Weili Chen

Single nanoparticle detection using photonic crystal enhanced microscopy

We demonstrate a label-free biosensor imaging approach that utilizes a photonic crystal (PC) surface to detect surface attachment of individual dielectric and metal nanoparticles through measurement of localized shifts in the resonant wavelength and resonant reflection magnitude from the PC. Using a microscopy-based approach to scan the PC resonant reflection properties with 0.6 μm spatial resolution, we show that metal nanoparticles attached to the biosensor surface with strong absorption at the resonant wavelength induce a highly localized reduction in reflection efficiency and are able to be detected by modulation of the resonant wavelength. Experimental demonstrations of single-nanoparticle imaging are supported by finite-difference time-domain computer simulations. The ability to image surface-adsorption of individual nanoparticles offers a route to single molecule biosensing, in which the particles can be functionalized with specific recognition molecules and utilized as tags.

Enhanced sandwich immunoassay using antibody-functionalized magnetic iron-oxide nanoparticles for extraction and detection of soluble transferrin receptor on a photonic crystal biosensor

Iron deficiency anemia (IDA) has detrimental effects on individuals and societies worldwide. A standard sandwich assay (SA) for the detection of soluble transferrin receptor (sTfR), a biomarker of IDA, on a photonic crystal (PC) biosensor was established, but it was susceptible to non-specific signals from complex matrixes. In this study, iron-oxide nanoparticles (fAb-IONs) were used as magnetic immuno-probes to bind sTfR and minimize non-specific signals, while enhancing detection on the PC biosensor. This inverse sandwich assay (IA) method completely bound sTfR with low variability (<4% RSD) in buffer and allowed for its accurate and precise detection in sera (Liquichek™ control sera) on the PC biosensor using two certified ELISAs as reference methods. A linear dose-response curve was elicited at the fAb-IONs concentration in which the theoretical binding ratio (sTfR:fAb-IONs) was calculated to be <1 on the IA. The LoDs for sTfR in the SA and IA were similar (P>0.05) at 14 and 21 μg/mL, respectively. The inherent imprecision of the IA and reference ELISAs was σ(δ)=0.45 µg/mL and the mean biases for Liquichek™ 1, 2 and 3 were 0.18, 0.19 and -0.04 µg/mL, respectively. Whereas the inherent imprecision of the SA and reference ELISAs was σ(δ)=0.52 µg/mL and the biases for Liquichek™ 1, 2 and 3 were 0.66, 0.14 and -0.67 µg/mL, respectively. Thus, unlike the SA, the IA method measures sTfR with the same bias as the reference ELISAs. Combined magnetic separation and detection of nutrition biomarkers on PC biosensors represents a facile method for their accurate and reliable quantification in complex matrixes.

Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy

We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives.

Smartphone-Imaged HIV-1 Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) on a Chip from Whole Blood

Viral load measurements are an essential tool for the long-term clinical care of hum an immunodeficiency virus (HIV)-positive individuals. The gold standards in viral load instrumentation, however, are still too limited by their size, cost, and sophisticated operation for these measurements to be ubiquitous in remote settings with poor healthcare infrastructure, including parts of the world that are disproportionately affected by HIV infection. The challenge of developing a point-of-care platform capable of making viral load more accessible has been frequently approached but no solution has yet emerged that meets the practical requirements of low cost, portability, and ease-of-use. In this paper, we perform reverse-transcription loop-mediated isothermal amplification (RT-LAMP) on minimally processed HIV-spiked whole blood samples with a microfluidic and silicon microchip platform, and perform fluorescence measurements with a consumer smartphone. Our integrated assay shows amplification from as few as three viruses in a ~ 60 nL RT-LAMP droplet, corresponding to a whole blood concentration of 670 viruses per µL of whole blood. The technology contains greater power in a digital RT-LAMP approach that could be scaled up for the determination of viral load from a finger prick of blood in the clinical care of HIV-positive individuals. We demonstrate that all aspects of this viral load approach, from a drop of blood to imaging the RT-LAMP reaction, are compatible with lab-on-a-chip components and mobile instrumentation.

Hands-free smartphone-based diagnostics for simultaneous detection of Zika, Chikungunya, and Dengue at point-of-care

Infectious diseases remain the world’s top contributors to death and disability, and, with recent outbreaks of Zika virus infections there has been an urgency for simple, sensitive and easily translatable point-of-care tests. Here we demonstrate a novel point-of-care platform to diagnose infectious diseases from whole blood samples. A microfluidic platform performs minimal sample processing in a user-friendly diagnostics card followed by real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) on the same card with pre-dried primers specific to viral targets. Our point-of-care platform uses a commercial smartphone to acquire real-time images of the amplification reaction and displays a visual read-out of the assay. We apply this system to detect closely related Zika, Dengue (types 1 and 3) and Chikungunya virus infections from whole blood on the same pre-printed chip with high specificity and clinically relevant sensitivity. Limit of detection of 1.56e5 PFU/mL of Zika virus from whole blood was achieved through our platform. With the ability to quantitate the target nucleic acid, this platform can also perform point-of-care patient surveillance for pathogen load or select biomarkers in whole blood.

Protein-protein binding detection with nanoparticle photonic crystal enhanced microscopy (NP-PCEM).

We demonstrate a novel microscopy-based biosensing approach that utilizes a photonic crystal (PC) surface to detect protein-protein binding with the functionalized nanoparticles as tags. This imaging approach utilizes the measurement of localized shifts in the resonant wavelength and resonant reflection magnitude from the PC biosensor in the presence of individual nanoparticles. Moreover, it substantially increases the sensitivity of the imaging approach through tunable localized surface plasmon resonant frequency of the nanoparticle matching with the resonance of the PC biosensor. Experimental demonstrations of photonic crystal enhanced microscopy (PCEM) imaging with single nanoparticle resolution are supported by Finite-Difference Time-Domain (FDTD) computer simulations. The ability to detect the surface adsorption of individual nanoparticles as tags offers a route to single molecule biosensing with photonic crystal biosensor in the future.

Multiplexed detection of infectious diseases with microfluidic loop-mediated isothermal amplification and a smartphone

New tools are needed to enable rapid detection, identification, and reporting of infectious viral and microbial pathogens in a wide variety of point-of-care applications that impact human and animal health. We report the design, construction, and characterization of a platform for multiplexed analysis of disease-specific DNA sequences that utilizes a smartphone camera as the sensor in conjunction with a handheld instrument that interfaces the phone with a silicon-based microfluidic chip. Utilizing specific nucleic acid sequences for four equine respiratory pathogens as representative examples, we demonstrated the ability of the system to use a single 15-μL droplet of test sample to perform selective positive/negative determination of target sequences, including integrated experimental controls, in approximately 30 minutes. The system achieves detection limits comparable to those obtained by laboratory-based methods and instruments.

Photonic crystal enhanced microscopy

By modifying a microscope to perform hyperspectral imaging of reflectance from a photonic crystal, we describe a new microscopy approach that enables quantitative, spatially resolved imaging of the interaction of cells and nanoparticles with surfaces.

Detection of single nanoparticles using photonic crystal enhanced microscopy

We demonstrate a label-free biosensor imaging approach that utilizes a photonic-crystal surface to detect attachment of individual nanoparticles down to ~65×30×30nm3. Matching nanoparticle plasmon resonant-frequency to the photonic-crystal resonance substantially increases sensitivity of the approach.