Label-free Optical Detection Method Research Articles

Ultrasensitive, dynamic, and online monitoring photonic sensors for protein conformation

The monitoring of molecular dynamic transition and protein conformation changes is important for some neurodegenerative diseases (NDs) and has attracted much attention due to its ability to reveal the pathological mechanism of several NDs. Here, a label-free optical detection method with an ultrahigh refractive index sensitivity (S) and dynamical S tuning range was developed to provide a novel technology roadmap for the online monitoring of protein conformation. The real-time conformation variation process was transduced into a visualization optical signal, allowing it to be monitored. The biosensor prototype was constructed by the cross-overlapping of two rows of square capillaries on a gold mirror to form two microcavities with different free spectral ranges (denoted as the sensing and reference cavities). The coupling of the resonance mode between the two microcavities induced the generation of the Vernier effect and the magnifying of S, presented in the form of a modulated envelope in the spectrum. S values ranged from −17,256 nm/RIU to 93,937 nm/RIU, indicating that the biosensor could not only realize large dynamic tuning ranges but also an ultrahigh S. The conformation changes of silk proteins from the random coil to the β–sheet were sensitively monitored by the output spectrum after it was stimulated with ethanol solution. This ultrasensitive biosensor (which is both label-free and optical) provides a novel and supplementary technique to monitor the structural and conformation changes process of proteins. The results reveal its significant potential for clinical diagnosis, healthcare, pharmaceutical screening, and food security.

Docking and Activity of DNA Polymerase on Solid-State Nanopores.

Integration of motor enzymes with biological nanopores has enabled commercial DNA sequencing technology; yet studies of the similar principle applying to solid-state nanopores are limited. Here, we demonstrate the real-life monitoring of phi29 DNA polymerase (DNAP) docking onto truncated-pyramidal nanopore (TPP) arrays through both electrical and optical readout. To achieve effective docking, atomic layer deposition of hafnium oxide is employed to reduce the narrowest pore opening size of original silicon (Si) TPPs to sub-10 nm. On a single TPP with pore opening size comparable to DNAP, ionic current measurements show that a polymerase–DNA complex can temporally dock onto the TPP with a certain docking orientation, while the majority become translocation events. On 5-by-5 TPP arrays, a label-free optical detection method using Ca2+ sensitive dye, are employed to detect the docking dynamics of DNAP. The results show that this label-free detection strategy is capable of accessing the docking events of DNAP on TPP arrays. Finally, we examine the activity of docked DNAP by performing on-site rolling circle amplification to synthesize single-stranded DNA (ssDNA), which serves as a proof-of-concept demonstration of utilizing this docking scheme for emerging nanopore sensing applications.

Label-free optical detection of cells grown in 3D silicon microstructures

We demonstrate high aspect-ratio photonic crystals that could serve as three-dimensional (3D) microincubators for cell culture and also provide label-free optical detection of the cells. The investigated microstructures, fabricated by electrochemical micromachining of standard silicon wafers, consist of periodic arrays of silicon walls separated by narrow deeply etched air-gaps (50 μm high and 5 μm wide) and feature the typical spectral properties of photonic crystals in the wavelength range 1.0-1.7 μm: their spectral reflectivity is characterized by wavelength regions where reflectivity is high (photonic bandgaps), separated by narrow wavelength regions where reflectivity is very low. In this work, we show that the presence of cells, grown inside the gaps, strongly affects light propagation across the photonic crystal and, therefore, its spectral reflectivity. Exploiting a label-free optical detection method, based on a fiberoptic setup, we are able to probe the extension of cells adherent to the vertical silicon walls with a non-invasive direct testing. In particular, the intensity ratio at two wavelengths is the experimental parameter that can be well correlated to the cell spreading on the silicon wall inside the gaps.

