large and superhydrophilic surface, thus offering exclusive advantages for biosensing, particularly for immunoassay (Kong et al 2016;Kong et al 2017;Ren et al 2013;Squire et al 2018). The biosensor fabrication begins with cost-effective algae cultivation to grow diatoms.…”

Recent Advances on Diatom-Based Biosensors

“…The hierarchical, quasi ordered disposition of porous nanostructures in diatom frustules has been exploited by the Rorrer group for demonstrating a photonic crystal enhanced fluorescence imaging immunoassay able to detect target analytes for cardiovascular disease and IgG molecules [50,51]. The pores of the diatom Pinnularia sp.…”

Frustules of Amphora sp. as a photonic crystal with photoluminescent CdS nanoparticles

“…[ 12–14 ] Specifically, the cadmium sulfide nanoparticle (CdS‐NP)/diatom biosilica system may have application as sensors, photonic band gap materials, or photocatalysts for dye degradation or hydrogen production. [ 13,15 ] Of specific interest, in the current context of the infectious disease Covid‐19, one study in which the authors profited from enhancing PL intensity produced by the Purcell effect allowed single molecule detection of rhodamine‐coupled immunoglobulin G for immunoassay [ 16 ] using diatoms frustules, a cheap and abundant material (diatomite or diatomaceous earth). This was despite the low quantum yield and weak fluorescence of CdS‐NP.…”