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Rapid, simple, inexpensive, accurate, and sensitive point-of-care (POC) detection of viral pathogens in bodily fluids is a vital component of controlling the spread of infectious diseases. The predominant laboratory-based methods for sample processing and nucleic acid detection face limitations that prevent them from gaining wide adoption for POC applications in low-resource settings and self-testing scenarios. Here, we report the design and characterization of an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a 2-stage microfluidic cartridge for sample processing and nucleic acid amplification, and a clip-on detection instrument that interfaces with the image sensor of a smartphone. The cartridge is designed to release viral RNA from Zika virus in whole blood using chemical lysis, followed by mixing with the assay buffer for performing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) reactions in six parallel microfluidic compartments. The battery-powered handheld detection instrument uniformly heats the compartments from below, and an array of LEDs illuminates from above, while the generation of fluorescent reporters in the compartments is kinetically monitored by collecting a series of smartphone images. We characterize the assay time and detection limits for detecting Zika RNA and gamma ray-deactivated Zika virus spiked into buffer and whole blood and compare the performance of the same assay when conducted in conventional PCR tubes. Our approach for kinetic monitoring of the fluorescence-generating process in the microfluidic compartments enables spatial analysis of early fluorescent "bloom" events for positive samples, in an approach called "Spatial LAMP" (S-LAMP). We show that S-LAMP image analysis reduces the time required to designate an assay as a positive test, compared to conventional analysis of the average fluorescent intensity of the entire compartment. S-LAMP enables the RT-LAMP process to be as short as 22 minutes, resulting in a total sample-to-answer time in the range of 17–32 minutes to distinguish positive from negative samples, while demonstrating a viral RNA detection as low as 2.70 × 102 copies per μl, and a gamma-irradiated virus of 103 virus particles in a single 12.5 μl droplet blood sample. [ABSTRACT FROM AUTHOR]

One of the frontiers in the field of biosensors is the ability to quantify specific target molecules with enough precision to count individual units in a test sample, and to observe the characteristics of individual biomolecular interactions. Technologies that enable observation of molecules with "digital precision" have applications for in vitro diagnostics with ultra-sensitive limits of detection, characterization of biomolecular binding kinetics with a greater degree of precision, and gaining deeper insights into biological processes through quantification of molecules in complex specimens that would otherwise be unobservable. In this review, we seek to capture the current state-of-the-art in the field of digital resolution biosensing. We describe the capabilities of commercially available technology platforms, as well as capabilities that have been described in published literature. We highlight approaches that utilize enzymatic amplification, nanoparticle tags, chemical tags, as well as label-free biosensing methods. [ABSTRACT FROM AUTHOR]

Rapid, sensitive and specific detection and reporting of infectious pathogens is important for patient management and epidemic surveillance. We demonstrated a point-of-care system integrated with a smartphone for detecting live virus from nasal swab media, using a panel of equine respiratory infectious diseases as a model system for corresponding human diseases such as COVID-19. Specific nucleic acid sequences of five pathogens were amplified by loop-mediated isothermal amplification on a microfluidic chip and detected at the end of reactions by the smartphone. Pathogen-spiked horse nasal swab samples were correctly diagnosed using our system, with a limit of detection comparable to that of the traditional lab-based test, polymerase chain reaction, with results achieved in ∼30 minutes. [ABSTRACT FROM AUTHOR]

