Erich A. Lidstone

Nanostructured optical photonic crystal biosensor for HIV viral load measurement

Detecting and quantifying biomarkers and viruses in biological samples have broad applications in early disease diagnosis and treatment monitoring. We have demonstrated a label-free optical sensing mechanism using nanostructured photonic crystals (PC) to capture and quantify intact viruses (HIV-1) from biologically relevant samples. The nanostructured surface of the PC biosensor resonantly reflects a narrow wavelength band during illumination with a broadband light source. Surface-adsorbed biotarget induces a shift in the resonant Peak Wavelength Value (PWV) that is detectable with <10 pm wavelength resolution, enabling detection of both biomolecular layers and small number of viruses that sparsely populate the transducer surface. We have successfully captured and detected HIV-1 in serum and phosphate buffered saline (PBS) samples with viral loads ranging from 104 to 108 copies/mL. The surface density of immobilized biomolecular layers used in the sensor functionalization process, including 3-mercaptopropyltrimethoxysilane (3-MPS), N-gamma-Maleimidobutyryl-oxysuccinimide ester (GMBS), NeutrAvidin, anti-gp120, and bovine serum albumin (BSA) were also quantified by the PC biosensor.

Cytotoxicity screening of Bangladeshi medicinal plant extracts on pancreatic cancer cells

BackgroundThere has been a long standing interest in the identification of medicinal plants and derived natural products for developing cancer therapeutics. Our study focuses upon pancreatic cancer, due to its high mortality rate, that is attributed in part to the lack of an effective chemotherapeutic agent. Previous reports on the use of medicinal plant extracts either alone or alongside conventional anticancer agents in the treatment of this cancer have shown promising results. This work aims to investigate the therapeutic properties of a library of medicinal plants from Bangladesh.Methods56 extracts of 44 unique medicinal plants were studied. The extracts were screened for cytotoxicity against the pancreatic adenocarcinoma cell line Panc-1, using a label-free biosensor assay. The top cytotoxic extracts identified in this screen were tested on two additional pancreatic cancer cell lines (Mia-Paca2 and Capan-1) and a fibroblast cell line (Hs68) using an MTT proliferation assay. Finally, one of the most promising extracts was studied using a caspase-3 colorimetric assay to identify induction of apoptosis.ResultsCrude extracts of Petunia punctata, Alternanthera sessilis, and Amoora chittagonga showed cytotoxicity to three cancer cell lines with IC50 values ranging between 20.3 - 31.4 μg/mL, 13.08 - 34.9 μg/mL, and 42.8 - 49.8 μg/mL, respectively. Furthermore, treatment of Panc-1 cells with Petunia punctata was shown to increase caspase-3 activity, indicating that the observed cytotoxicity was mediated via apoptosis. Only Amoora chittagonga showed low cytotoxicity to fibroblast cells with an IC50 value > 100 μg/mL.ConclusionBased upon the initial screening work reported here, further studies aimed at the identification of active components of these three extracts and the elucidation of their mechanisms as cancer therapeutics are warranted.

Identifying Modulators of Protein−Protein Interactions Using Photonic Crystal Biosensors

Inhibitors and activators of protein-protein interactions are valuable as biological probes and medicinal agents but are often difficult to identify. Herein we describe a high-throughput assay, based upon photonic crystal (PC) biosensors, for the identification of modulators of protein-protein interactions. Through the use of a d-biotin-tris-NTA (BTN) hybrid compound, any His6-tagged protein can be immobilized on the surface of a PC biosensor. Binding of the bound protein to its cognate partner is detected via a shift in the peak wavelength value. We demonstrate this assay with three protein-protein pairs (caspase-9-XIAP, caspase-7-XIAP, FKBP12-FRB) and their small molecule modulators.

