High contrast cleavage detection for enhancing porous silicon sensor sensitivity

Abstract

Using porous silicon (PSi) interferometer sensors, we show the first experimental implementation of the high contrast cleavage detection (HCCD) mechanism. HCCD makes use of dramatic optical signal amplification caused by cleavage of high-contrast nanoparticle labeled reporters instead of the capture of low-index biological molecules. An approximately 2 nm reflectance peak shift was detected after cleavage of DNA-quantum dot reporters from the PSi surface via exposure to a 12.5 nM DNase enzyme solution. This signal change is 20 times greater than the resolution of the spectrometer used for the interferometric measurements, and the interferometric measurements agree with the response predicted by simulations and fluorescence measurements. These proof of principle experiments show a clear path to achieving a real-time, highly sensitive readout for a broad range of biological diagnostic assays that generate a signal via nucleic acid cleavage triggered by specific molecular binding events.

Roadmap on Universal Photonic Biosensors for Real-Time Detection of Emerging Pathogens

“…Interestingly, both low-and high-quality-factor photonic biosensors can be re-engineered to work efficiently under the probe-cleavage detection scenario owing to the dramatic signal amplification provided by the high-contrast probes and collateral cleavage multiplication factors [182,184,185]. This offers opportunities for multiplexing photonic biosensors with spectroscopic techniques and making use of the surface-plasmon signal enhancement.…”

“…As cleavage-based sensing is still a relatively new approach, there are numerous novel applications being developed that make use of the unique combination of the biological recognition and amplification mechanisms provided by the CRISPR technology with the optical signal amplification techniques provided by photonic and plasmonic biosensing platforms. Nonspecific collateral cleavages activated by precise crRNA target matching to viral RNA and DNA are being investigated as a transduction mechanism in photonic biosensors of various configurations, including planar porous biosensors with Fabry-Perot spectra and out-of-plane readout (Figure 5a) [182], integrated microresonator sensors with in-plane readout [183,184], and diffractive biosensors inverse-designed for cleavageenabled sensing with simple single-frequency readout (Figure 5b) [185]. The new approach makes use of the biosensor surface pre-test functionalization with high-index nanoscale reporters (e.g., quantum dots, semiconductor or noble-metal nanoparticles), which are then cleaved from the surface by a cleaving agent (e.g., a CRISPR complex) activated by a target biomolecule in the sample.…”

Botulinum Neurotoxin C Dual Detection through Immunological Recognition and Endopeptidase Activity Using Porous Silicon Interferometers

“…The unbound molecules were removed from PSiO 2 by thorough rinsing and washing with HEPES buffer. Next, the proteolytic activity was optically amplified by the attachment of ZnO nanoparticles (ZnO-NPs) onto the recombinant substrate . Briefly, 12.5 mg of ZnO-NPs was dissolved in 2 mL of EtOH and uniformly dispersed by continuous sonication for 30 min.…”

“…Two independent PSi Fabry–Pérot interferometers were designed to assess minute BoNT/C concentrations available in complex media by immunorecognition response (as noted earlier) and in addition assessing its endopeptidase activity using substrate-specific cleavage. The proteolytic activity included a general BoNT peptide substrate, synaptosomal-associated protein (SNAP25B) linked to a vesicle-associated membrane protein (VAMP-2), which was optically amplified by the lateral addition of zinc oxide nanoparticles (ZnO-NPs) to augment the average refractive index contrast upon specific cleavage; see Scheme b . To enhance the sensitivity and durability of the platforms, covalent immobilization was applied for anchoring any biomolecule onto the PSi surfaces.…”

Porous Silicon-Based Aptasensors: Toward Cancer Protein Biomarker Detection

“…Their inferior performance is mainly attributed to mass transfer limitations, − and several strategies have been implemented to enhance their sensitivity while still detecting the analyte in a direct and label-free manner. These include the optimization of the porous nanostructure and surface chemistry, − the design of a flow-through PSi biosensor, , microfluidic integration, , the decoration of the PSi with gold nanoparticles for an improved optical signal, , signal amplification, the application of an electrokinetic focusing of the target on top of the biosensors, , and novel signal processing techniques. , Specifically, we have demonstrated the integration of PSi biosensors with the isotachophoresis (ITP) technique for on-chip analyte preconcentration and demonstrated a nanomolar detection limit for both DNA and protein targets. , Furthermore, we have recently presented a novel signal processing technique, which reduces system noise by applying Morlet wavelet convolution to filter spectra, resulting in an improved limit of detection …”