We demonstrate a rapid, 2-step, and ultrasensitive assay approach for quantification of target protein molecules from a single droplet test sample. The assay is comprised of antibody-conjugated gold nanoparticles (AuNPs) that are "activated" when they are mixed with the test sample and bind their targets. The resulting liquid is passed through a microfluidic channel with a photonic crystal (PC) biosensor that is functionalized with secondary antibodies to the target biomarker, so that only activated AuNPs are captured. Utilizing recently demonstrated hybrid optical coupling between the plasmon resonance of the AuNP and the resonance of the PC, each captured AuNP efficiently quenches the resonant reflection of the PC, thus enabling the captured AuNPs to be digitally counted with high signal-to-noise. To achieve a 2-step assay process that is performed on a single droplet test sample without washing steps or active pump elements, controlled single-pass flow rate is obtained with an absorbing paper pad waste reservoir embedded in a microfluidic cartridge. We use the activate capture and digital counting (AC + DC) approach to demonstrate HIV-1 capsid antigen p24 detection from a 40 μL spiked-in human serum sample at a one thousand-fold dynamic range (1–103 pg mL−1) with only a 35-minute process that is compatible with point-of-care (POC) analysis. The AC + DC approach allows for ultrasensitive and ultrafast biomolecule detection, with potential applications in infectious disease diagnostics and early stage disease monitoring. [ABSTRACT FROM AUTHOR]

We demonstrate a smartphone-integrated handheld detection instrument capable of utilizing the internal rear-facing camera as a high-resolution spectrometer for measuring the colorimetric absorption spectrum, fluorescence emission spectrum, and resonant reflection spectrum from a microfluidic cartridge inserted into the measurement light path. Under user selection, the instrument gathers light from either the white “flash” LED of the smartphone or an integrated green laser diode to direct illumination into a liquid test sample or onto a photonic crystal biosensor. Light emerging from each type of assay is gathered via optical fiber and passed through a diffraction grating placed directly over the smartphone camera to generate spectra from the assay when an image is collected. Each sensing modality is associated with a unique configuration of a microfluidic “stick” containing a linear array of liquid chambers that are swiped through the instrument while the smartphone captures video and the software automatically selects spectra representative of each compartment. The system is demonstrated for representative assays in the field of point-of-care (POC) maternal and infant health: an ELISA assay for the fetal fibronectin protein used as an indicator for pre-term birth and a fluorescent assay for phenylalanine as an indicator for phenylketonuria. In each case, the TRI-analyzer is capable of achieving limits of detection that are comparable to those obtained for the same assay measured with a conventional laboratory microplate reader, demonstrating the flexibility of the system to serve as a platform for rapid, simple translation of existing commercially available biosensing assays to a POC setting. [ABSTRACT FROM AUTHOR]

Biosensors are extensively employed for diagnosing a broad array of diseases and disorders in clinical settings worldwide. The implementation of biosensors at the point-of-care (POC), such as at primary clinics or the bedside, faces impediments because they may require highly trained personnel, have long assay times, large sizes, and high instrumental cost. Thus, there exists a need to develop inexpensive, reliable, user-friendly, and compact biosensing systems at the POC. Biosensors incorporated with photonic crystal (PC) structures hold promise to address many of the aforementioned challenges facing the development of new POC diagnostics. Currently, PC-based biosensors have been employed for detecting a variety of biotargets, such as cells, pathogens, proteins, antibodies, and nucleic acids, with high efficiency and selectivity. In this review, we provide a broad overview of PCs by explaining their structures, fabrication techniques, and sensing principles. Furthermore, we discuss recent applications of PC-based biosensors incorporated with emerging technologies, including telemedicine, flexible and wearable sensing, smart materials and metamaterials. Finally, we discuss current challenges associated with existing biosensors, and provide an outlook for PC-based biosensors and their promise at the POC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A form of microscopy that utilizes a photonic crystal biosensor surface as a substrate for cell attachment enables label-free, quantitative, submicron resolution, time-resolved imaging of cell–surface interactions without cytotoxic staining agents or temporally-unstable fluorophores. Other forms of microscopy do not provide this direct measurement of live cell–surface attachment localization and strength that includes unique, dynamic morphological signatures critical to the investigation of important biological phenomena such as stem cell differentiation, chemotaxis, apoptosis, and metastasis. Here, we introduce Photonic Crystal Enhanced Microscopy (PCEM), and apply it to the study of murine dental stem cells to image the evolution of cell attachment and morphology during chemotaxis and drug-induced apoptosis. PCEM provides rich, dynamic information about the evolution of cell–surface attachment profiles over biologically relevant time-scales. Critically, this method retains the ability to monitor cell behavior with spatial resolution sufficient for observing both attachment footprints of filopodial extensions and intracellular attachment strength gradients. [ABSTRACT FROM AUTHOR]