Label-free imaging of cell attachment with photonic crystal enhanced microscopy

We introduce photonic crystal enhanced microscopy (PCEM) as a label-free biosensor imaging technique capable of measuring cell surface attachment and attachment modulation. The approach uses a photonic crystal optical resonator surface incorporated into conventional microplate wells and a microscope-based detection instrument that measures shifts in the resonant coupling conditions caused by localized changes in dielectric permittivity at the cell-sensor interface. Four model systems are demonstrated for studying cancer cells, primary cardiac muscle cells, and stem cells. First, HepG2/C3 hepatic carcinoma cells were cultured and observed via PCEM in order to characterize cell adhesion in the context of growth and locomotion. Second, Panc-1 pancreatic cancer cells were used to verify that cell attachment density decreases in response to staurosporine, a drug that induces apoptosis. Third, we used PCEM to confirm the influence of integrin-mediated signaling on primary neonatal cardiomyocyte growth and development. Rounded cardiomyocytes consistently showed decreased cell attachment density as recorded via PCEM, while spreading cells exhibited greater attachment strength as well as increased contractility. Finally, PCEM was used to monitor the morphological changes and extracellular matrix remodeling of porcine adipose-derived stem cells subjected to a forced differentiation protocol. Each of these experiments yielded information regarding cell attachment density without the use of potentially cytotoxic labels, enabling study of the same cells for up to several days.

A Method for Identifying Small-Molecule Aggregators Using Photonic Crystal Biosensor Microplates

Small molecules identified through high-throughput screens are an essential element in pharmaceutical discovery programs. It is now recognized that a substantial fraction of small molecules exhibit aggregating behavior leading to false positive results in many screening assays, typically due to nonspecific attachment to target proteins. Therefore, the ability to efficiently identify compounds within a screening library that aggregate can streamline the screening process by eliminating unsuitable molecules from further consideration. In this work, we show that photonic crystal (PC) optical biosensor microplate technology can be used to identify and quantify small-molecule aggregation. A group of aggregators and nonaggregators were tested using the PC technology, and measurements were compared with those gathered by three alternative methods: dynamic light scattering (DLS), an α-chymotrypsin colorimetric assay, and scanning electron microscopy (SEM). The PC biosensor measurements of aggregation were confirmed by visual observation using SEM, and were in general agreement with the α-chymotrypsin assay. DLS measurements, in contrast, demonstrated inconsistent readings for many compounds that are found to form aggregates in shapes, very different from the classical spherical particles assumed in DLS modeling. As a label-free detection method, the PC biosensor aggregation assay is simple to implement and provides a quantitative direct measurement of the mass density of material adsorbed to the transducer surface, whereas the microplate-based sensor format enables compatibility with high-throughput automated liquid-handling methods used in pharmaceutical screening.

Recognition of apoptotic cells by viable cells is specific, ubiquitous, and species independent: analysis using photonic crystal biosensors

Apoptotic recognition is linked to profound anti-inflammatory and immunosuppressive responses. A sensitive photonic crystal biosensor method for the assessment of apoptotic recognition shows that apoptotic recognition occurs in a strikingly species-independent manner, with obligate cytoskeletal involvement.

A method for identifying small molecule aggregators using photonic crystal biosensor microplates

Small molecules identified through high-throughput screens are an essential element in pharmaceutical discovery programs. It is now recognized that a substantial fraction of small molecules exhibit aggregating behavior leading to false positive results in many screening assays, typically due to nonspecific attachment to target proteins. Therefore, the ability to efficiently identify compounds within a screening library that aggregate can streamline the screening process by eliminating unsuitable molecules from further consideration. In this work we show that photonic crystal (PC) optical biosensor microplate technology can be utilized to identify and quantify small molecule aggregation. A group of aggregators and nonaggregators were tested using the PC technology, and measurements were compared with those gathered by three alternative methods: dynamic light scattering (DLS), an α-chymotrypsin colorimetric assay, and scanning electron microscopy (SEM). The PC biosensor measurements of aggregation were confirmed by visual observation using SEM, and were in general agreement with the α-chymotrypsin assay. As a label-free detection method, the PC biosensor aggregation assay is simple to implement and provides a quantitative direct measurement of the mass density of material adsorbed to the transducer surface, while the microplate-based sensor format enables compatibility with high-throughput automated liquid handling methods used in pharmaceutical screening.

High resolution imaging of cell adhesion activity using photonic crystal biosensor surfaces

Photonic crystal surfaces combined with an imaging detection instrument are used for label-free quantification of cell adhesion with intra-cell spatial resolution. The adhesion response to chemical stimuli of cancer cells, cardiac cells, and stem cells are monitored.

Photonic Crystal Enhanced Microscopy: Multimode Imaging for Photonic Crystal Biosensors

Photonic Crystal Enhanced Microscopy (PCEM) utilizes the optical resonances of photonic crystal surfaces for label-free biosensor imaging and amplification of fluorescence. We describe the application of PCEM to biomolecular and cell-based assays.