Photonic Crystal-Enhanced Fluorescence Imaging Immunoassay for Cardiovascular Disease Biomarker Screening with Machine Learning Analysis.

When myocardial walls experience stress due to cardiovascular diseases, like heart failure, hormone N-terminal pro-B-type natriuretic peptide (NT-proBNP) is secreted into the blood. Early detection of NT-proBNP can assist diagnosis of heart failure and enable early medical intervention. A simple, cost-effective detection technique such as the widely used fluorescence imaging immunoassay is yet to be developed to detect clinically relevant levels of NT-proBNP. In this work, we demonstrate photonic crystal-enhanced fluorescence imaging immunoassay using diatom biosilica, which is capable of detecting low levels of NT-proBNP in solution with the concentration range of 0~100 pg/mL. By analyzing the fluorescence images in the spatial and spatial frequency domain with principle component analysis (PCA) and partial least squares regression (PLSR) algorithms, we create a predictive model that achieves great linearity with a validation R2 value of 0.86 and a predictive root mean square error of 14.47, allowing for good analyte quantification. To demonstrate the potential of the fluorescence immunoassay biosensor for clinical usage, we conducted qualitative screening of high and low concentrations of NT-proBNP in human plasma. A more advanced machine learning algorithm, the support vector machine classification, was paired with the PCA and trained by 160 fluorescence images. In the 40 testing images, we achieved excellent specificity of 93%, as well as decent accuracy and sensitivity of 78% and 65% respectively. Therefore, the photonic crystal-enhanced fluorescence imaging immunoassay reported in this article is feasible to screen clinically relevant levels of NT-proBNP in body fluid and evaluate the risk of heart failure.

Highly-porous diatom biosilica stationary phase for thin-layer chromatography.

This study showed that a nanostructured, highly-porous stationary phase composed of randomly-deposited biosilica frustules isolated from living cells of diatom Pinnularia sp. significantly improved the conventional thin-layer chromatography (TLC) based separation of the triphenylmethane dyes malachite green and fast green relative to silica gel by two mobile phases (9:1:1 v/v 1-butanol:ethanol:water, 5:1:2 v/v 1-butanol:acetic acid:water). Although both stationary phases were composed of amorphous silica rich in silanol groups with particle size of 10-12 µm, diatom biosilica frustules were highly porous, hollow shells with surface structure dominated by 200 nm pore arrays. Diatom biosilica significantly improved the mobility of both malachite green and fast green, enabling the resolution of these analytes. The diatom biosilica layer had a high void fraction of 96% but reduced the flow velocity and permeability constant by a factor of two relative to silica gel. TLC performance was enhanced, as evidenced by ten-fold reduction in theoretical plate height for both analytes using the 1-butanol:acetic acid:water mobile phase, and an increased difference in retention time between malachite green and fast green (ΔRf = 0.26) using the 1-butanol:ethanol:water mobile phase. Analysis of plate height vs. solvent front position by the modified van Deemter equation suggested that dispersive mass transfer was reduced, leading to improved analyte resolution, and that surface of the frustule decreased boundary layer resistance, leading to increased analyte flux. Overall, the basis for improved chromatographic performance is believed to be the unique nano- and microstructure of the diatom biosilica frustule.

Biological Photonic Crystal-Enhanced Plasmonic Mesocapsules: Approaching Single-Molecule Optofluidic-SERS Sensing.

Surface-enhanced Raman scattering (SERS) sensing in microfluidic devices, namely optofluidic-SERS, suffers an intrinsic trade-off between mass transport and hot spot density, both of which are required for ultra-sensitive detection. To overcome this compromise, photonic crystal-enhanced plasmonic mesocapsules are synthesized, utilizing diatom biosilica decorated with in-situ growth silver nanoparticles (Ag NPs). In our optofluidic-SERS testing, 100× higher enhancement factors and greater than 1,000× better detection limit were achieved compared with traditional colloidal Ag NPs, the improvement of which is attributed to unique properties of the mesocapsules. First, the porous diatom biosilica frustules serve as carrier capsules for high density Ag NPs that form high density plasmonic hot-spots. Second, the submicron-pores embedded in the frustule walls not only create a large surface-to-volume ratio allowing for effective analyte capture, but also enhance the local optical field through the photonic crystal effect. Last, the mesocapsules provide effective mixing with analytes as they are flowing inside the microfluidic channel. The reported mesocapsules achieved single molecule detection of Rhodamine 6G in microfluidic devices and were further utilized to detect 1 nM of benzene and chlorobenzene compounds in tap water with near real-time response, which successfully overcomes the constraint of traditional optofluidic sensing.