Enhancement of the fluorescent output of surface-based fluorescence assays by performing them upon nanostructured photonic crystal (PC) surfaces has been demonstrated to increase signal intensities by ＞8000×. Using the multiplicative effects of optical resonant coupling to the PC in increasing the electric field intensity experienced by fluorescent labels (“enhanced excitation”) and the spatially biased funneling of fluorophore emissions through coupling to PC resonances (“enhanced extraction”), PC enhanced fluorescence (PCEF) can be adapted to reduce the limits of detection of disease biomarker assays, and to reduce the size and cost of high sensitivity detection instrumentation. In this work, we demonstrate the first silicon-based PCEF detection platform for multiplexed biomarker assay. The sensor in this platform is a silicon-based PC structure, comprised of a SiO2 grating that is overcoated with a thin film of high refractive index TiO2 and is produced in a semiconductor foundry for low cost, uniform, and reproducible manufacturing. The compact detection instrument that completes this platform was designed to efficiently couple fluorescence excitation from a semiconductor laser to the resonant optical modes of the PC, resulting in elevated electric field strength that is highly concentrated within the region ＜100 nm from the PC surface. This instrument utilizes a cylindrically focused line to scan a microarray in ＜1 min. To demonstrate the capabilities of this sensor-detector platform, microspot fluorescent sandwich immunoassays using secondary antibodies labeled with Cy5 for two cancer biomarkers (TNF-α and IL-3) were performed. Biomarkers were detected at concentrations as low as 0.1 pM. In a fluorescent microarray for detection of a breast cancer miRNA biomarker miR-21, the miRNA was detectable at a concentration of 0.6 pM. [ABSTRACT FROM AUTHOR]

This paper reports the design, fabrication and validation of a microfluidic well plate for combinatorial screening applications. Each well within the array is comprised of two 200 picoliter compartments that each contain a photonic crystal biosensor to enable the on-chip, in situdetection of (bio-) molecular binding events. This microfluidic chip utilizes arrays of Actuate-to-Open valves to isolate all compartments, which allows the chip to be decoupled from pneumatic control lines and thus to be transported freely between filling, sensing and characterization platforms. A proof-of-principle 4 × 4 protein/antibody binding assay was performed to demonstrate the discrete mixing and on-chip sensing capabilities. [ABSTRACT FROM AUTHOR]

Utilizing its integrated camera as a spectrometer, we demonstrate the use of a smartphone as the detection instrument for a label-free photonic crystal biosensor. A custom-designed cradle holds the smartphone in fixed alignment with optical components, allowing for accurate and repeatable measurements of shifts in the resonant wavelength of the sensor. Externally provided broadband light incident upon an entrance pinhole is subsequently collimated and linearly polarized before passing through the biosensor, which resonantly reflects only a narrow band of wavelengths. A diffraction grating spreads the remaining wavelengths over the camera's pixels to display a high resolution transmission spectrum. The photonic crystal biosensor is fabricated on a plastic substrate and attached to a standard glass microscope slide that can easily be removed and replaced within the optical path. A custom software app was developed to convert the camera images into the photonic crystal transmission spectrum in the visible wavelength range, including curve-fitting analysis that computes the photonic crystal resonant wavelength with 0.009 nm accuracy. We demonstrate the functionality of the system through detection of an immobilized protein monolayer, and selective detection of concentration-dependent antibody binding to a functionalized photonic crystal. We envision the capability for an inexpensive, handheld biosensor instrument with web connectivity to enable point-of-care sensing in environments that have not been practical previously.