Monitoring Stepwise Proteolytic Degradation of Peptides by Supramolecular Domino Tandem Assays and Mass Spectrometry for Trypsin and Leucine Aminopeptidase

A label-free optical detection method has been designed that allows direct monitoring of enzymatic peptide digestion in vitro. The method is based on the addition of a reporter pair, composed of the macrocyclic host cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO), to detect the proteolytic degradation of peptides. The enzymatic activity of trypsin and leucine aminopeptidase (LAP) was investigated using H-LSRFSWGA-OH as a substrate. The substrate as well as the intermediary and final products (i.e., H-FSWGA-OH and phenylalanine) formed during its enzymatic hydrolysis differ in their binding affinity to the receptor CB7, which results in varying degrees of dye displacement and, therefore, different fluorescence intensities. CB7 showed a relatively weak binding constant of K approximately 10(4) M(-1) with the substrate, a relatively strong binding constant of K > or = 10(6) M(-1) with H-FSWGA-OH (which is a final product formed by trypsin digestion and the intermediary product formed during the enzymatic activity of LAP), and a moderate binding constant of K < or = 10(5) M(-1) with phenylalanine. Owing to this differential binding affinity of CB7 with the substrate and the corresponding products, the digestion of a peptide by trypsin was followed as a decrease in fluorescence signal, while the complete degradation of the peptide by LAP was monitored as a decrease and a subsequent increase in fluorescence signal. The k(cat)/K(M) value for trypsin (2.0 x 10(7) min(-1) M(-1)) was derived from the change in fluorescence signal with time. Additionally, the complete degradation of the peptide by LAP was also followed by mass spectrometry. The use of a supramolecular sensing ensemble (macrocyclic host and dye) as a fluorescent reporter pair gives this method the flexibility to adapt for monitoring the stepwise degradation of different biologically relevant peptides by other proteases.

Label-free detection of H1N1 virus for point of care testing

An assay for the detection of H1N1 virus was established using Reflectometric Interference Spectroscopy (RIfS), a label-free optical detection method. A direct assay format was used to detect the virus by the corresponding antibody immobilized on the chip surface. The used surface chemistry showed low non-specific binding of the virions and gave reproducible signals. The binding of H1N1 virus was monitored in RIfS and verified by atomic force microscopy (AFM) measurements. The proposed assay has the potential to be integrated in portable and miniaturized devices based on RIfS, which can be used in point of care testing at e.g. airports to improve homeland security.

A label-free optical detection method for biosensors and microfluidics

A label-free detection method is developed for biosensors and for microfluidic applications. Multiple optical modes, including a conventional surface plasmon resonance (SPR) mode and many bulk modes, are created in a microfluidic channel with metallic coatings on top and bottom. The bulk modes are bulk sensitive and detect analytes in the channel with 20 times higher sensitivity than the SPR, which is particularly suitable for microfluidic devices. The method can be operated either in the SPR mode for detection of molecular binding on a surface or in one of the bulk optical modes for monitoring analytes in the channel.

Label-free parallel screening of combinatorial triazine libraries using reflectometric interference spectroscopy.

The parallel reflectometric interference spectroscopy is presented as a label-free optical detection method. A new setup was adapted to accommodate sample carriers in a 96-well microplate. It allows for the first time simultaneous plate imaging by a CCD camera for the parallel detection of specific biomolecular interaction in the microplate wells at heterogeneous phase using direct optical monitoring. The detection of binding events with time resolution enables a highly parallel functional biomolecular interaction analysis (BIA). The combination of this new screening setup with combinatorial solid-phase synthesis is performed in the wells of glass-bottom microplates to accomplish the synthesis and the screening platform within one device. As a model system for a solid-phase substance library, synthesis of a triazine library and the subsequent BIA with four different antibodies were carried out. The presented setup enables a time resolution of 18 s with a total screening time of less than 35 min including baseline adjustment, BIA, and regeneration of the screening device for 96 samples in parallel. The binding studies reveal a fast classification of the different monoclonal and polyclonal antibodies and enable the detection of triazines with high binding affinity. The presented prototype is the first parallelized optical label-free detection system for biomolecular interaction analysis that is suitable for a high-throughput screening based on the 96-well microplate format.