Utilizing a tunable photonic crystal resonant reflector as a mirror of an external cavity laser cavity, we demonstrate a new type of label-free optical biosensor that achieves a high quality factor through the process of stimulated emission, while at the same time providing high sensitivity and large dynamic range. The photonic crystal is fabricated inexpensively from plastic materials, and its resonant wavelength is tuned by adsorption of biomolecules on its surface. Gain for the lasing process is provided by a semiconductor optical amplifier, resulting in a simple detection instrument that operates by normally incident noncontact illumination of the photonic crystal and direct back-reflection into the amplifier. We demonstrate single-mode, biomolecule-induced tuning of the continuous-wave laser wavelength. Because the approach incorporates external optical gain that is separate from the transducer, the device represents a significant advance over previous passive optical resonator biosensors and laser-based biosensors.

In medical facilities, there is strong motivation to develop detection systems that can provide continuous analysis of fluids in medical tubing used to either deliver or remove fluids from a patient's body. Possible applications include systems that increase the safety of intravenous (IV) drug injection and point-of-care health monitoring. In this work, we incorporated a surface-enhanced Raman scattering (SERS) sensor comprised of an array of closely spaced metal nanodomes into flexible tubing commonly used for IV drug delivery and urinary catheters. The nanodome sensor was fabricated by a low-cost, large-area process that enables single use disposable operation. As exemplary demonstrations, the sensor was used to kinetically detect promethazine (pain medication) and urea (urinary metabolite) within their clinically relevant concentration ranges. Distinct SERS peaks for each analyte were used to demonstrate separate detection and co-detection of the analytes.

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.

Biosensor technologies that have been primarily used in the past for characterizing biomolecular interactions are now being used to develop new approaches for performing cell-based assays. Biosensors monitor cell attachment to a transducer surface, and thus provide information that is fundamentally different from that provided by microscopy, as the sensor is capable of monitoring temporal evolution of integrin-surface interactions that are difficult to measure by other means. Label-free biosensor technologies are especially advantageous for monitoring the behavior of cells because they do not require stains that typically result in cell death, and are not subject to effects such as photobleaching. As a result, cells can be quantitatively monitored in their culture environment over an extended period of time while processes such as proliferation, apoptosis, cytotoxicity, chemotaxis, ion channel activation, and membrane-bound protein activation are modulated by the introduction of a variety of chemical or biological stimuli. This review describes the application of photonic crystal optical biosensor microplates to a variety of cell-based assays. Detection instruments for photonic crystals measure the aggregate behavior of large cell populations, or, using recently developed biosensor imaging detection, independent monitoring of individual cells. These technological developments offer the ability to perform assays with a limited number of available cells for applications such as high throughput screening with primary cells or stem cells., (This journal is © The Royal Society of Chemistry 2011)

A nanoreplica molding process has been used to produce polymer microfluidic channels, with integrated label-free photonic crystal biosensors as the bottom surface of the channels. Multiple flow channels are gathered in parallel so that an imaging detection instrument may simultaneously monitor the binding kinetics of many biomolecular interactions. In this work, the flow channel pattern has been adapted to a 96-well microplate format in which, for each 12-element row of the microplate, a single well serves as a common access port for 11 flow channels that are connected to separate microplate wells. Application of pneumatic pressure or suction to the common well serves to drive forward or backward flow to the channels. The system is demonstrated by measuring the kinetic binding interaction of protein A with IgG molecules of high, medium, and low affinity. The approach offers a means for minimizing the volume of reagent required to functionalize the biosensor surface, while retaining compatibility with the microplate assay fluid-handling methods that are most commonly used in biological research.

A method for simultaneously integrating label-free photonic crystal biosensor technology into microfluidic channels by a single-step replica molding process is presented. By fabricating both the sub-micron features of the photonic crystal sensor structure and the >10 microm features of a flow channel network in one step at room temperature on a plastic substrate, the sensors are automatically self-aligned with the flow channels, and patterns of arbitrary shape may be produced. By measuring changes in the resonant peak reflected wavelength from the photonic crystal structure induced by changes in dielectric permittivity within an evanescent field region near its surface, detection of bulk refractive index changes in the fluid channel or adsorption of biological material to the sensor surface is demonstrated. An imaging detection instrument is used to characterize the spatial distribution of the photonic crystal resonant wavelength, gathering thousands of independent sensor readings within a single fluid channel.